Nghien Cuu Gen Tren Ecoli

I HC THI NGUYN TRNG I HC S PHM --------------------------------------------
HONG PH HIP
NGHIN CU BIU HIN GEN M HA CHT HOT HA PLASMINOGEN M CA NGI TRONG VI KHUN Echerichia coli
LUN VN THC S SINH HC
Thi Nguyn 2008
I HC THI NGUYN TRNG I HC S PHM --------------------------------------------
HONG PH HIP
NGHIN CU BIU HIN GEN M HA CHT HOT HA PLASMINOGEN M CA NGI TRONG VI KHUN Echerichia coli
Chuyn ngnh : Di truyn hc M s : 60.42.70

LUN VN THC S SINH HC
Ngi hng dn khoa hc: TS. L TH THU HIN
Thi Nguyn - 2008
MC LC
Trang Bng vit tt ............................................................................................................. 1 Danh mc cc hnh ................................................................................................... 2 M u .................................................................................................................... 3 Chng 1: Tng quan ti liu ............................................................................... 5 1.1. Bnh tim mch ............................................................................................. 5 1.2. Cht hot ha plasminogen.......................................................................... 5 1.3. Cht hot ha plasminogen m ngi ......................................................... 9 1.4. Vai tr ca cht hot ha plasminogen m trong qu trnh lm tan mu ng ............................................................................................. 12 1.5. Nghin cu ng dng sn xut cht hot ha plasminogen m ngi ......................................................................................................... 14 1.6. Tnh hnh nghin cu Vit Nam ............................................................... 18 Chng 2: Vt liu v phng php nghin cu ................................................ 19 2.1. Vt liu, ha cht v thit b ........................................................................ 19 2.1.1. Vt liu ..................................................................................................... 19 2.1.2. Ha cht .................................................................................................... 20 2.1.3. Thit b ...................................................................................................... 20 2.2. Phng php nghin cu ............................................................................. 20 2.2.1. in di DNA trn gel agarose .................................................................. 20 2.2.2. in di protein trn gel polyacrylamide ................................................... 21 2.2.3. Phn ng dy chuyn polymerase (Polymerase Chain Reaction - PCR) ........................................................................................ 22 2.2.4. X l DNA bng enzyme hn ch ............................................................ 23 2.2.5. Ghp ni DNA .......................................................................................... 23 2.2.6. Bin np DNA plasmid vo t bo vi khun E. coli bng phng php sc nhit .............................................................................. 24 2.2.6.1. Chun b t bo kh bin E. coli ............................................................ 24
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2.2.6.2. Bin np DNA plasmid vo t bo E. coli............................................. 24 2.2.7. Tch chit v tinh sch DNA plasmid ...................................................... 25 2.2.8. Xc nh trnh t gen ................................................................................ 26 2.2.9. Biu hin protein trong vi khun E. coli................................................... 27 Chng 3: Kt qu v tho lun ........................................................................... 28 3.1. Thit k vector biu hin mang cDNA m ha h-tPA................................. 28 3.1.1. Nhn on cDNA m ha h-tPA bng k thut PCR ............................... 28 3.1.2. Ghp ni cDNA m ha h-tPA vo vector pGEX6p1 v pET21a(+) ..................................................................................................... 30 3.1.2.1. X l sn phm PCR cDNA m ha h-tPA bng enzyme hn ch .......................................................................................................... 30 3.1.2.2. X l vector pGEX6p1 v pET21a(+) bng enzyme hn ch ..................... 31 3.1.2.3. Ghp ni cDNA m ha h-tPA vo vector pGEX6p1 v pET21a(+) ..................................................................................................... 32 3.1.3. Chn dng pGEX6p1 v pET21a(+) cha cDNA m ha h-tPA ................. 32 3.1.3.1. Bin np sn phm lai vo t bo E. coli bng phng php sc nhit ................................................................................................ 32 3.1.3.2. Chn dng pGEX6p1 v pET21a(+) cha cDNA m ho h-tPA ............................................................................................................. 33 3.1.4. Xc nh v phn tch trnh t cDNA m ha h-tPA ............................... 37 3.2. Biu hin gen ............................................................................................... 43 3.2.1. Tng hp protein h-tPA ti t hp ........................................................... 43 3.2.2. Kim tra protein bng phng php in di trn SDS-PAGE ................. 43 Kt lun v ngh ................................................................................................ 47 Ti liu tham kho ................................................................................................. 48 Ph lc ..................................................................................................................... 54
BNG VIT TT Amp Bp CIAP ddNTP DNA Ampicillin Cp baz (Base pair) Calf Intestinal Alkaline Phosphatase Dideoxynucleoside triphosphate Deoxyribonucleic acid Deoxynucleoside triphosphate Escherichia coli Ethylene Diamine Tetraacetic Acid Vng nhn t sinh trng biu b (Epidermal growth factor region) Ethidium bromide Vng ngn tay (Finger) Glutathione S-transferase Cht hot ha plasminogen m ngi (Human tissue plasminogen activator) kb LB P PA PAI Kilo base Luria Bertani Vng serine protease (Serine protease) Cht hot ha plasmingen (Plasminogen activator) Cht c ch cht hot ha plasminogen (Plasminogen activator inhibitor) Phn ng dy chuyn polymerase (Polymerase Chain Reaction) DNA ti t hp (Recombinant DNA) Ribonucleic acid Ribonuclease Cht hot ha urokinase si n (single chain urokinase - type plasminogen activator) Sodium Dodecyl Sulphate Streptokiase Tris-acetate- EDTA Cht hot ha plasminogen m (Tissue - type plasminogen activator) Cht hot ha urokinase (Urokinase - type plasminogen activator) Vng/ pht
dNTP E. coli EDTA EGF
PCR rDNA RNA RNase
EtBr F GST h- tPA
scuPA SDS SK TAE
IPTG
Isopropyl b-D thiogalactoside tPA
K1 K2
Vng Kringle 1 Vng Kringle 2
uPA v/p
DANH MC CC HNH Hnh 1.1 1.2 1.3 1.4 1.5 2.1 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 Tn hnh Cu trc ca tPA v uPA Cc vng ca tPA Cu trc h-tPA Qu trnh phn hy huyt khi Con ng phn gii t mu (Fibrin) Bn vector pET21a(+) v pGEX6p1 Sn phm PCR nhn cDNA m ha h-tPA Kt qu x l cDNA m ha h-tPA v cc vector biu hin bng enzyme hn ch Chn dng pGEX6p1 mang cDNA m ha h-tPA (pGEX6p1/h-tPA) Chn dng pET21a(+) mang cDNA m ha h-tPA (pET21a(+)/h-tPA) Kim tra cc vector ti t hp mang cDNA m ha h-tPA bng enzyme hn ch Kim tra cc vector ti t hp mang cDNA m ha h-tPA bng k thut PCR So snh trnh t nucleotide ca h-tPA trong pGEX6p1/htPA v pET21a(+)/h-tPA vi NM_000930 Kim tra kim tra protein h-tPA trong pGEX6p1/h-tPA p3 Kim tra protein h-tPA trong pET21a(+)/h-tPA p1 42 44 45 37
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M U
1. L do chn ti Bt u t nhng nm u thp nin 1970, cc thnh tu v sinh hc phn t v k thut di truyn gip chng ta c th hiu r bn cht phn t ca cc bnh xut hin trn ng vt, thc vt v c bit l trn ngi. Gio s Paul Berg thuc trng i hc Stanford (Hoa K), ngi c nhn gii Nobel ha hc nm 1980, l ngi u tin to ra DNA ti t hp (recombinant DNA- rDNA) vo nm 1972. K t n nay, cng ngh rDNA v nhng ng dng trong ngnh cng nghip dc phm thc y s pht trin mnh m ca cc cng ty cng ngh sinh hc v cc dc phm to ra nh cng ngh rDNA (dc phm sinh hc) nhm phc v v bo v sc khe con ngi [44]. Trong lnh vc nghin cu to cc loi dc phm lm tan cc cc mu ng iu tr huyt khi v cc ri lon huyt khi tc mch, cui nhng nm 1980, thng qua cng ngh rDNA, cc nh khoa hc nghin cu v to ra mt s loi dc phm sinh hc l cc protein ti t hp c kh nng lm tan cc cc mu ng c hiu. Mt trong nhng protein , cht hot ha plasminogen m ca ngi (human tissue plasminogen activator- h-tPA) c nhiu nhm tc gi trn th gii nghin cu v a vo ng dng trong y dc. DNA b sung (complementary DNA- cDNA) ca gen m ha h-tPA c phn lp, to dng, biu hin nhiu loi t bo nh t bo trng chut ng (Chinese hamster ovary- CHO), t bo thn chut cha trng thnh... v sn xut di dng dc phm ti t hp [39]. Vit Nam, nghin cu to cc dc phm bng cng ngh sinh hc ang bt u c tip cn. Vic nghin cu biu hin gen m ha cht hot ha plasminogen m trong vi khun Escherichia coli tin ti sn xut dc phm sinh hc phc v bo v sc khe con ngi c ngha khoa hc v thc tin cao. Xut pht t cc l do trn, chng ti tin hnh ti: Nghin cu biu hin gen m ha cht hot ha plasminogen m ca ngi trong vi khun Escherichia coli.
2. Mc tiu nghin cu S dng cc k thut sinh hc phn t v cng ngh rDNA thit k vector v biu hin gen m ha h-tPA trong vi khun E. coli nhm tin ti sn xut protein h-tPA ti t hp c gi tr s dng trong y dc. 3. Ni dung nghin cu - To dng gen m ha h-tPA trong cc vector biu hin thch hp. - Biu hin gen m ha h-tPA trong vi khun E. coli. 4. Nhng im mi ca ti Kt qu nghin cu ca ti l c s khoa hc tip tc nghin cu nhm tin ti ng dng sn xut cht hot ha plasminogen m ca ngi - mt dc phm cng ngh sinh hc c gi tr hon ton cha c nghin cu v sn xut nc ta.
CHNG 1:
1.1. Bnh tim mch
TNG QUAN TI LIU
Bnh tim mch l cc bnh lin quan n nhng ri lon nh hng ti tim v cc mch mu bao gm bnh mch vnh, bnh mch mu no, bnh mch mu ngoi bin v tng huyt p. Bnh tim mch l nguyn nhn hng u gy t vong trn th gii. Mi nm, bnh tim mch l nguyn nhn gy t vong cho 17,5 triu ngi v d on s c khong 25 triu ngi b bnh tim mch t vong vo nm 2020. Bnh tim mch cng c d on s l nguyn nhn ln nht gy tn ph cho ngi vo nm 2020. Theo T chc Y t Th gii (WHO), c mi 2 giy c 1 ngi cht v bnh tim mch. C mi 5 giy th c 1 trng hp nhi mu c tim v mi 6 giy th c mt trng hp t qu. Hin c n 300 yu t nguy c kt hp vi bnh mch vnh v t qu s dn n bnh tim mch [60]. Mt trong nhng nguyn nhn sinh l quan trng dn ti cc bnh tim mch l do thnh mch b tn thng, dn ti hnh thnh huyt khi bn trong mch mu. Do vy, iu tr huyt khi l mt trong nhng phng php hiu qu gip lm gim nguy c t vong v cc bin chng cc bnh nhn mc bnh tim mch, c bit l cc bnh nhn t qu v nghn mch mu. Trong phc iu tr huyt khi v bnh tim mch, cht hot ha plasminogen (plasminogen activator, PA) ng vai tr quan trng v c xem l thuc iu tr c hiu qu cao. Cht hot ha plasmminogen c vai tr bin i plasminogen thnh plasmin- l cht phn hy huyt khi (thrombolytic). Plasmin sau s ha tan dn fibrin v fibrinogen t lm tan huyt khi. Bng cng ngh gen, cc PA c nghin cu v a vo sn xut nhm to ra thuc c hiu trong iu tr huyt khi v bnh tim mch. Tuy nhin, do quy trnh sn xut phc tp nn gi thnh ca sn phm ny kh cao. 1.2. Cht hot ha plasminogen Cht hot ha plasminogen c tc dng quan trng trong qu trnh lm tan khi mu ng. Cht hot ha plasminogen l nhm enzyme duy nht chuyn cht
xc tc plasminogen t dng bt hot sang dng hot ng - plasmin. V c tnh ny, mt vi loi plasminogen khc nhau c s dng nhm iu tr cc chng bnh tim mch. Cht hot ha plasminogen gm bn nhm chnh. Mt nhm l cht hot ha plasminogen urokinase (urokinasetype plasminogen activator, uPA). Cht ny c lin quan n khng th urokinase v khng kt hp vi t mu. Bi vy cht hot ha ny c cho rng c th c lin quan n hot ha plasminogen trong pha lng ca huyt thanh. Loi th hai l cht hot ha plasminogen m (tissue-type plasminogen activator, tPA), c kh nng tng tc vi khng th cht hot ha m v kt hp vi t mu. Loi th ba l streptokinase (SK). SK hot ha plasminogen bng c ch trc tip. Cui cng l enzyme phn hy t mu (fibrinolysis) c gii phng nhanh khi huyt tng b tn thng v gii phng mt s cht hot ha vo thnh mch [18], [47]. Cht hot ha plasminogen urokinase l sn phm chnh ca thn, tn ti di dng phn t cht hot ha urokinase si n (single chain urokinase - type plasminogen activator - scuPA) khng hot ng. Cht hot ha plasminogen urokinase c phn lp c hai dng: dng c trng lng phn t cao (khi lng 54,7kDa) v dng c trng lng phn t thp (khi lng 31,5kDa) [18]. Chng c hai chc nng: chc nng chnh l tham gia trong phn gii protein m lin kt v chc nng th hai c bit n vi vai tr iu khin ca tPA nh cht hot ng sinh l trong mu [30]. S hot ha ca scuPA do s phn hy xung quanh bi plasmin trong cu trc hai si dn ti vic tng hot tnh ca plasminogen ln. Thng qua qu trnh ny, mt s lng nh ca plasmin c th xc tc sn phm uPA hot ng, nh hng ti hnh dng ca nhiu plasmin. Cht hot ha plasminogen urokinase c th ch hot ha plasminogen vi s c mt ca fibrin. Tuy nhin, n s khng kt hp vi fibrin v s khng phn hy fibrin. Trong huyt tng ngi, nng khng th uPA t 2 n 7ng/ml. Gi tr cao nht thng c tm thy trong bnh nhn gan mn tnh v ung th gan [9].
Hnh 1.1. Cu trc ca tPA v uPA [9]
Gen m ha tPA v protein ny c nhiu nhm tc gi trn th gii nghin cu v a vo ng dng trong y- dc [31], [40]. Nhng nghin cu gn y cho thy tPA cn ng vai tr quan trng trong h thng thn kinh [49], [56]. ngi v cc ng vt khc, tPA ng vai tr quan trng trong h thng tiu hy fibrin [53], [54]. i vi cc trng hp mc bnh huyt khi, sau khi tPA c a vo c th, di tc dng ca tPA, plasmin s c to ra ch yu ti v tr hnh thnh huyt khi, nn trnh c tnh trng phn hy nhng vng khc trong c th, tuy nhin hot tnh chn lc ny ch l tng i [21]. Streptokinase l mt cht hot ha plasminogen ngoi sinh c khi lng 47kDa. Hin nay, Streptokinase c thu ch yu t nui cy vi khun Streptococci B-hemolytic. Streptokinase c chc nng tng t vi plasminogen ngi. Chc nng chnh l ph thm vo thay i cu trc trong phn t plasminogen, lm trung tm hot ng ca phn t plasminogen ny hot ng hot ha phn t plasminogen th hai bng cch ct phn t plasminogen th hai ti v tr Agr 560-
Val561. Tip theo, plasminogen trong hn hp Streptokinase- plasminogen s c chuyn thnh plasmin, cui cng, hn hp tch nhau hnh thnh dng Streptokinase v plasmin t do. C hai loi ca hn hp Streptokinase u nh hng ngang nhau ti s hot ha ca plasminogen: Streptokinase + plasminogen Streptokinase-plasminogen Streptokinase- plasminogen + plasminogen streptokinase- plasmin + plasmin Streptokinase- plasmin + plasminogen streptokinase- plasmin + plasmin Streptokinase l mt cht hot ha plasminigen khng c hiu. Vic dng Streptokinase dn ti tnh trng phn hy h thng. Mt tc dng khng mong mun ca vic dng Streptokinase l gy cm ng vic p ng ca cht tr ng thng qua vic tng gii phng thrombin. Mc d p ng tr ng xy ra vi tt c cc thuc tan huyt khi, nhng cc s liu in vitro cho thy tc dng ny ln nht vi Streptokinase. Tng ng vi cc nhm hot ha plasminogen, hin nay trn th trng c 5 loi thuc tan huyt khi c php lu hnh: Alteplase tng ng vi cht hot ha plasminogen m, c ngun gc t DNA ti t hp do hng Genentech sn xut [8]; Streptase tng ng vi streptokinase c ngun gc t cu khun tan mu beta c sn xut bi hng Aventis Pharma; Urokinase bit n vi tn l Abbokinase c sn xut bi hng ImaRx Therapeutics, urokinase c ngun gc t cht chit t bo thn ngi; phc cht hot ha Streptokinase plasminogen anisoylat ha (APSAC); v Tenecteplase, mt dng bin i ca cht ha ha plasminogen m ngi gn vi fibrin v lm bin i plasminogen thnh plasmin. Tt c cc dng thuc ny c dng iu tr nhi mu c tim, huyt khi tnh mch su, nghn mch phi, ti thng mch. Tuy nhin, ty tng trng hp, cc loi thuc khc nhau c s dng iu tr mt cch hiu qu v t tn km nht. V d, Streptase l thuc r nht nhng ch c dng cho nhng trng hp c bit v nhiu l do nh thiu tnh c hiu vi si huyt, hiu qu thp, thi gian dng thuc ko di... Cn Alteplase c s dng nhiu hn trong iu tr nghn ng mch phi ln. 8
