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TIU LUN K THUT GEN

ti : Phng php pht hin v phng chng HIV


GVHD: PGS.TS Khut Hu Thanh SVTH : Phm Anh Ton Nguyn Xun Knh

HIV
Hi

chng suy gim min dch ngi Ngun gc 1900 ,1 th sn nhim AIDS virus t Pan troglodytes chimpanzee 1981-2006 ,25 tr ngi 0,4% nhim HIV ,2009

HIV
H

Ritrovirus Chi Lentivirus Di truyn ngc chiu t RNA sang DNA HIV-1 , HIV-2

TI LNG VIRUT
o

s lng bn sao ca virut trong mu Copies/ml Gi tr nn Chu k ngng (CT) ng nn (base line)

CC K THUT PHN T
K thut real time PCR K thut bDNA K thut NASBA

O TI LNG VIRUT

K THUT REAL TIME PCR


X

l huyt tng thu RNA tinh sch RNA virut phin m ngc thnh cDNA Mu d Taq man bt cp vi cDNA Taq polymerase ct b mu d , nh sng hunh quang pht ra

X L HUYT TNG THU DNA RNA TINH SCH


trng ni ly mu bng cn 70 S dng cht chng ng mu EDTA Ly tm ng cha mu 2000v/ pht thu huyt tng
St

TCH CHIT RNA TNG S T MU MU BNG KIT HNG


INVITROGEN m bo thu c Rna tinh sch ,cn loi b Rnase bm trn dng c thit b bng cch ngm dng c trong dung dch DEPC 1% trong 8-12 h ,hp kh trng vi nhit p sut cao .ly 200 ul mu cho vo ng eppendorf ,b sung proteinase K .lc nh 30s , 20 pht 37 .li tm 6 pht tc 12000 vng /pht thu dch ,thm 400 ul iso propan v trn u .chuyn dch sang ct S N A P .Li tm 1 pht tc 12000v/pht ,b dch li tm v ra ct b dch li tm v ra ct 2 ln bng dung dch ra 1X ,trong 2 pht S dng 120 ul nc kh ion thu axit nucleic tng s .Loi DNA bng cch cho thm 15ul m 10X cho Dnase , 37 c trong 8 -10 pht ,thm 300ul Iso propan v o ng 4 -5 ln Chuyn dch vo 1 ct S N A P mi .Li tm trong 1 -2 pht tc 12000v/pht ,b dch ly tm v ra ct 2 ln bng dung dch ra 1x .s dng 120 ul nc kh ion thu RNA tng s tinh sch
K thut gen v nguyn l ng dng PGS.TS KHUT HU THANH page 106

KIM TRA TINH SCH RNA


Kim tra tinh sch RNA bng in di trn gel agarose Kim tra tinh sch ca RNA bng quang ph k

MU D TAQ MAN BT CP VI CDNA

KIT TH HIV

K THUT NASBA (NUCLEIC ACID SEQUENCE BASED AMPLIFICATION)


K thut NASBA dng cho cc tc nhn c b gene l RNA mch n Huyt tng ca bnh nhn s qua bc x l v thu RNA tinh sch Trnh t RNA ca HIV s c phin m ngc thnh cDNA Mch RNA trong phn t RNA-DNA lai s b hy b bi enzyme RNAse H Cui cng mi xui ,enzyme T7 RNA polymerase mch RNA mi s c tng hp trn khun mu ca mch cDNA Qu trnh ny c lp li nhiu ln, kt qu l 1 lng ln RNA s c to ra

1.Khuch i chui acid nucleic ca hn 109 bn cth c thc hin ch trong 90 pht hnh ng ba enzyme. 2. Thit b t tin thermocycling l khng cn thit nh l cc phn ng xy ra isothermally 41 C 3. N gip trong phn ng RT-PCR tt hn v n cung cp ng hc khuch i nhanh hn. 4. Retrovirus c c pht hin v nh lng bng cch khuch i ch c b gen RNA v khng phi bn sao DNA proviral. 5. N c th o sao chp ca virus DNA bng cch pht hin cc biu hin mRNA cui. 6. N h tr vic pht hin cc trnh t mRNA con ngi m khngc cc nguy c nhim DNA

II.LIU PHP GEN TRONG PHNG CHNG HIV/AIDS


Gy c ch hot ng gen ca HIV, hoc bin i gen ca HIV a. Vecto liu php -Vect c bn cht virus thng -.ng dng l vect thit k t adenovirus AAV v HIV-1. -Vect khng c bn cht virus thng s dng cc oligoncleotid, polycalion. liposom... -AAV viral vecto c thit k mang mt gen liu php 4,7 kb nhm hn ch ti bn HIV trong t bo, tuy nhin kh nng gn gen liu php vo nhim sc th s 19 ca ngi rt thp. v hu nh khng c tc dng (Chaterjee v cng s, 1992).
1.

ADENOVIRAL TRONG LIU PHP PHNG CHNG HIV

-Vect liu php thit k t adenovirus l mt vect lai cha ba loi gen: gen ca adenovirus l (on CCD Ad5; gen ca HIV l gen gag v tpa bin i ca HIV ; gen ca ngi l promoter CMV (hCMV - gen human cytomegavirus). to nn hai vect liu php gen phng chng HIV l vect Ad5FLgag (Ad5 HIVI gag) v Ad5tpagag (Ad CMVI- ipaHIVgag). Cc vect liu php gen Ad5FLgag vi Ad5tpagag d c th nghim trn kh v trn chui cho kt qu rt kh quan, t l t bo nhim HIV ca kh dc iu tr gim nhiu so vi kh khng c iu tr. Cc vect ny l nhng vect trin vng c th s dng phng v chng HIV/AIDS cho ngi trong thi gian sp ti.