1.3.
Cht hot ha plasminogen m ngi Cht hot ha plasminogen m ngi l dng ch yu ca dng hot ng
ni sinh ca plasminogen trong mu. N c to ra di dng phn t si n t bo thnh mch trong v c gi trong huyt tng mt cch lin tc hoc gii thot mt cch nhanh chng bng phn ng kch thch ca cht cm ng ca t bo thnh mch mu [39]. Dng hot ng plasminogen c s dng lm tan huyt khi v phng din lm sng cho cc qu trnh iu tr tc mch phi v chng nhi mu c tim [9], [27], [46]. Khng ging nh nhiu protease serine khc, h-tPA hot ng di dng si n, c bit khi c mt ca fibrin hoc fibrinogen. ngi, gen m ha h-tPA l mt gen n bn c v tr 8p12 nm trn nhim sc th s 8 [20]. chut, gen ny cng nm trn nhim sc th s 8 v c v tr 24p22. Cu trc v chc nng ca gen m ha cho h-tPA c xc nh bng kt hp nhn bn invitro ca DNA hng cu t nhng c th ring bit v phn lp c cc dng t th vin gen ngi. Nhng dng ny c xc nh bng bn enzyme hn ch, lai Southern blot v gii trnh t DNA [13], [23]. Kt qu xc nh v phn tch trnh t gen cho thy, gen m ha cht hot ha plasminogen c chiu di 36.594bp, bao gm 32.720bp t v tr khi u n v tr ui polyadenyl, c thm khong 353 v 344 bp ca hai u 5 v 3, mi ba intron xen gia mi bn exon, kch thc trung bnh ca cc exon l 914bp, trong khi ca intron t 111 n 14.257bp [20], [37 ], [41]. Thnh phn base v tn sut dinucleotide trong mRNA ca gen nh sau: A:26,4%; C:23,6%; G:25,3% v T:24,8% v trong trnh t ngc: A:24,8%; C:26,3%; G:22,1% v T:26,9%. Trnh t cp nucleotide nh sau: AA(7,5%), AC(5,2%), AG(8,1%), AT(5,6%), CA(7,7%), CC(6,8%), CG(1,9%), CT(7,3%), GA(6,8%), GC(5,8%), GG(7,6%), GT(5,1%), TA(4,3%), TC(5,8%), TG(7,7%) v TT(6,9%) [20]. Cc kt qu phn tch cho thy cu trc protein h-tPA bao gm 5 vng thuc hai chui: chui nng ca h-tPA (hay cn gi chui A, c khi lng 39.000 kDa) c xc nh u amino, cn chui nh (hay cn gi chui B, khi lng 33.000 9
kDa) u carboxyl. Trong , chui nng cha ba vng: vng ngn tay (F vng finger ) gm 47 amino acid t v tr acid amin th 4 - 50, ngi ta cng thy cu trc tng t trong fibronectin; vng nhn t sinh trng biu b (epidermal growth factor region - EGF) t amino acid c v tr 51 - 87; vng th ba bao gm hai cu trc kringle, vng kringle 1 (K1 - kringle one region) t amino acid 88 176, vng kringle 2 (K2 - kingle two region) t v tr amino acid 177 n 262. Cc cu trc cng c tm thy trong prothrombin, plasminogen, urokinase v nhn t XII [14], [19], [20], [23], [38], [45], [52]. Chui nh ca h-tPA, cha v tr tm hot ng (gm nm exon c ngn cch bi bn intron), vng serine protease (P serine protease domain) c v tr t amino acid 267 n 527 v tng ng vi chui xc tc ca enzyme phn hy protein serine khc [38], [45].
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Hnh1.2. Cc vng ca tPA
Cc vng ny c th nm k tip nhau hoc c ngn cch vi nhau bi cc vng ni ngn. Cc vng ny gp phn to nn cc c tnh sinh hc c hiu ca c phn t. Vng F ng vai tr ch yu to nn tnh i lc cao vi t mu. Hot tnh ny th hin tnh c hiu cao ca h-tPA trong qu trnh phn gii cc khi mu ng giu t mu. Vng EGF th hin hot tnh gn h-tPA vi b mt t bo, chc nng ny c 10
bo th trong rt nhiu protein tPA ng vt c v. Vng EGF cha 6 cysteine, y l thnh phn to nn cu disulfide trong domain. Vng kringle trong protease serine rt quan trng trong mi tng tc protein-protein trong qu trnh hot ha plasminogen v phn gii t mu, hoc qu trnh chuyn i prothrombin-thrombin. Vng kringle 2 ca h-tPA cng lin quan cht ch vi qu trnh gn t mu v c kh nng kch thch hot tnh h-tPA i vi fibrin nn c vai tr quan trng trong s kt hp gia h-tPA v t mu [16], [55]. Vng kringle 1 khng c xem nh c lin quan n trong c tnh kt hp vi t mu ca h-tPA mc d trnh t amino acid ca kringle 1 v kringle 2 c tnh tng ng cao [52]. Vng P m nhim chc nng phn ct plasminogen bng enzyme to ra plasmin [37].
Hnh 1.3. Cu trc h-tPA [38]
Protein h-tPA c tng hp v tn ti trong t bo mng trong mch di dng chui polypeptide si n, tuy nhin h-tPA trng thnh l mt si n glycoprotein c 530 amino acid; 32 amino acid trnh t u s c ct khi h-tPA
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trc khi h-tPA c a ra bn ngoi t bo. Plasmin, kallikrein huyt tng, hoc nhn t Xa hot ha h-tPA bng cch ct h-tPA v tr Arg 275- Ile 276 thnh hai si (A v B), hai si ny lin kt vi nhau bi cu disulfide [15], [20], [28], [34], [45], [52]. Trong t nhin, h-tPA tn ti dng si n. Dng hai si quan st c trong nui cy t bo ung th t bo hc t ngi. Nghin cu cho thy h-tPA dng mt si hot ng c hiu qu hn dng hai si. in di trn SDS (sodium dodecyl sulfate) cho thy, h-tPA dng mt si c chuyn thnh dng hai si trong qu trnh phn gii huyt khi [45]. Theo Berg, khi b glycosyl ha v tr Asn- 184 s gy c ch trung gian i vi plasmin chuyn h-tPA t phn t dng si n sang dng si i, v khi glycosyl ha v tr Asn- 117 v/hoc Asn- 448 s lm gim kch thch hot ha ca h-tPA kt hp vi t mu v hot ng phn gii huyt khi [11]. 1.4. Vai tr ca cht hot ha plasminogen m trong qu trnh lm tan mu ng S ng mu l mt qu trnh phc tp qua hn ch qu trnh mt mu ca c th. Khi thnh mch mu b tn thng, mu c cm nh ch tn thng c che ph bi huyt khi cha tiu cu v si huyt. C ch ng mu c bo th kh bn vng trong tin ha; lp th, h thng ng mu bao gm hai thnh phn: t bo (tiu cu) v protein (cc yu t ng mu). Phn ng ng mu c kch hot ngay sau chn thng lm tn hi n ni mc mch mu. Tiu cu lp tc to nt chn cm mu ti vt thng; y chnh l qu trnh cm mu ban u. Qu trnh cm mu th pht din ra ng thi; cc yu t ng mu trong huyt tng p ng trong mt chui phn ng to cc si huyt c vai tr cng c nt chn tiu cu.
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Hnh 1.4. Qu trnh phn hy huyt khi [9]
Thnh phn quan trng nht trong h thng phn gii t mu l glycoprotein plasminogen, l cht do gan sinh ra v c mt trong huyt tng v c mt hu ht ngoi thnh mch. Plasminogen l dng tin enzyme (zymogen), l cht c vng d phn chia, di tc dng ca cht hot ha plasminogen s c chuyn i thnh dng hot ng v l dng phn gii protein, plasmin. Mc tiu u tin ca plasmin l t mu, nhng plasmin c th lm gim mt vi thnh phn ca hn hp ngoi thnh mch v chuyn mt s tin hormone v tin cytokine thnh dng hot ng ca chng. Plasmin thng lin quan n s di cn ca bnh ung th. S pht sinh ca plasmin xy ra trc tin trn b mt t mu, thng c im kt hp cho plasminogen v yu t c bn hot ha ca n trong mu, tPA [59]. Qu trnh lm gim v phn hy t mu s c cn bng trong qu trnh sa cha thnh mch mu b tn thng. T bo thnh mch mu b tn thng s gii phng cht hot ho plasminogen (tPA, uPA), hot ha h
Hnh1.5. Con ng phn gii t mu (Fibrin) [58]
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thng phn gii t mu. Cht hot ha plasminogen ct plasminogen to thnh plasmin, l cht phn hy huyt khi. Nguyn tc phn hy t mu: bn thn qu trnh phn hy t mu c iu ha bi cc cht c ch cht hot ho plasminogen (plasminogen activator inhibitors - PAIs) v cht c ch plasmin (2-antiplasmin), y l nhng cht lm chm qu trnh phn gii t mu. PAI-1, mt cht quan trng ca PAI, lm cho tPA v uPA khng hot ng v c gii thot khi t bo thnh mch v hot ha tiu cu. Cht c ch tin plasmin l 2-antiplasmin, rt nhanh gii phng plasmin t do khng hot ha thot khi huyt khi. Mt vi 2- antiplasmin thng lin kt vi nhn t XIIIa vi t mu trong qu trnh hnh thnh cc mu ng, n c th ngn nga qu mc plasmin hot ng trong cc mu. C tPA v uPA u nhanh chng c phn hy bi thn, mt b phn ca ngn nga phn hy huyt khi qu mc. 1.5. Nghin cu ng dng sn xut cht hot ha plasminogen m ngi Nh trnh by, cht hot ha plasminogen m ngi c thng mi ha vi tn gi l Alteplase do hng Genentech sn xut. y l thuc lm tan huyt khi c hiu (ngha l chng kt hp chn lc vi plasminogen gn vi t mu) [43], [44]. Dc phm ny c C quan Qun l Thc phm v Dc phm Hoa K (Food and Drug Administration - FDA) cng nhn nh l mt dng thuc cha bnh t qu vo nm 1996. V phng din lm sng, h-tPA c xem l thuc tan huyt khi c hiu (chng kt hp chn lc vi plasminogen gn vi t mu) v c la chn iu tr nhi mu c tim, tc mch phi, t qu, tc ng mch ngoi bin v cc bnh khc lin quan n tan huyt khi [36], [40]. Trong nhng nm t cui 1990 n u nm 2000, t l bnh nhn b t qu c iu tr bng h-tPA ch chim 2%. Khong 9 nm tr li y, t l bnh nhn mc bnh tim mch v b t qu c iu tr bng h-tPA tng ln ng k, chim khong 10- 20%. Thuc ny c u im l khng c tc dng ph, khng gy chy mu h thng v hin ang thuc loi c doanh thu rt cao trn th trng. T nm 1998 n nm 2003, doanh thu mi nm t khong 200 triu USD. D kin, ti nm 2010 doanh thu t c ln ti 600 USD [22], [58].
14
Trong nhng nghin cu ng dng u tin v h-tPA, cc t bo u nui cy c s dng lm ngun sn sinh h-tPA cho cc mc ch tr bnh [26]. Tuy nhin, cc ng dng lm sng i hi quy trnh sn xut protein thch hp vi sn lng v tinh sch cao. V vy, nghin cu to h-tPA ti t hp s dng cc t bo ng vt c v cng c trin khai. Cc t bo bung trng ca chut ng Trung Quc (Chinese hamster ovary - CHO) c chuyn gen h-tPA tng hp protein htPA ti t hp [17]. Cc sn phm DNA ti t hp to ra t h thng ln men mi trng nui cy t bo ng vt c v cng c thu nhn v lm sch. Nhng n lc xy dng quy trnh n gin to h-tPA ti t hp hiu qu vi gi thnh h t vi sinh vt, c bit l t vi khun, v c th hn l t E. coli, rt c quan tm nghin cu [23], [40]. Vi khun E. coli: Gen m ha h-tPA c nghin cu v biu hin trn vi khun E. coli t nhng nm 1983 [23], [40]. Trong nghin cu ca Pennica v ng tc gi (tg), lng h-tPA thu c ch t khong 50- 80 g/l dch nui, tng ng vi 1500- 2400 phn t h-tPA c hot tnh/ t bo [40]. n nm 1998, sau khi tinh sch, hm lng protein h-tPA thu c vo khong 180 g/l dch nui [42]. Gn y, Zhu v tg biu hin thnh cng protein h-tPA trong vi khun E. coli vi hm lng protein h-tPA chim 30% tng s protein ca vi khun [57]. Nm men: khc phc nhng nhc im ca vi khun ni chung v E. coli ni ring, ngi ta cng c th dng h thng biu hin l cc sinh vt nhn chun bc thp lm vt ch tch dng, nh nm men (v d, Saccharomyces cerevisiae, Hansenulla polymorpha, Pichia pastoris) hay to. u im ca nm men l chng c th tin hnh rt nhiu dng bin i (cu trc) protein v ging nh vi khun, chng c tc sinh sn tng i nhanh, nhu cu dinh dng kh n gin, khng sn sinh ni c t v c kh nng thch ng cao vi sn xut quy m ln. S. cerevisiae l nm men c s dng rng ri nht nhm sn xut cc protein ti t hp. Hin nay, nm P. pastoris cng c s dng rng ri, hn 120 protein ti t hp c biu hin t bo ch ny trong rt nhiu gen c ngun gc t ngi v ng vt.
15
Cht hot ha plasminogen m ngi l mt trong nhng protein ng vt c tng hp trn nm Aspergillus nidulans [51], S. cerevisiae [35] v Aspergillus niger. i vi nm A. niger, hm lng h-tPA c tng hp khong 0,07mg/g sinh khi v tng ln 1,9mg khi b sung peptone. Cht hot ha plasminogen m ngi c to ra trong A. niger b ct thnh dng hai si c trng lng phn t ging vi dng h-tPA hai si ca khi u ngi, dng hai si ny thng xut hin sau 16h nui cy t bo. Tuy nhin, thng thng ch di 1% sn phm h-tPA to ra trong A. niger c hot tnh, v dng h-tPA hot ho khng b mt khi dch trong sut qu trnh nui cy. Dng h-tPA hot ho khng xut hin b mt trong giai on tnh ca b nui cy, c th do qu trnh tng hp khng ng hoc do qu trnh phn hy protein xy ra vng phn hy protein ca protein h-tPA. T bo ng vt c v: Do yu cu a s cc sn phm protein ti t hp cn s bin i v cu trc nn cc t bo prokaryote khng p ng c. Cc t bo eukaryote bc thp nh nm men hay t bo cn trng cng khng p ng c i vi cc qu trnh bin i tng t ng vt c v nh phn gii protein, lin kt cc tiu n v hoc nhiu phn ng kt hp khc nhau nh glycosylation, methylation, carboxylation, amidation, hnh thnh cc cu ni disulfide hoc phosphoryl ha cc gc amino acid, cho nn h thng t bo ng vt c v nh t bo trng chut ng Trung Quc (Chinese hamster ovary - CHO) v t bo thn chut cha trng thnh (baby hamster kidney - BHK) thng c la chn sn xut cc protein tr liu cho ngi. Khong 60% protein ti t hp dng lm dc phm c sn xut t cc h thng t bo vt ch ny. Tuy nhin, vic s dng t bo ng vt lm t bo ch c mt s nhc im nh: Tc sinh trng ca t bo ng vt rt chm so vi t bo vi sinh vt, v th, sn lng ca chng kh thp v vic duy tr iu kin nui cy v trng trong mt thi gian di thng gp nhiu kh khn hn. Cc t bo ng vt c bao bc bi mng huyt tng, mng hn nhiu so vi thnh t bo dy chc thng thy vi sinh vt hoc t bo thc vt v kt qu l chng rt d b bin dng v v. Trong khi nhu cu dinh dng ca t bo ng vt cha c xc nh mt cch y , v mi trng nui cy thng i hi b sung huyt thanh mu rt t tin. 16
Do vy gi thnh sn xut cc sn phm s dng cc h thng ny kh cao. S la chn h thng biu hin trn c th nh hng n c tnh, cht lng v gi thnh ca sn phm cui cng [12]. ng - thc vt bin i gen: Hin nay, ng vt v thc vt bin i gen cng c a vo ng dng sn xut cc protein tr liu [24], [25], [32], [50]. ng vt bin i gen c thit k sao cho c th tit protein vo cc dch th d thu nhn nh sa (b, cu, d...). Ngi ta tnh ton v thy rng s dng cc ng vt bin i gen tng hp cc protein tr liu ti t hp s c th r hn bn n nm ln so vi s dng cc t bo ng vt c v nui cy. Bn cnh , thc vt, c bit l ng, cng c nghin cu v kh nng sn xut cc protein ti t hp. T nm 1990, protein ti t hp c sn xut t cy thuc l v khoai ty chuyn gen phc v cho y- dc bt u c thng mi ha. Mi lm nm tip theo, cc loi DNA ti t hp c chuyn vo thc vt thu nhn protein cha bnh nh khng sinh, sn phm mu, cytokine, hormone sinh trng, enzyme ti t hp, vaccine ngi v ng vt [33]. Thc vt c nhiu u im hn so vi ng vt trong vic to ra cc protein ti t hp. Ging nh nm men, thc vt c kh nng t thc hin nhiu khu to ra cc protein phc tp vi chi ph cho trng trt kh thp. S dng thc vt bin i gen khng pht sinh cc tranh ci v o c, cng nh c th trnh c cc nguy c lin quan n cc virus, ng vt, cc cht c vi khun. Tuy nhin, h thng biu hin l thc vt bin i gen vn cn mt vi hn ch. Th nht, sn lng protein ti t hp do h thng biu hin ny sinh ra cn tng i thp. Th hai, tc dng tr liu ca cng mt sn phm protein c ngun gc thc vt v ng vt l khc nhau. V d, nhiu protein ca ngi thng b glycosyl ha, ngha l sau khi c to ra, cc protein thng c gn thm mt s gc ng c trng gip chng kim sot hot ng chc nng mt cch bnh thng. Cc nh nghin cu thao tc vt qua c hng ro ny bng cch to ra cc thc vt bin i gen c th tin hnh qu trnh glycosyl ha protein. Cc thc vt bin i gen ny mang cc gen ca ngi m ha cho cc enzyme xc tc qu trnh gn cc phn
17
t ng vo protein. Mt nhm nghin cu H Lan tin hnh bin i vt cht di truyn ca mt cy thuc l v sau lai cy thuc l bin i gen ny vi mt cy c thit k sinh ra mt loi khng th ca chut. Kt qu l khng th ny cng c dng glycosyl ha, rt ging vi khng th sinh ra bi chut hn bt k khng th thc vt no c sinh ra trc . C th thy r rng, khoa hc hin i c vai tr rt to ln trong vic hon thin cc h thng biu hin protein ti t hp. 1.6. Tnh hnh nghin cu Vit Nam Vit Nam, hng nghin cu gen/ protein c gi tr s dng trong y dc, tin ti biu hin sn xut protein ti t hp l rt cn thit v mi c tip cn nghin cu nc ta trong thi gian gn y. Mt s phng th nghim tin hnh phn lp, xc nh trnh t mt s gen t cc ngun sinh vt khc nhau, trong c c gen ngi nghin cu ng dng sn xut dc phm cng ngh sinh hc. Trong , Vin Cng ngh sinh hc thnh cng trong nghin cu tng hp Trihobakin, protein ti t hp c ngun gc thc vt c kh nng c ch cc dng t bo ung th cng nh thnh cng trong vic tinh ch protein bt hot ribosome (RIP) phn lp t cy mp ng [2], [3]. Cng ti Vin Cng ngh sinh hc, gen m ha interleukin-2 ca ngi, tc nhn iu bin min dch, c s dng trong iu tr ung th, HIV, cc bnh nhim trng, d ng c tch dng v biu hin [5], [6]; iterleukin-2 ca ngi rh-LL2MM b t bin cng c biu hin thnh cng [4]. Ngoi ra, cc nh khoa hc ti Trng i hc Khoa hc T nhin, i hc Quc gia thnh ph H Ch Minh cng triu khai to dng biu hin miniproinsulin ca ngi trong E. coli [7]. Bn cnh , cc nghin cu to vaccine ti t hp cng c quan tm nghin cu nhiu phng th nghim [1]. Tuy nhin, cho n nay cha c mt cng b no v nghin cu sn xut h-tPA ti t hp nc ta, v vy ti ny s lm tin cho qu trnh nghin cu v sn xut cht hot ha plasminogen ti t hp.
18
CHNG 2:
VT LIU V PHNG PHP NGHIN CU
2.1. 2.1.1.
Vt liu, ha cht v thit b Vt liu
Trong nghin cu ny, chng ti s dng vt liu ban u l cDNA m ha h-tPA c chn dng trong vector pUC18 (pUC18/ h-tPA) trong nghin cu trc y [10]. Vector biu hin gm hai loi: pET21a(+) c mua t Hng Novagen (M) v pGEX6p1 c mua t Hng Pharmacia. Bn hai vector c trnh by trong hnh 2.1:
Hnh 2.1. Bn vector pET21a(+) v pGEX6p1
Chng E.coli DH5 c mua t Hng Invitrogen (M) v chng E.coli BL21(DE3) pLysS Competent Cells t Hng Promega (M).
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2.1.2.
Ha cht
Cc ha cht s dng trong nghin cu c t mua t nhiu hng khc nhau. Danh sch cc ha cht cn thit, cc dung dch s dng v trnh t mi c trnh by tng ng cc Bng 1, 3 v 4 trong phn Ph lc. 2.1.3. Cc thit b
Cc thit b c s dng thuc Phng Th nghim Trng im Cng ngh Gen v phng Cng ngh ADN ng dng, Vin Cng ngh sinh hc, Vin Khoa hc v Cng ngh Vit Nam c mua t nhiu hng khc nhau. Cc thit b thng s dng c lit k Bng 2 phn Ph lc. 2.2. Phng php nghin cu 2.2.1. in di DNA trn gel agarose Mc ch: in di gip phn tch cc on DNA c kch thc khc nhau, t xc nh c s c mt cc on DNA quan tm. Nguyn tc: Nguyn tc ca phng php ny da trn c tnh tch in ca hu ht cc phn t. Cc phn t dng ion khc nhau v mc ion ha, kch thc phn t c th tch ring bit theo kh nng di ng trong trng in t v hai cc m (-) v dng (+). Cc phn t DNA c khi lng v in tch khc nhau c tch ra khi di chuyn t cc m sang cc dng ca my in di trong mt in trng c in th v cng thch hp. Ha cht in di: agarose 0,8- 1,5% (ph thuc vo kch thc phn t DNA. y, chng ti s dng agarose nng 0,8% bi nng ny ph hp vi kch thc ca on gen), dung dch m TAE 1X. Quy trnh in di: - Chun b gel agarose: ha tan 0,8 gram agarose vo 100ml dung dch TAE 1X, un si cho dung dch tan hon ton. nhit xung khong 50- 60oC, dung dch agarose vo khay gel in di c ci sn lc thch hp. Sau 30- 60 pht, khi gel ng cng, g lc ra v t vo b in di, m TAE 1X ngp cch mt gel t 1- 2 mm [48].
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- Mu DNA c trn vi 3- 5l m tra mu v c tra vo cc ging trn gel. Chy in di vi hiu in th 100V, 60- 80mA trong khong 30 pht. - Bn gel c ly ra khi khun v ngm 5 pht trong dung dch EtBr nng 0,5g/ml, sau ra sch bng nc. - Quan st v chp nh trn my Bio- Rad vi tia UV c bc sng 320nm. 2.2.2. in di protein trn gel polyacyamide Nguyn tc: Cc tiu phn protein c xc nh da vo tc di chuyn khc nhau ca cc tiu phn protein trong trng in. mt s iu kin nht nh, protein dng ion v c mc ion ha khc nhau, nn c th tch ring bit chng theo kh nng di ng trong trng in t v hai cc m (-) v dng (+). Kt qu nhn c ph cc vch protein khc nhau. Phng php in di l mt phng php nghin cu sinh ha thng dng v rt tin li. Phng php in di protein thng c tin hnh trn cht mang dng gel l li polymer. Li polymer lm chm tc di chuyn cc phn t protein theo khi lng, kch thc, mang in ca chng. Phng php in di protein trn gen polyacrylamit c cha SDS c tin hnh theo phng php ca Laemmli [29]. Thnh phn gel c trnh by Bng 11 phn Ph lc. Quy trnh in di nh sau: - Pha mu vi Protein Sample Buffer 4X v bin tnh mu 95 oC trong 5 pht. Dng 10 l mu tra vo tng ging v tin hnh in di trn gel polyacrylamide 10% gm gel c 4% v gen tch 10%. - dung dch chy (Running buffer) SDS- Page 1x v chy in di: Chy hiu in th 110V trong 2- 3 gi. Theo di n khi thy mu dung dch m mu bt u thot ra ngoi dung dch m chy th kt thc. - Bn gel c nhum trong dung dch nhum (Comasive Brilliant Blue R250) trong khong t 30 pht n 1 gi. - Ty gel bng cch ngm bn gel nhum vo dung dch ty (acid acetic 7%, metanol 20%) n khi bn gel mu trng th kt thc qu trnh ty. Thi gian ty khong 30 pht. Sau gel c bo qun v chp nh.
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2.2.3. Phn ng dy chuyn polymerase (Polymerase Chain ReactionPCR) Phng php PCR nhm mc ch khuych i in vitro mt on DNA khi bit trnh t hai u. Nguyn tc ca k thut PCR l nh s xc tc ca enzyme DNA polymerase chu nhit khuych i mt on trnh t DNA c hiu in vitro trong mi trng c cc dNTP v cp mi c hiu [48]. Trong nghin cu ny, chng ti s dng 2 cp mi l Cp 1: h-tPA-NdeI F2, h-tPA-XhoI R3; Cp 2: h-tPA-BamHI F2, h-tPA-XhoI R4. Cc thnh phn ca PCR: on DNA khun (cDNA m ha h-tPA c to dng trong vector pUC18), hai cp mi c hiu c chiu di 15- 30 nucleotide, bn loi dNTP (dATP, dTTP, dGTP, dCTP), DNA polymerase. PCR bao gm cc giai on: Giai on bin tnh: DNA c bin tnh nhit khong 94oC- 95oC trong thi gian mt vi pht, khi cc lin kt hydro b t v si DNA dng si kp thnh hai si n; Giai on gn mi: nhit t 40oC- 65oC trong khong 30 giy n 1 pht, hai on mi chuyn bit s bt cp vi si DNA khun theo nguyn tc b sung hai u on DNA cn nhn. Nhit ca bc gn mi ty thuc vo tng loi mi c th, c tnh ton da trn nhit nng chy (Tm) ca mi; Giai on ko di chui: nhit phn ng c nng ln 70oC- 80oC (trung bnh khong 720C) trong khong thi gian thch hp (ty thuc chiu di on DNA cn tng hp), enzyme Taq polymerase hot ng v qu trnh tng hp DNA din ra; Quy trnh phn ng c thc hin trn my PCR GeneAmp PCR System 9700 vi thnh phn phn ng c trnh by Bng 5 phn Ph lc, chu trnh nhit phn ng nh sau: Chu trnh nhit trn my GenAmp PCR System 9700 nh sau: 94oC 3 94oC 30 60oC 30 72oC 130 x30ck 72oC 10 4oC
22
Sn phm PCR c kim tra bng phng php in di trn gel agarose 0,8%, sau c tinh sch DNA theo kit Wizard SV Gel and Clean-Up System. 2.2.4. X l DNA bng enzyme hn ch Nguyn tc: Enzyme hn ch l mt nuclease ni bo c kh nng nhn bit cc on DNA vi cc trnh t nucleotide nht nh, bm vo on DNA v ct c hai si ca phn t DNA. Enzyme hn ch ct DNA hnh thnh hai dng u: to u bng v to u dnh. i vi sn phm PCR chng ti tin hnh ct bng hai cp enzyme BamHI/XhoI v NdeI/XhoI. Vector pET21a(+) c x l bng NdeI/XhoI v pGEX6p1 c ct bng BamHI/XhoI. Thnh phn phn ng c trnh by Bng 6 phn Ph lc. Hn hp phn ng c trn u, nhit v thi gian thch hp (thng 370C, 2 gi). Sn phm phn ng c kim tra bng in di trn gel agarose 0,8%; Sau khi ct bng enzyme hn ch, cc vector c x l vi phosphatase loi nhm phosphate ra khi u 5 nhm gim qu trnh ni li ca vector. Thnh phn phn ng c trnh by Bng 7 phn Ph lc. Hn hp c 37oC trong 1h sau tip tc 75oC trong 15 pht loi CIP [48]. 2.2.5. Ghp ni DNA Nguyn tc: on cDNA v vector c x l bng cng mt loi enzyme hn ch to u b sung, cc u ny c trnh t bt cp vi nhau. Di tc dng ca enzyme T4 DNA ligase, on DNA quan tm s c gn vo vector. Thnh phn phn ng ghp ni c trnh by Bng 8 Phn Ph lc. Hn hp phn ng c trn u v 14- 24 gi, 16oC. Sn phm phn ng c bin np vo E. coli v chn lc trn mi trng LB c b sung Ampicillin (Amp), nng 50g/ml [48].
23
2.2.6. Bin np DNA plasmid vo t bo vi khun E. coli bng phng php sc nhit Ty tng mc ch khc nhau m chng ti chun b cc chng vi khun khc nhau. Chng t bo dng to dng l vi khun E. coli chng DH5. biu hin, chng ti s dng vi khun E. coli chng BL21. Quy trnh chun b v bin np i vi hai chng l tng t nhau [48]. 2.2.6.1. Chun b t bo kh bin E. coli Chng vi khun ly t ng gi chng c cy ria trn mi trng LB c v nui qua m 37oC; Ngy hm sau ly 1 khun lc ri t a cy ria, nui lc 200vng/pht (v/p) 37oC trong mi trng LB lng t 1- 2h; Ngy tip theo cy tri dch nui ra a cha mi trng LB c. Nui qua m 37oC; Chn 1 khun lc t a, cy chuyn vo 1,5 ml mi trng LB lng, nui
qua m 37oC; Cy chuyn 200l dch nui sang 30 ml LB lng. Nui lc trong 3h 37oC; Chuyn dch nui sang ng ly tm. Gi lnh trn trong 10 pht; Ly tm 4200 v/p trong 10 pht, 4oC. b dch ni; sau b sung 15 ml
dung dch CaCl2 0,1M. Lm tan ht ta t bo trong dung dch CaCl 2. trn trong 40 pht; Ly tm 4200 v/p trong 10 pht 4 oC. b dch ni; b sung 1,5 ml dung
dch CaCl2 0,1 M. Ly tm nh ha tan ta t bo vo dung dch CaCl2; Chia ra cc ng eppendorf v b sung 20 l glycerol 100%. Bng nh trn
u cc thnh phn vi nhau (thao tc trong box). Gi t bo kh bin -80oC [48]. 2.2.6.2. Bin np DNA plasmid vo t bo E. coli T bo kh bin E. coli c gi trn khong 30 pht, sau b sung 5l
ca sn phm ghp ni vo ng t bo kh bin (khong 100l). Gi trong khong 15 pht;
24
Sc nhit 42oC trong 70 giy, sau chuyn ngay sang bnh v gi B sung 300l mi trng LB lng; Nui lc 200 v/p 37oC trong 1gi; Cy tri ht dch t bo trn a mi trng LB c c b sung Amp
trong 5 pht; -
(50g/ml), nui 37oC qua m [48]. 2.2.7. Tch chit v tinh sch DNA plasmid Nguyn tc: Tch chit plasmid da trn hai nguyn ngc c bn l DNA nhim sc th ca E. coli c kch thc ln hn rt nhiu so vi DNA plasmid; do trng lng, kch thc khc nhau gia cc dng DNA E. coli m hu ht DNA plasmid tch ra di dng vng ng [48]. Ha cht tch chit DNA plasmid gm: Sol I, Sol II, Sol III, dung dch loi protein (hn hp chloroform: isoamylalcohol = 24:1), dung dch TE 0,01M, pH 8,0. Quy trnh tch chit DNA plasmid ca E. coli Nui cy lc qua m t bo E. coli trong 1,7ml mi trng LB (c khng sinh Amp nng 50g/ml) 370C, 200 v/p; Ngy hm sau, ly tm dch nui cy trn 6.000 v/p, 7 pht; loi b dch; Ho cn t bo vi khun trong 150l dung dch I lnh, voltex lm tan cn B sung 150l dung dch II, o u ng Eppendorf nh nhng v gi trn Thm 150l dung dch III lnh, o nh v trong t 3 5 pht; Thm 500 l dung dch chloroform: isoamyl alcohol (t l th tch 24:1). Ly tm 12.000 v/p trong 15 pht 4 oC. Chit v chuyn pha lng pha trn t bo, sau gi trong 5 pht; 10 pht; -
o u hn hp; sang mt ng eppendorf mi; Thm 50 l CH3COONa 3 M (pH 5,2) v 1 ml cn 100 %. o u v gi lnh -200C trong 3 gi; Ly tm 12.000 v/p trong 15 pht 4oC thu ta DNA;
25
Ra cn bng 0,2ml cn 70%, ly tm 12.000 v/p trong 15 pht, lm kh v
ho cn trong 40l TE 0,01M, gi -20oC; Sau b cn. Lm kh ta bng my SpeedVac trong 5 pht; Ha tan ta DNA trong 50 l nc cha RNase (0,01 mg/ml). Bo qun Cc dng mang vector ti t hp sau khi c xc nh s c kim tra bng enzyme hn ch v PCR kim tra. Thnh phn ct kim tra c nu Bng 9 phn Ph lc. Hn hp c 37oC trong 2h. Thnh phn v chu trnh nhit ca phn ng PCR kim tra ging vi qu trnh nhn gen. Sn phm ct v PCR kim tra c in di trn gel agarose 0,8%. 2.2.8. Xc nh trnh t gen Trnh t gen c xc nh trn my xc nh trnh t t ng ABI PRISM 3100 Genetic Analyzer kt hp vi s dng b kit BigDye Terminator v3.1 Cycle Sequencing. Thnh phn hn hp s dng trong xc nh trnh t DNA bao gm c dNTPs v ddNTPs, l cc nucleotide c nh du bng cc cht pht hunh quang khc nhau. Mi c trnh t i vi vector pET21a(+) l cp mi T7, i vi vector pGEX6p1 l cp mi PEXF/PEXR. Phn ng gn cc nucleotide nh du vo si DNA tng hp trn si khun c tin hnh trn my PCR. Qu trnh tng hp ko di chui s khng tip tc din ra khi ddNTP c lp vo, kt qu l to ra mt b cc on c chiu di hn km nhau mt nucleotide. Sau sn phm PCR c in di trn gel c phn gii cao, cho php phn bit c cc si n hn km nhau mt nucleotide [48]. Thnh phn PCR xc nh trnh t c trnh by Bng 10 phn Ph lc. Chu trnh nhit trn my GenAmp PCR System 9700 nh sau: 96oC 1 96oC 10 50oC 5
x25ck
DNA -20oC; Kim tra DNA bng gel agarose 0,8 %.
60C 4
4oC
Tip , sn phm PCR c tinh sch bng phng php ta cn/EDTA:
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B sung 4l EDTA 125mM c pH 8,0 v 60l cn 100%. o nh hn hp
v nhit phng trong 15 pht. Ly tm 12.000 v/p trong 15 pht 4 oC. Loi b cn. Ra ta bng 60l cn 70%, ly tm 12.000 v/p trong 10 pht v lm kh. B sung 10l HiDiTM Formamide v bin tnh 95oC trong 5 pht. Cc
mu c cho vo cc ging ca khay ng mu. in di trong ng vi mao qun 80 cm x 50 m cha POP- 4TM. D liu c x l bng phn mm ABI PRISM 3100 Data Collection v2.0, Seqscape v2.5 v BioEdit v7.0.5. 2.2.9. Biu hin protein trong vi khun E. coli Sau khi plasmid ti t hp mang gen m ha h-tPA c bin np thnh cng vo t bo biu hin E.coli chng BL21, protein ti t hp c tin hnh biu hin v kim tra theo cc bc sau: Chn mt khun lc (E. coli BL21) nui lc 200 v/p trong 5ml LB lng c Ngy tip theo, ht 2ml dch vi khun trn vo 100 ml LB lng c b sung Amp Cm ng biu hin protein bng IPTG nng 1mM. (T l 100 l IPTG cho Kim tra biu hin: ht 1ml dung dch nui cy sang ng eppendoft mi. Ly tm thu t bo v b sung 100 l loading dye. Voltex ph t bo, bin tnh protein trong 5 pht 95oC; Dng 10l mu in di SDS-PAGE cng vi thang chun protein v mu i chng m l dch ph mng ca E.coli BL21 v dch ph mng E.coli BL21cha vector rng c cm ng IPTG; Phn dch nui cy cn li c ly tm thu t bo. Gi t bo -20oC ch bc tinh ch protein. b sung Amp (100g/ml) 37oC qua m; (100g/ml). Tip tc nui lc 200 v/p 37oC n OD600= 0,6- 0,8; 100ml dch nui). t bo tip tc sinh trng trong 3h;
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Chng 3:
3.1.
KT QU V THO LUN
Thit k vector biu hin mang cDNA m ha h-tPA 3.1.1. Nhn on cDNA m ho h-tPA bng k thut PCR Gen m ha h-tPA s dng trong nghin cu c to dng trong vector
pUC18. kim tra cDNA m ha h-tPA, chng ti tin hnh xc nh trnh t cDNA m ha h-tPA trong vector pUC18 s dng cp mi M13, sau trnh t ny c phn tch v so snh vi trnh t NM000930 trn Ngn hng Gen Quc T. Kt qu phn tch v so snh cho thy, trnh t cDNA trong vector pUC18 c tng ng ti 99% vi trnh t so snh, cc im t bin gia h-tPA vi trnh t NM000930 khng lm thay i trnh t acid amin. Trnh t bao gm y cc thnh phn cn thit cho biu hin gen nh m m u, m kt thc, ng khung c... Sau khi xc nh trnh t cDNA m ha h-tPA, chng ti tin hnh PCR vi hai cp mi c hiu v khun l vector pUC18 ti t hp to dng cDNA ny trong vector biu. Hai cp mi c chng ti thit k mang cc trnh t nhn bit ca enzyme hn ch s dng cho cc qu trnh ghp ni gen v kim tra sn phm. Vic thit k thm trnh t nhn bit ca cc enzyme hn ch vo mi tun theo mt s nguyn tc nh trnh t nhn bit ch c mt v tr trong vng gn a v ca vector v khng c mt trong gen cn to dng; s nucleotide pha ngoi trnh t nhn bit t 3- 6 nucleotide nhm bo v cc trnh t ny v gip cc enzyme ct tt hn. Trong nghin cu ny, i vi vector pGEX6p1 chng ti s dng hai enzyme l BamHI v XhoI, v chng c mt trong vng ct gn a v ca vector biu hin pGEX6p1; i vi vector pET21a(+), chng ti s dng hai enzyme NdeI v XhoI. Cc enzyme ny u khng c im ct trn cDNA m ho h-tPA. Trnh t hai cp mi nh sau: Cp 1: htPA-NdeI F2: 5-GTG AAG CAC ATA TGG ATG CAA TGA AGA GAG G-3 htPA-XhoI R3: 5-GTG GTG CTC GAG CGG TCG CAT GTT GTC-3
28
Cp 2: htPA-BamHI F2: 5-ATT CCG TGG ATC CAC CAT GGA TGC AAT GAG G-3 htPA-XhoI R4: 5-GTG GTG CTC GAG TCA CGG TCG CAT GTT GTC-3 K thut PCR l phng php c s dng nhm nhn mt on DNA nm gia hai vng bit trnh t. Hai on oligonucleotide c s dng nh mi cho mt lot phn ng tng hp c xc tc bi enzyme DNA polymerase. Cc on oligonucleotide c trnh t khc nhau v b sung cho trnh t nm hai u ca trnh t DNA mu c nhn ln. DNA mu b bin tnh bi nhit trong s c mt ca hai on oligonucleotide v bn loi dNTPs. Hn hp phn ng sau c h xung ti nhit cho php mi oligonucleotide bm vo trnh t ch, tip theo mi c ko di bi DNA polymerase. S chu k c nhc li nhiu ln t 25 n 35 chu k. Bi v sn phm ca mt vng ca qu trnh nhn l mu cho qu trnh tip theo, mi chu k cho hai sn phm DNA nn s lng sn phm l phn ng m mt on i DNA c xc nh bi u cui 5 ca on mi oligonucleotide v chiu di c xc nh bi khong cch gia hai mi. Hai cp mi ny c th c thit k theo yu cu ca nghin cu. Vic nhn gen bng PCR t vector d hn so vi nhn gen t h gen nn thng thu c lng sn phm ln, c hiu do s im bm ca mi trn vector ti t hp t, t l bm mi cao... S dng cp mi c hiu, chng ti tin hnh phn ng nhn cDNA m ha h-tPA. Thnh phn v chu trnh nhit c trnh by phn 2.2.3. Sn phm PCR c kim tra bng in di trn gel agarose 0,8%. Kt qu in di c th hin hnh 3.1.
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Hnh 3.1. Sn phm PCR nhn cDNA m ha h-tPA M: Marker 1kb 1: Sn phm PCR h-tPA vi cp mi 1 2: Sn phm PCR h-tPA vi cp mi 2 Kt qu in di cho thy, sn phm PCR thu c c kch thc phn t khong 1,7kb, kch thc ny ph hp vi kch thc tnh ton l thuyt. Bng chnh u sng, m, r nt v s c tinh sch trc khi s dng tin hnh ghp ni gen. 3.1.2. Ghp ni cDNA m ho h-tPA vo vector pGEX6p1 v pET21a(+) 3.1.2.1. X l sn phm PCR cDNA m ho h-tPA bng enzyme hn ch Sau khi thu c sn phm PCR, chng ti tin hnh chit v lm sch sn phm qua ct theo kit Wizard SV Gel and Clean- Up System. H thng ny da trn kh nng kt hp ca DNA vi mng silica trong s c mt ca hn hp mui, h thng mng c th kt hp vi 40g DNA v cho php thu hi sn phm PCR trong 15 pht. Qu trnh tinh sch ny gip loi b khi sn phm PCR cc thnh phn khng mong mun nh cc on DNA v tinh, cc NTPs... Sau khi tinh sch, hai mu sn phm PCR c x l bng hai cp enzyme hn ch tng ng l
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NdeI/XhoI v BamHI/XhoI. Thnh phn v qu trnh x l c trnh by mc 2.2.4. Phn ng c kim tra bng in di trn gel agarose 0,8%. 3.1.2.2. X l vector pGEX6p1 v pET21a(+) bng enzyme hn ch
Sau khi nghin cu ti liu v iu kin vt cht phng th nghim, chng ti tin hnh biu hin protein h-tPA trn vi khun E. coli chng BL21 vi hai vector biu hin l pET21a(+) v pGEX6p1 mang cDNA m ha h-tPA. Vector pET21a(+) l vector mnh to dng v biu hin protein ti t hp trong E. coli. Gen ch c to dng trong plasmid pET21a(+) c iu khin bi tn hiu dch m tt ca phage T7, biu hin di s tng hp ca T7 RNA polymerase trong t bo ch. Vector pET21a(+) l vector mang trnh t T7 u N v trnh t ui His u C. Vector ny mang ch th chn lc l gen khng khng sinh (khng Amp v Kanamycin). Vng to dng/ biu hin c iu khin bi T7 polymerase. Trnh t ny c xc nh, gii trnh t khi s dng mi u T7. H thng biu hin ny c cm ng bi IPTG hoc lactose trong qu trnh nui cy. Vector pGEX6p1 iu khin tng hp protein ngoi li bi promoter tac, promoter ny c cm ng bi isopropyl b-D thiogalactoside (IPTG). Vector pGEX6p1 c thit kt gm c mt gen lacIq, sn phm gen lacIq l protein c ch kt hp vi vng operator trn promoter tac, lm ngn cn qu trnh biu hin cho ti khi cm ng bi IPTG, qua iu khin qu trnh biu hin ca gen c thm vo. u 3 vng a ni c cha on gen m ho Glutathione S- Transferase - GST, y l mt protein gip qu trnh tinh sch protein ti t hp d hn. Ngoi ra, vector pGEX6p1 c mt s u im nh: mc biu hin cao, iu kin tinh sch protein n gin, nh hng ca khng nguyn v chc nng ca protein l nh nht. Qu trnh ct m vng hai vector cng s dng hai cp enzyme l NdeI/XhoI v BamHI/XhoI tng t nh i vi sn phm PCR. Thnh phn v qu trnh x l i vi vector pET21a(+) v pGEX6p1 c trnh by phn 2.2.4. Sau khi ct vi enzyme, vector c x l vi phosphatase loi nhm phosphate ra khi u 5, lm gim qu trnh t ni ca vector. Phn ng c kim tra bng in di trn gel agarose 0,8%.
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M: Marker 1kb 1: Vector pGEX6p1/BamHI+XhoI 2: Vector pET21a(+)/NdeI+XhoI 3: cDNA h-tPA/NdeI+XhoI 4: cDNA h-tPA/BamHI+XhoI
Hnh 3.2. Kt qu x l cDNA m ha h-tPA v cc vector biu hin bng enzyme hn ch Kt qu in di trn hnh 3.2 cho thy sn phm ct tng i sch v iu kin tin hnh phn ng ghp ni. 3.1.2.3. Ghp ni cDNA m ho h-tPA vo vector pGEX6p1 v pET21a(+) Chng ti tin hnh ghp ni cDNA m ha h-tPA vi cc vector pET21a(+) v pGEX6p1 to cc vector ti t hp mang gen m ho h-tPA. Do c sn phm PCR v vector u c x l bng hai enzyme tng ng nn di tc dng ca enzyme T4 ligase, sn phm PCR v vector c th ni li vi nhau to vector ti t hp. Thnh phn v phn ng ghp ni gia vector v sn phm PCR trnh by phn 2.2.5. Phn ng ghp ni c tin hnh 16oC, qua m. 3.1.3. Chn dng pGEX6p1 v pET21a(+) cha cDNA m ho h-tPA 3.1.3.1. Bin np sn phm lai vo t bo E. coli bng phng php sc nhit Sn phm lai gia vector v sn phm PCR c bin np vo t bo E. coli chng DH5 bng phng php sc nhit trong 70 giy 42 oC. Cc t bo ny sau c nui cy trn mi trng LB c c b sung Amp (50g/ ml) 37C qua
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m. Kt qu, chng ti thu c rt nhiu khun lc, l cc dng t bo vi khun khc nhau. Do trong mi trng c cht khng sinh nn ch c nhng khun lc no mang vector mi mc c, cc dng t bo vi khun ny gm 2 loi t bo khc nhau: t bo mang vector nhng khng mang on chn v t bo mang vector v on chn. V vy xc nh dng t bo no mang vector ti t hp, chng ti tin hnh sng lc thng qua tch chit plasmid, x l bng enzyme hn ch, PCR v xc nh trnh t. 3.1.3.2. Chn dng pGEX6p1 v pET21a(+) cha cDNA m ho h-tPA
Tch chit DNA plasmid Plasmid c tch chit nhm xc nh vector no mang on chn v vector no khng mang on chn. Phng php tch plasmid c trnh by mc 2.2.7. tch chit DNA plasmid, mt s khun lc c nui cy trong mi trng LB lng c b sung Amp 37oC t 12- 14h. Phng php tch chit plasmid gm 3 bc chnh: thu nhn v phn gii t bo, lm sch plasmid v kt ta DNA. Dung dch I (sol I) c tc dng ra sch t bo, dung dch II (sol II) c cha SDS v NaOH c tc dng ph mng t bo v c ch hot ng ca nuclease, khng cho chng phn gii DNA do Mg2+ (mt nhn t cn thit cho hot ng ca cc nuclease) b lin kt vi EDTA, khi cho tip dung dch III (sol III) c cha axetate v axetic acid th pH mi trng chuyn thnh acid yu gn vi im ng in ca DNA, v vy, DNA plasmid d b kt ta bng cn v d dng tch c nh ly tm. Do trong t bo vi khun c cha hai dng DNA l DNA nhim sc th v DNA plasmid nn vic tch v tinh sch DNA plasmid l rt quan trng, DNA plasmid c tch ring da trn s khng ch thi gian x l cc dng dch I, II, III. Do DNA nhim sc th c kch thc phn t ln v lin kt cht ch vi protein nn khong thi gian ngn gia cc ln thm dung dch khng kp thot ra ngoi. DNA plasmid thu c sau khi tch chit t t bo vi khun thng tn ti 3 dng: dng siu xon c to ra do lin kt hydro gia mt s phn ca phn t DNA plasmid hay do lc ht tnh in gia cc phn ca phn t vi nhau, dng vng m do b t gy mt mch n ca chui DNA mch kp v dng mch
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thng khi c hai mch DNA du b t gy. Dng siu xon c cu trc khng gian gn nn khi in di chng thng chy nhanh nht, tip n l mch thng v cui cng l mch vng. DNA sau c ho tan trong dung dch RNase 0,01mg/ml nhm loi b RNA v c in di kim tra trn gel agarose 0,8%. Theo l thuyt khi mt DNA plasmid mang gen ngoi lai, chng s c kch thc bng tng kch thc ca plasmid v kch thc ca gen ngoi lai. Do vy trong in di , chng s chy chm hn (thp hn) so vi i chng m (cc plasmid cha m vng), da vo chng ta c th kt lun s b c nhng plasmid ny c kch thc ln hn v c th chng mang gen ngoi lai.
Hnh 3.3. Chn dng pGEX6p1 mang cDNA m ha h-tPA (pGEX6p1/h-tPA)
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C: i chng m 1- 7: DNA plasmid ca cc dng pGEX6p1 Kt qu in di cho thy tt c cc ng chy u xut hin 2 bng chy trc r nt hn, iu chng t lng DNA plasmid ca chng ti thu c tn ti dng siu xon nhiu hn dng thng. Hnh 3.3 cho thy, i vi cc dng mang vector pGEX6p1, dng 1 v 3 (k hiu pGEX/htPA p1 v pGEX/htPA p3) l hai dng c kch thc ln hn cc dng khc v ln hn so vi i chng m; do vy rt c th cc dng ny mang cDNA m ha h-tPA. i vi cc dng mang vector pET21a(+), cc dng 1,2,3 c th mang cDNA m ha h-tPA (k hiu: pET/htPA p1, pET/htPA p2, pET/htPA p3).
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Hnh 3.4. Chn dng pET21a(+) mang cDNA m ha h-tPA (pET21a(+)/h-tPA) C: i chng m (vector pET21a(+) khng mang h-tPA) 1- 5: DNA plasmid ca cc dng pET21a(+) Tng t, i vi cc dng mang vector pET21a(+), cc dng 1,2,3 c kch thc ln hn cc dng khc v i chng m; do vy rt c th cc dng ny mang cDNA m ha h-tPA (k hiu: pET/htPA p1, pET/htPA p2, pET/htPA p3).
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chng minh chnh xc xem on xen vo plasmid c phi l on gen m ho h- tPA hay khng, chng ti tip tc kim tra bng enzyme hn ch, tin hnh PCR v gii trnh t. Kim tra vector ti t hp mang cDNA m ha h-tPA bng x l enzyme hn ch Hai cp enzyme NdeI/XhoI v BamHI/XhoI c s dng to u b sung cho vector v on chn nn kim tra, chng ti cng s dng hai cp enzyme ny nhm kim tra cc dng c chn c mang on chn hay khng. Theo tnh ton l thuyt, sn phm x l enzyme ca plasmid ti t hp bao gm hai on gen, mt on c kch thc tng ng vi vector gc (vector m vng) v mt on c kch thc tng ng vi on chn (cDNA m ha h-tPA). Thnh phn v quy trnh phn ng c tin hnh nh trnh by mc 2.2.4. Cc sn phm x l enzyme c in di trn gel agarose 0,8%.
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M: Marker 1kb 1: pGEX/htPA p1 2: pGEX/htPA p3
Hnh 3.5. Kim tra cc vector ti t hp mang cDNA m ha h-tPA bng enzyme hn ch Kt qu trn hnh 3.5 cho thy, sau khi x l cc vector ti t hp bng hai cp enzyme tng ng, chng ti thu c hai bng c kch thc khc nhau; i
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vi vector pGEX6p1, chng ti thu c mt bng c kch thc khong 5kb (tng ng vi kch thc ca vector pGEX6p1 khi x l bng hai enzyme BamHI/ XhoI) v mt bng c kch thc 1,7kb (tng ng vi kch thc cDNA m ho htPA). i vi vector pET21a(+), chng ti cng thu c mt bng c kch thc khong 5,5kb (tng ng vi kch thc vector pET21a(+)) v mt bng c kch thc khong 1,7kb (tng ng vi kch thc cDNA m ho h-tPA). Nh vy, chng ti s b kt lun l chn c mt s dng plasmid ti t hp mang on gen ph hp vi kch thc ca sn phm PCR h-tPA. Kim tra vector ti t hp mang cDNA m ha h-tPA bng k thut PCR khng nh li cc dng plasmid ti t hp c chn c mang ng on gen m ha h-tPA, chng ti tin hnh PCR vi vic s dng cc plasmid c chn lm khun nhn on h-tPA vi cc cp mi c hiu. DNA mu chy PCR l pGEX/htPA p3 v pET/htPA p1. Kt qu kim tra cc vector ti t hp bng k thut PCR c trnh by trn hnh 3.6.
Hnh 3.6. Kim tra cc vector ti t hp mang cDNA m ha h-tPA bng k thut PCR M: Marker 1kb 1: PCR s dng khun l pET/htPA p1 2: PCR s dng khun l pGEX/htPA p3
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Kt qu in di cho thy, sn phm PCR ca cc dng mang kim tra ny u xut hin bng vi kch thc khong 1,7 kb, ng vi kch thc cDNA m ha cho h-tPA. Nh vy, cc dng c chn lm khun trong th nghim PCR u c mang cDNA m ha h-tPA. Cc dng ny c chng ti tinh sch phc v cho xc nh trnh t. 3.1.4. Xc nh v phn tch trnh t cDNA m ho h-tPA Mc d c xc nh trnh t trong vector to dng pUC18, tuy nhin, khi nhn li cDNA ny bng enzyme Taq DNA polymerase c kh nng pht sinh cc im t bin. V vy, sau khi chn c cc dng mang cDNA m ho h-tPA, trc khi tin hnh biu hin, chng ti tinh sch v mt ln na xc nh trnh t c hai chiu on cDNA bng hai cp mi: cp mi T 7 i vi vector pET21a(+) v cp mi PEXF/PEXR i vi vector pGEX6p1 trn my xc nh trnh t t ng. Trnh t DNA sau c x l trn cc phn mm: BioEdit, Sequencing analysis 7.0. Sau khi s l s liu, chng ti thu c on gen c kch thc khong 1700bp. Ph trnh t ca cc dng plasmid u r rng, khng c tn hiu nhiu. Kt qu phn tch v so snh vi cc trnh t cng b trn Ngn hng Gen Quc t nh sau:
10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| atggatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtcttcgtt MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGlyAlaValPheVal ............................................................ MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGlyAlaValPheVal ............................................................ MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGlyAlaValPheVal 70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| tcgcccagccaggaaatccatgcccgattcagaagaggagccagatcttaccaagtgatc SerProSerGlnGluIleHisAlaArgPheArgArgGlyAlaArgSerTyrGlnValIle ............................................................ SerProSerGlnGluIleHisAlaArgPheArgArgGlyAlaArgSerTyrGlnValIle ............................................................ SerProSerGlnGluIleHisAlaArgPheArgArgGlyAlaArgSerTyrGlnValIle 130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| tgcagagatgaaaaaacgcagatgatataccagcaacatcagtcatggctgcgccctgtg CysArgAspGluLysThrGlnMetIleTyrGlnGlnHisGlnSerTrpLeuArgProVal
NM_000930 pET21a(+) pGEX6p1
NM_000930
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pET21a(+) pGEX6p1
............................................................ CysArgAspGluLysThrGlnMetIleTyrGlnGlnHisGlnSerTrpLeuArgProVal ............................................................ CysArgAspGluLysThrGlnMetIleTyrGlnGlnHisGlnSerTrpLeuArgProVal 190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| ctcagaagcaaccgggtggaatattgctggtgcaacagtggcagggcacagtgccactca LeuArgSerAsnArgValGluTyrCysTrpCysAsnSerGlyArgAlaGlnCysHisSer ............................................................ LeuArgSerAsnArgValGluTyrCysTrpCysAsnSerGlyArgAlaGlnCysHisSer ............................................................ LeuArgSerAsnArgValGluTyrCysTrpCysAsnSerGlyArgAlaGlnCysHisSer 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| gtgcctgtcaaaagttgcagcgagccaaggtgtttcaacgggggcacctgccagcaggcc ValProValLysSerCysSerGluProArgCysPheAsnGlyGlyThrCysGlnGlnAla ............................................................ ValProValLysSerCysSerGluProArgCysPheAsnGlyGlyThrCysGlnGlnAla ............................................................ ValProValLysSerCysSerGluProArgCysPheAsnGlyGlyThrCysGlnGlnAla 310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| ctgtacttctcagatttcgtgtgccagtgccccgaaggatttgctgggaagtgctgtgaa LeuTyrPheSerAspPheValCysGlnCysProGluGlyPheAlaGlyLysCysCysGlu ............................................................ LeuTyrPheSerAspPheValCysGlnCysProGluGlyPheAlaGlyLysCysCysGlu ............................................................ LeuTyrPheSerAspPheValCysGlnCysProGluGlyPheAlaGlyLysCysCysGlu 370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| atagataccagggccacgtgctacgaggaccagggcatcagctacaggggcacgtggagc IleAspThrArgAlaThrCysTyrGluAspGlnGlyIleSerTyrArgGlyThrTrpSer ............................................................ IleAspThrArgAlaThrCysTyrGluAspGlnGlyIleSerTyrArgGlyThrTrpSer ............................................................ IleAspThrArgAlaThrCysTyrGluAspGlnGlyIleSerTyrArgGlyThrTrpSer 430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....| acagcggagagtggcgccgagtgcaccaactggaacagcagcgcgttggcccagaagccc ThrAlaGluSerGlyAlaGluCysThrAsnTrpAsnSerSerAlaLeuAlaGlnLysPro ............................................................ ThrAlaGluSerGlyAlaGluCysThrAsnTrpAsnSerSerAlaLeuAlaGlnLysPro ............................................................ ThrAlaGluSerGlyAlaGluCysThrAsnTrpAsnSerSerAlaLeuAlaGlnLysPro 490 500 510 520 530 540 ....|....|....|....|....|....|....|....|....|....|....|....| tacagcgggcggaggccagacgccatcaggctgggcctggggaaccacaactactgcaga TyrSerGlyArgArgProAspAlaIleArgLeuGlyLeuGlyAsnHisAsnTyrCysArg ....................T....................................... TyrSerGlyArgArgProAspAlaIleArgLeuGlyLeuGlyAsnHisAsnTyrCysArg ....................T....................................... TyrSerGlyArgArgProAspAlaIleArgLeuGlyLeuGlyAsnHisAsnTyrCysArg
40
550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....| aacccagatcgagactcaaagccctggtgctacgtctttaaggcggggaagtacagctca AsnProAspArgAspSerLysProTrpCysTyrValPheLysAlaGlyLysTyrSerSer ............................................................ AsnProAspArgAspSerLysProTrpCysTyrValPheLysAlaGlyLysTyrSerSer ............................................................ AsnProAspArgAspSerLysProTrpCysTyrValPheLysAlaGlyLysTyrSerSer 610 620 630 640 650 660 ....|....|....|....|....|....|....|....|....|....|....|....| gagttctgcagcacccctgcctgctctgagggaaacagtgactgctactttgggaatggg GluPheCysSerThrProAlaCysSerGluGlyAsnSerAspCysTyrPheGlyAsnGly ............................................................ GluPheCysSerThrProAlaCysSerGluGlyAsnSerAspCysTyrPheGlyAsnGly ............................................................ GluPheCysSerThrProAlaCysSerGluGlyAsnSerAspCysTyrPheGlyAsnGly 670 680 690 700 710 720 ....|....|....|....|....|....|....|....|....|....|....|....| tcagcctaccgtggcacgcacagcctcaccgagtcgggtgcctcctgcctcccgtggaat SerAlaTyrArgGlyThrHisSerLeuThrGluSerGlyAlaSerCysLeuProTrpAsn ............................................................ SerAlaTyrArgGlyThrHisSerLeuThrGluSerGlyAlaSerCysLeuProTrpAsn ............................................................ SerAlaTyrArgGlyThrHisSerLeuThrGluSerGlyAlaSerCysLeuProTrpAsn 730 740 750 760 770 780 ....|....|....|....|....|....|....|....|....|....|....|....| tccatgatcctgataggcaaggtttacacagcacagaaccccagtgcccaggcactgggc SerMetIleLeuIleGlyLysValTyrThrAlaGlnAsnProSerAlaGlnAlaLeuGly ............................................................ SerMetIleLeuIleGlyLysValTyrThrAlaGlnAsnProSerAlaGlnAlaLeuGly ............................................................ SerMetIleLeuIleGlyLysValTyrThrAlaGlnAsnProSerAlaGlnAlaLeuGly 790 800 810 820 830 840 ....|....|....|....|....|....|....|....|....|....|....|....| ctgggcaaacataattactgccggaatcctgatggggatgccaagccctggtgccacgtg LeuGlyLysHisAsnTyrCysArgAsnProAspGlyAspAlaLysProTrpCysHisVal ............................................................ LeuGlyLysHisAsnTyrCysArgAsnProAspGlyAspAlaLysProTrpCysHisVal ............................................................ LeuGlyLysHisAsnTyrCysArgAsnProAspGlyAspAlaLysProTrpCysHisVal 850 860 870 880 890 900 ....|....|....|....|....|....|....|....|....|....|....|....| ctgaagaaccgcaggctgacgtgggagtactgtgatgtgccctcctgctccacctgcggc LeuLysAsnArgArgLeuThrTrpGluTyrCysAspValProSerCysSerThrCysGly ............................................................ LeuLysAsnArgArgLeuThrTrpGluTyrCysAspValProSerCysSerThrCysGly ............................................................ LeuLysAsnArgArgLeuThrTrpGluTyrCysAspValProSerCysSerThrCysGly
41
910 920 930 940 950 960 ....|....|....|....|....|....|....|....|....|....|....|....| ctgagacagtacagccagcctcagtttcgcatcaaaggagggctcttcgccgacatcgcc LeuArgGlnTyrSerGlnProGlnPheArgIleLysGlyGlyLeuPheAlaAspIleAla ............................................................ LeuArgGlnTyrSerGlnProGlnPheArgIleLysGlyGlyLeuPheAlaAspIleAla ............................................................ LeuArgGlnTyrSerGlnProGlnPheArgIleLysGlyGlyLeuPheAlaAspIleAla 970 980 990 1000 1010 1020 ....|....|....|....|....|....|....|....|....|....|....|....| tcccacccctggcaggctgccatctttgccaagcacaggaggtcgcccggagagcggttc SerHisProTrpGlnAlaAlaIlePheAlaLysHisArgArgSerProGlyGluArgPhe ............................................................ SerHisProTrpGlnAlaAlaIlePheAlaLysHisArgArgSerProGlyGluArgPhe ............................................................ SerHisProTrpGlnAlaAlaIlePheAlaLysHisArgArgSerProGlyGluArgPhe 1030 1040 1050 1060 1070 1080 ....|....|....|....|....|....|....|....|....|....|....|....| ctgtgcgggggcatactcatcagctcctgctggattctctctgccgcccactgcttccag LeuCysGlyGlyIleLeuIleSerSerCysTrpIleLeuSerAlaAlaHisCysPheGln ............................................................ LeuCysGlyGlyIleLeuIleSerSerCysTrpIleLeuSerAlaAlaHisCysPheGln ............................................................ LeuCysGlyGlyIleLeuIleSerSerCysTrpIleLeuSerAlaAlaHisCysPheGln 1090 1100 1110 1120 1130 1140 ....|....|....|....|....|....|....|....|....|....|....|....| gagaggtttccgccccaccacctgacggtgatcttgggcagaacataccgggtggtccct GluArgPheProProHisHisLeuThrValIleLeuGlyArgThrTyrArgValValPro ............................................................ GluArgPheProProHisHisLeuThrValIleLeuGlyArgThrTyrArgValValPro ............................................................ GluArgPheProProHisHisLeuThrValIleLeuGlyArgThrTyrArgValValPro 1150 1160 1170 1180 1190 1200 ....|....|....|....|....|....|....|....|....|....|....|....| ggcgaggaggagcagaaatttgaagtcgaaaaatacattgtccataaggaattcgatgat GlyGluGluGluGlnLysPheGluValGluLysTyrIleValHisLysGluPheAspAsp ............................................................ GlyGluGluGluGlnLysPheGluValGluLysTyrIleValHisLysGluPheAspAsp ............................................................ GlyGluGluGluGlnLysPheGluValGluLysTyrIleValHisLysGluPheAspAsp 1210 1220 1230 1240 1250 1260 ....|....|....|....|....|....|....|....|....|....|....|....| gacacttacgacaatgacattgcgctgctgcagctgaaatcggattcgtcccgctgtgcc AspThrTyrAspAsnAspIleAlaLeuLeuGlnLeuLysSerAspSerSerArgCysAla ............................................................ AspThrTyrAspAsnAspIleAlaLeuLeuGlnLeuLysSerAspSerSerArgCysAla ............................................................ AspThrTyrAspAsnAspIleAlaLeuLeuGlnLeuLysSerAspSerSerArgCysAla 1270 1280 1290 1300 1310 1320 ....|....|....|....|....|....|....|....|....|....|....|....| caggagagcagcgtggtccgcactgtgtgccttcccccggcggacctgcagctgccggac GlnGluSerSerValValArgThrValCysLeuProProAlaAspLeuGlnLeuProAsp
NM_000930
42
pET21a(+) pGEX6p1
............................................................ GlnGluSerSerValValArgThrValCysLeuProProAlaAspLeuGlnLeuProAsp ............................................................ GlnGluSerSerValValArgThrValCysLeuProProAlaAspLeuGlnLeuProAsp 1330 1340 1350 1360 1370 1380 ....|....|....|....|....|....|....|....|....|....|....|....| tggacggagtgtgagctctccggctacggcaagcatgaggccttgtctcctttctattcg TrpThrGluCysGluLeuSerGlyTyrGlyLysHisGluAlaLeuSerProPheTyrSer ............................................................ TrpThrGluCysGluLeuSerGlyTyrGlyLysHisGluAlaLeuSerProPheTyrSer ............................................................ TrpThrGluCysGluLeuSerGlyTyrGlyLysHisGluAlaLeuSerProPheTyrSer 1390 1400 1410 1420 1430 1440 ....|....|....|....|....|....|....|....|....|....|....|....| gagcggctgaaggaggctcatgtcagactgtacccatccagccgctgcacatcacaacat GluArgLeuLysGluAlaHisValArgLeuTyrProSerSerArgCysThrSerGlnHis ............................................................ GluArgLeuLysGluAlaHisValArgLeuTyrProSerSerArgCysThrSerGlnHis ............................................................ GluArgLeuLysGluAlaHisValArgLeuTyrProSerSerArgCysThrSerGlnHis 1450 1460 1470 1480 1490 1500 ....|....|....|....|....|....|....|....|....|....|....|....| ttacttaacagaacagtcaccgacaacatgctgtgtgctggagacactcggagcggcggg LeuLeuAsnArgThrValThrAspAsnMetLeuCysAlaGlyAspThrArgSerGlyGly ............................................................ LeuLeuAsnArgThrValThrAspAsnMetLeuCysAlaGlyAspThrArgSerGlyGly ............................................................ LeuLeuAsnArgThrValThrAspAsnMetLeuCysAlaGlyAspThrArgSerGlyGly 1510 1520 1530 1540 1550 1560 ....|....|....|....|....|....|....|....|....|....|....|....| ccccaggcaaacttgcacgacgcctgccagggcgattcgggaggccccctggtgtgtctg ProGlnAlaAsnLeuHisAspAlaCysGlnGlyAspSerGlyGlyProLeuValCysLeu ............................................................ ProGlnAlaAsnLeuHisAspAlaCysGlnGlyAspSerGlyGlyProLeuValCysLeu ............................................................ ProGlnAlaAsnLeuHisAspAlaCysGlnGlyAspSerGlyGlyProLeuValCysLeu 1570 1580 1590 1600 1610 1620 ....|....|....|....|....|....|....|....|....|....|....|....| aacgatggccgcatgactttggtgggcatcatcagctggggcctgggctgtggacagaag AsnAspGlyArgMetThrLeuValGlyIleIleSerTrpGlyLeuGlyCysGlyGlnLys ............................................................ AsnAspGlyArgMetThrLeuValGlyIleIleSerTrpGlyLeuGlyCysGlyGlnLys ............................................................ AsnAspGlyArgMetThrLeuValGlyIleIleSerTrpGlyLeuGlyCysGlyGlnLys 1630 1640 1650 1660 1670 1680 ....|....|....|....|....|....|....|....|....|....|....|....| gatgtcccgggtgtgtacaccaaggttaccaactacctagactggattcgtgacaacatg AspValProGlyValTyrThrLysValThrAsnTyrLeuAspTrpIleArgAspAsnMet ............................................................ AspValProGlyValTyrThrLysValThrAsnTyrLeuAspTrpIleArgAspAsnMet ............................................................ AspValProGlyValTyrThrLysValThrAsnTyrLeuAspTrpIleArgAspAsnMet
43
....|.... cgaccgtga ArgProEnd ......--ArgPro ......... ArgProEnd
Hnh 3.7. So snh trnh t nucleotide ca h-tPA trong pGEX6p1/h-tPA v pET21a(+)/h-tPA vi NM_000930 T kt qu so snh trn, chng ti nhn thy trnh t cDNA m ha h-tPA c to dng trong hai vector biu hin hon ton khng thay i so vi trnh t cDNA xc nh c trong vector to dng pUC18. So vi NM_000930, trnh t cDNA ca chng ti khng c s thay i no ng k, ch c mt sai khc nm v tr 499 ca cDNA m ha h-tPA. trnh t NM_000930, v tr 499 nucleotitde ny l C c thay th bng nucleotide T trong trnh t chng ti ang nghin cu biu hin. Tuy nhin trnh t ny khng lm thay i trnh t acid amin, v vy khng nh hng ti cu trc v chc nng ca protein. vector pET21a(+), khng c trnh t kt thc do chng ti thit k mi PCR nhm gn ui His vo gip qu trnh tinh sch protein h-tPA sau ny c d hn. 3.2. Biu hin gen 3.2.1. Tng hp protein h-tPA ti t hp Trong nghin cu ny, sau khi thu c cc dng plasmid mang gen cDNA m ho h-tPA, biu hin trong vi khun E. coli, chng ti tin hnh bin np hai vector ti t hp (pET21a(+) v pGEX6p1) mang gen m ho h-tPA vo t bo vi khun E. coli chng BL21(DE3) plysS. Cc dng t bo ny sau c cy chuyn v tip tc nui lc 200 v/p trong mi trng LB lng c b sung Amp 37oC cho ti khi OD= 0,6 th tin hnh cm ng IPTG 0,1mM. i vi vector biu hin pGEX6p1, theo tnh ton l thuyt, protein h-tPA ti t hp c khi lng phn t khong 95kDa (bng tng khi lng ca protein h-tPA (70 kDa) v on GST (26,4 kDa)); cn i vi vector biu hin pET21a(+), khi lng phn t protein ti t hp thu c khong 72kDa.
44
Sau mi gi t khi cm ng IPTG chng ti tin hnh thu 1ml dch t bo nhm kim tra biu hin ca t bo. Kt thc th nghim, chng ti thu c dch t bo c cha protein ti t hp. kim tra kt qu biu hin chng ti tin hnh in di protein trn gel polyacrylamide 10%. 3.2.2. Kim tra protein bng phng php in di trn SDS-PAGE i vi vector biu hin pGEX6p1 mang h-tPA, kt qu phn tch SDSPAGE cho thy cc mu th nghim xut hin mt bng protein mi c kch thc khong 95kDa so vi cc mu i chng. y rt c th l dng protein dung hp gia h-tPA (72kDa) vi ui GST (26,4kDa) ca vector pGEX6p1. Mu i chng m chng ti s dng l dch ph mng ca t bo BL21 c cha vector pGEX6p1 gc (ging 5) v dch ph mng ca E.coli BL21 khng mang vector (ging 7) c cm ng IPTG. Cc mu i chng m ny cng xut hin bng nhng kch thc nh v khng r nt. C th nhng mu ny, t bo ch c tng hp mt s protein c cng kch thc nhng hm lng thp. ging 5 xut hin bng m c kch thc 26,4kDa, y l bng GST c tng hp tuy nhin do plasmid khng mang gen cDNA m ha h-tPA nn trong qu trnh tng hp protein, h-tPA khng c tng hp.
45
Hnh 3.8. in di kim tra protein h-tPA trong pGEX/h-tPA p3 M: Marker 1, 2, 3, 4: Mu thu c sau 0h, 1h, 2h, 3h cm ng IPTG 5: Dng t bo mang vector pGEX6p1 6: Mu thu c sau 3h cm ng IPTG 7: Dng t bo khng mang vector xc nh c thi im ti lng protein c tng hp l cao nht, chng ti tin hnh th nghim trong 3h sau khi cm ng. Sau khong thi gian 0h, 1h, 2h, 3h chng ti tin hnh thu mu. Kt qu cho thy kch thc v
46
m ca bng ny tng theo thi gian sau khi cm ng IPTG v m nht ti thi im 3h sau cm ng. Kt qu ny cho thy dng t bo E.coli BL21 c mang plasmid pGEX6p1 ti t hp c kh nng tng hp protein dung hp GST-htPA. Tuy nhin hm lng protein ti t hp cn thp so vi tng lng protein c tng hp. Song song vi th nghim biu hin h-tPA trong vector pGEX6p1, chng ti cng tin hnh biu hin protein h-tPA trong vector pET21a(+) trn t bo ch E.coli BL21. Sau khi thu c dch protein, chng ti in di trn gel polyacrylamide 10% vi thang protein chun v cc i chng m. Cc i chng m y cng l dch ph mng t bo E. coli BL21 khng mang vector v c mang vector pET21a(+) u c cm ng IPTG.
47
Hnh 3.9. in di protein h-tPA trong pET21/h-tPA p1 M: Marker 1: Dng t bo BL21 2: Dng t bo BL21 mang vector pET21a(+) gc 3, 4, 5, 6: Mu thu c sau 0h, 1h, 2h, 3h cm ng IPTG Sau khi thu c dch protein, chng ti in di trn gel polyacrylamide 10% vi thang protein chun v cc i chng m. Cc i chng m y cng l
48
dch ph mng t bo E. coli BL21 khng mang vector v c mang vector pET21a(+) u c cm ng IPTG. Kt qu in di cho thy khng c s khc bit nhiu v s lng v m nht bng gia cc dng E.coli BL21 mang vector pET21a(+)/h-tPAvi cc i chng m (ging 1 v ging 2). v tr 72kDa c xut hin bng nhng gia cc mu in di l ging nhau. Nu h-tPA khng c biu hin th bng 72kDa ny l protein c cng khi lng ca t bo E.coli c tng hp. Nu gen h-tPA c tng hp th hm lng protein c tng hp l rt thp so vi tng lng protein ca t bo ch. c nhng kt lun chnh xc v qu trnh biu hin ca cDNA m ha htPA, cn c nhng nghin cu khc nh: lai Western Blot, hay k thut Elisa... nhm khng nh protein h-tPA c tng hp. Mt khc, cc iu kin th nghim khc cng cn c nghin cu ti u qu trnh biu hin cho lng protein htPA l ln nht v tin ti ng dng quy trnh biu hin. Cng cn nhn mnh rng, mc d h thng biu hin gen ca sinh vt bc cao trn i tng vi khun E. coli bn cnh nhiu u im nh: mi trng nui cy r; d iu khin qu trnh biu hin; hm lng protein thu c ln (n 30% tng protein t bo).... cn tn ti mt vi nhc im. y l mt qu trnh phc tp v kh thc hin do h thng ny ch biu hin tt i vi protein c kch thc nh hn 80 acid amin, i vi cc protein c kch thc ln th h thng biu hin E.coli c th tng hp c protein nhng protein ny c th khng cun xon; b ba m ha E.coli c s khc bit nhiu vi b ba m ha sinh vt bc cao; cc qu trnh ci bin sau dch m thng khng c; kh nng biu hin mt protein n l khng tt..... i vi h-tPA, c nhiu cng trnh cng b biu hin c trn E. coli nhng hm lng protein thu c cha cao. V vy, vic nghin cu biu hin trn cc i tng khc nh nm men, t bo cn trng, t bo ng vt cng c quan tm nhiu phng th nghim.
49
KT LUN V NGH
Kt lun 1. to c hai vector biu hin pGEX6p1 v pET21a(+) mang cDNA m ha h-tPA c kch thc khong 1,7kb. 2. xc nh trnh t nucleotide ca cDNA m ha h-tPA. So snh trnh t nucleotide ca cDNA m ha h-tPA vi trnh t gen trn Ngn hng Gen Quc t, m s NM_000930 cho thy tng ng t 99%. So vi NM_000930, trnh t cDNA nghin cu khng c s thay i no ng k, ch c mt sai khc nm v tr 499 ca cDNA m ha h-tPA. trnh t NM_000930, v tr 499 nucleotitde ny l C c thay th bng nucleotide T. Tuy nhin, s khc bit ny khng lm thay i trnh t acid amin, v vy khng nh hng ti cu trc v chc nng ca protein. 3. tin hnh biu hin cDNA m ha h-tPA trn hai vector pGEX6p1 v pET21a(+) v thu c protein h-tPA khi biu hin trong vector pGEX6p1. ngh Tip tc nghin cu biu hin cDNA m ha h-tPA trong vector pET21a(+) trn vi khun E.coli chng BL21. Ti u ho qu trnh biu hin i vi cDNA m ha h-tPA trong pGEX6p1 nhm thu c hm lng protein ln nht.
50
TI LIU THAM KHO

Ting Vit
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L Trn Bnh, Cao Huyn Trang (2005), Nghin cu v pht trin sn xut vaccine n c trong thc vt, Tp ch Cng ngh Sinh hc, 3(2), tr. 133- 142. Nguyn nh Cng, Nguyn Thy Dng, L Th Thu Hin, Lng Th Thu Hng, Trn Th Phng Lin, Nguyn Huy Hong, Phan Vn Chi, Nng Vn Hi (2003), Biu hin gen m ha protein bt hot ribosome ca cy mp ng vi khun Escherichia coli, Tp ch Cng ngh Sinh hc, 1(4), tr. 451- 460.
3.
Nguyn Thy H, Nguyn Bch Nhi, Khc Hiu, Phan Vn Chi (2003), Kh nng c ch cc dng t bo ung th nui cy in vitro ca Trichobakin ti t hp, Y hc Vit Nam, 284(5), tr. 26- 30.
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Bi Th Huyn, ng Thnh Nam, Nguyn Nam Long, Nguyn Bch Nhi, Hong Quc Trng, Phan Vn Chi (2005), Biu hin v xc nh interleukin- 2 ca ngi, Tp ch Cng ngh Sinh hc, 3(2), tr. 143- 148.
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Nguyn Nam Long, Nguyn Bch Nhi, Phan Vn Chi (2005), Tch dng gen m ha interleukin- 2 ca ngi, Y hc Vit Nam, 310(5), tr. 26- 30. Chu K Nam, Hong Vn Quc Chng, Trn Linh Thc (2005), To dng v biu hin mini-proinsulin ca ngi ti t hp trong Escherichia coli, Tp ch Cng ngh Sinh hc, 3(2), tr. 155- 160.
8.
Phm Thip, V Ngc Thy, Nguyn Hu Lc (1992), Thuc, bit dc v cch s dng, Nh xut bn Khoa hc v K thut.
51
Ting Anh
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Axelsson F. (1995), Plasminogen, Product monograph. Becker C., Nong V.H., Bumlein H., Le T.X., Farouk A., Will H., Bassner R. (1993), Synthesis and accumulation of human tissue-type plasminogen activator (h-tPA) in transgenic tobaco seeds, Seed storage compounds 35, pp. 326- 331.
11.
Berg D.T., Burck P.J., Grinnell B.W. (1993), Kringle glycosylation in a modified human tissue plasminogen activator improves functional properties, Blood 81(5), pp. 1312- 1322.
12. 13.
Bhopale G.M., Nanda R.K. (2005), Recombinant DNA expression products for human therapeutic use, Curr. Scie. 89(4), pp. 614- 622. Booyse F.M., Scheinbuks J., Lin P.H., Traylor M., Bruce R. (1988), Isolation and interrelationships of the multiple molecular tissue-type and urokinasetype plasminogen activator forms produced by cultured human umbilical vein endothelial cells, J. Biol. Chem. 263(29), pp. 15129- 15138.
14.
Bulleid N.J., Bassel- Duby R.S., Freedman R.B., Sambrook J.F., Gething M.H. (1992), Cell-free synthesis of enzymically active tissue-type plasminogen activator, Biochem. J. 286, pp. 275- 280.
15.
Burck P.J., Berg D.H., Warrick M.W., Berg D.T., Walls J.D., Jaskunas S.R., Crisel R.M., Weigel B., Vlahos C.J., McClure D.B., Grinnell B.W. (1990), Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain, J. Biol. Chem. 265(9), pp. 5170- 5177.
16.
Carlie V.C., Veerman H., Pannekoek H. (1989), Identification of the domains of tissue-type plasminogen activator involved in the augmented binding to fibrin after limited digestion with plasmin, J. Biol. Chem. 264(21), pp. 12604- 12610.
17. 18.
Cartwright T. (1992), Production of t-PA from animal cell culture, Animal. Cell Biotechnol. 5, pp. 218- 245. Castellio F.J. (1983), Plasminogen activator, BioScience 33(11), pp. 647- 650. 52
19. 20. 21.
Collen D., Lunen R. (2004), Tissue- type plasminogen activator: a historical perspective and personal account, J. Thromb. Haemost 2, pp. 541- 546. Degeng S.J.F., Rajput B., Reich E. (1986), The human tissue plasminogen activator gene, J. Biol. Chem. 261(15), pp. 6972- 6985. Demaerschalk B.M., Yip T.R. (2005), Economic benefit of increasing utilization of intravenous tissue plasminogen activator for acute ischemic stroke in the United States, Stroke 36, pp. 2500- 2503.
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Website
58. 59. 60.
http://www.i-s-b.org http://www.merck.com http://thuocbietduoc.com.vn
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PH LC Bng 1. Ha cht s dng Ha cht Cc loi enzyme hn ch Taq DNA polymerase T4 ligase Thang DNA chun, protein chun dNTP Kit Wizard SV Gel and PCR Clean-up System Agarose Phenol Chloroform Isoamylalcohol Tris acetic acid EDTA Ampicillin LB BigDye Terminator Cn
Hng sn xut MBI Fermentas (M) MBI Fermentas (M) MBI Fermentas (M) MBI Fermentas (M) MBI Fermentas (M) Promega (M) Gibco (M) Merck (c) Merck (c) Merck (c) Merck (c) Merck (c) Serva (c) US Biological (M) Applied Biosciences (M) Merck (c)
Bng 2. Thit b s dng Thit b My ly tm 5415R My PCR 9700 My in di My soi DNA My ht chn khng Speed Vac My xc nh trnh t t ng ABI 3100 My chun pH Hng sn xut Eppendorf (c) Applied Biosystems (M) Mupid- 2Plus (Nht) Bio- Rad (M) Savant (M) Applied Biosystems (M) Mettler Toledo (Thy S)
57
Bng 3. Dung dch cn pha Dung dch m chy in i DNA TAE 1X m tra mu DNA (Loading buffer) Ha cht Tris-acetate EDTA Bromophenol blue Xylene cyanol FF Sucrose trong H2O Tris base Glycine SDS H2O Tris- HCl pH= 6,8 Glycerol SDS DTT Bromophenol Blue Comasive R250 Metanol Acid acetic H2O Acid acetic 7% Metanol 20% H2O Tris- HCl, pH 8,0 EDTA, pH 8,0 Glucose NaOH SDS CH3COOK, pH 5,5 CH3COOH Tris- HCl, pH 8,0 EDTA, pH 8,0 58 Nng 40mM 1mM 0,25% 0,25% 40% 3g 14,4 g 1g 1l 0,5M 60% 20% 1M 0,2% 0,3 g 120 ml 30 ml 150 ml 35 ml 100 ml 365 ml 25mM 10mM 50mM 0,2N 1% 3,0M 11,5% 0,1mM 0,01mM
m chy in di protein (1X Tris- Glycine pH 8,3)
m tra mu protein (Protein sample buffer 4X)
Dung dch nhum protein (Comasive Brilliant Blue R250)
Dung dch ty gel protein
Dung dch I (Sol I) (Kh trng, gi 4oC) Dung dch II (Sol II) (Pha mi trc khi s dng) Dung dch III (Sol III) (Kh trng, gi 4oC) Dung dch TE 0,01M, pH 8,0
Bng 4. Cc cp mi s dng Mi T7 promoter T7 terminator M13 forward M13 reverse Trnh t mi

5-TAA TAC GAC TCA CTA TAG G-3 5-GCT AGT TAT TGC TCA GCG G-3 5-TGT AAA ACG ACG GCC AGT-3 5-CAG GAA ACA GCT ATG ACC-3
Bng 5. Thnh phn PCR nhn cDNA m ha h-tPA Thnh phn H2O Buffer PCR MgCl2 dNTP Primer F(Mi xui) Primer R(Mi DNA Taq DNA polymerase Tng th tch:
ngc)
Nng 10X 25mM 10mM 10pmol/ul 10pmol/ul 6g/ml 5u/l
Th tch (l) 14,25 2,5 2,5 2,5 1,0 1,0 1,0 0,25 25,0
59
Bng 6. Thnh phn phn ng x l DNA bng enzyme hn ch Thnh phn Buffer Tango BamHI (NdeI) XhoI DNA H2O Tng th tch Nng 10X 10u/l 10u/l 1 g/l Th tch (l) 1 0,5 0,5 2 6 10
Bng 7. Thnh phn phn ng loi phosphate vector Thnh phn Buffer for CIAP CIAP Vector ( x l bng enzyme hn ch) H2O Tng th tch Nng 10X 1u/l 1g/l Th tch (l) 2,0 1,5 15,0 1,5 20
Bng 8. Thnh phn phn ng ghp ni DNA vo vector Thnh phn T4 Buffer Vector Sn phm PCR T4 ligase Tng th tch Nng 10X 10ng/l 10ng/l 5u/l Th tch (l) 1,0 2 5 1,0 10,0
60
Bng 9. Thnh phn PCR xc nh trnh t Thnh phn Big Dye Mi Buffer DNA plasmid H2O Tng th tch Nng 0,01 g/l 2,5X 10ng/l Th tch (l) 3 1,275 3 3 4,725 15
Bng 10. Thnh phn gel polyacrylamide Gel c 4% (Stacking gel) 0,75 ml 30 l 0,4 ml 1,8 ml 15 l 3 l 3 ml Gel tch 10% (Separating gel) 2,25 ml 90 l 3,02 ml 3,55 ml 45 l 4,5 l 9 ml
Thnh phn Tris- HCl, pH=6,8 Tris- HCl, pH= 8,8 SDS Acrylamide H2O APS TEMED Tng th tch
Nng 0,5M 1,5M 10% 30% 10% -
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Nghien Cuu Gen Tren Ecoli

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I HC THI NGUYN TRNG I HC S PHM --------------------------------------------

LUN VN THC S SINH HC

Thi Nguyn 2008

I HC THI NGUYN TRNG I HC S PHM --------------------------------------------

Chuyn ngnh : Di truyn hc M s : 60.42.70

Ngi hng dn khoa hc: TS. L TH THU HIN

Thi Nguyn - 2008

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

dNTP E. coli EDTA EGF

PCR rDNA RNA RNase

EtBr F GST h- tPA

scuPA SDS SK TAE

Isopropyl b-D thiogalactoside tPA

Vng Kringle 1 Vng Kringle 2

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Formatted: Space Before: 0 pt, After: 0 pt Formatted: Font: Bold

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S ha bi Trung tm Hc liu i hc Thi Nguyn

TNG QUAN TI LIU

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Hnh 1.1. Cu trc ca tPA v uPA [9]

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Hnh1.2. Cc vng ca tPA

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Hnh 1.3. Cu trc h-tPA [38]

S ha bi Trung tm Hc liu i hc Thi Nguyn

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Hnh 1.4. Qu trnh phn hy huyt khi [9]

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VT LIU V PHNG PHP NGHIN CU

Vt liu, ha cht v thit b Vt liu

Hnh 2.1. Bn vector pET21a(+) v pGEX6p1

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ca sn phm ghp ni vo ng t bo kh bin (khong 100l). Gi trong khong 15 pht;

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S ha bi Trung tm Hc liu i hc Thi Nguyn

Ra cn bng 0,2ml cn 70%, ly tm 12.000 v/p trong 15 pht, lm kh v

DNA -20oC; Kim tra DNA bng gel agarose 0,8 %.

Tip , sn phm PCR c tinh sch bng phng php ta cn/EDTA:

S ha bi Trung tm Hc liu i hc Thi Nguyn

B sung 4l EDTA 125mM c pH 8,0 v 60l cn 100%. o nh hn hp

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S ha bi Trung tm Hc liu i hc Thi Nguyn