-Vect liu php thit k t virus HIV-1 c thit k v th nghim t nm 1991 (Poznansky v cng s, 1991; Shimada v cng s 1991). C s thit k vect liu php phng chng HIV -1 t b gen HIV - 1 bin di, v c ci gn thm mi s gen khc c chc nng c ch ti bn hoc hot ng ca HIV. n nay, c nhi loi vecto phng chng HIV c s dng. Trong c mt s vect th nghim c hiu qu.

-V d: Vect pCMVAR9 bao gm b gen virus HIV-1 d ct bt mt s gen, v ci thm promoter hCMV ca b gen ngi (hCMV gen human cytomegavirus) v tn hiu ng gi SD (packaging signal); Vect m hoa protein v ca virus (env-coding plasmid), plasmid ny dc thit k gm cc gen m ha v ca virus MLV (Moloney murine leukemia virus); Vecto HIV chuyn dch (transfer vector), vect ny mang cc gen GA, RRE v promoter CMV ca b gen ngi c chc nng ngn cn ti bn gen ca HIV trong t bo ng thi hn ch kh nng gn b gen HIV vi b gen t bo....

-Cc vect HIV-1 phng chng nhim HIV/AIDS c th nghim trn chut, c hiu qu hn ch pht trin ca virus HIV trong t bo. Tuy nhin khi s dng viral vect HIV-1 c hin tng ti t hp gen gia vect liu php vi virus HIV, dn n cc t bin gen v cc hu qu khng mong mun. Do vy vect liu php 111V-1 cho n nay cn l vn cn tip tc nghin cu, th nghim trn ng vt v trn cc t bo ngi in vitro.

B. S DNG OLIGO- AIITISEN V RIBOZYM TRONG PHNG CHNG HIVI AIDS


-Oligo-antisen c th c ch qu trnh dch m ca HIV

hoc c ch hot ng phin m ca nhiu gen khc nhau ca HIV nh c ch gen gag (Lori v cng s, 1994 ), km hm gen rev (Vandedriessche v cng s, 1995) gen em ((uotoku. J v cng s, 1991)... -Ribozvm c dng tm n RNA thng tin (mRNA) ca HIV , thc hin phn ng ct c hiu, lm mt hot tnh mRNA ca HIV (Dropulic, 1994). Virus HIV-1 ribozym c th tc ng v gy mt hot tnh mRNA ca mt s gen HIV nh cc gen gag, pol, tat (Chen v cng s, 1992; Dropulic, 1994)...

-Hin nay nhiu th nghim viral vect v nonviral

vecto phng chng HIV/AIDS, tuy nghin cn th nghim trn ngi thu c kt qu cn thp. Cn nghin cu tm ra cc vect liu c hiu qu thc s, gp phn hn ch tc ng ca i dch AIDS ton cu. -S dng cc liu php gen phng chng HIV/A1DS, cn la chn cc gii php thch hp vi nhng giai on khc nhau trong chu trnh xm nhim t bo v pht trin ca HIV, nhm m bo hiu qu ca liu php.

CC GIAI ON TRONG CHU TRNH XM NHIM


V PHT TRIN CA VIRUT HIV

MT S CHIN LC LIU PHP GEN CHNG HIV/AIDS


Tnh cht giai on
1.

Liu php thch hp


Tip cn t bo ti th th CD4

Oligo-antisens chng lin kt th th, khng th chng CCR,, ribozym Ribozym, oligo-antisens chng env transdominant gp41

2. Gn vi th th CD4

3. Phn li tch khi phn v 4. Phin m ngc to cDNA 5. B gen HIV gn vi b gen t ho 6. Phin m to mRNA virus 7.Dch m tng hp protein v 8.Hnh thnh v capsid 9. Ti bn b gen H1V 10. Lp ghp cc ht virion 11. Cc virus HIV mi ph t bo 12. Tip tc xm nhim t bo khc

Viral vect chng tat,att Ribozym, oligo-antisens chng pol

Transdominant rev, ribozym Viral vecto, transdominant gag

2. PHNG CHNG HIV/ AIDS BNG TNG CNG MIN


DCH CA T BO

S dng gen t bin Rev 10 - To mt loi pr t bin trong t bo bng gen t bin Rev 10 - S dng vect oligonucleotid mang Rev 10, chuyn vo t bo lympho TCD4 bo nh k thut bn gen. Kt qu cho thy, c s ti t hp vi b gen t bo to nn cc t bo lympho TCD4 mang gen t bin Rev 10, cc t bo ny c kh nng km hm s xm nhim ca HIV (Woffendin v cng s, 1996; Lim v cng s, 1993).
a.

B.S DNG GEN TNG CNG HOT NG CA

CC GEN CYTOKIN
-Cytokin : l cc pr , cc cht to nn bi h thng t bo min dch trong p ng min dch. -Trong trng hp t bo nhim HIV, do hot dng cc gen HIV lm suy yu kh nng sn sinh interferon ca t bo. Liu php gen vi chin lc a mt gen liu php tng cng hot ng gen sn sinh interferon, v ang c nghin cu v th nghim trn chut. -Gen IL-2 c tc dng kch thch s tng sinh ca cc t bo T CD4 ngn cn s ph hy h min dch do HIV gy ra, khng ch ti bn v nhn ln ca HIV. Vi vai tr quan trng ca gen IL-2 di vi h thng min dch chng HIV/AIDS, hin nay mt s th nghim liu php gen theo xu hng tng cng gen m ha IL-2 cho cc t bo nhim HIV, thu c nhiu kt qu bc u.

C. CHIN LC S DNG CC GEN T T CED) V GEN


C T (TOXIC GEN)

-Khi HIV mi xm nhim t bo, bng liu php a cc gen t t (Ced) thymidin kinase (TK) phi hp s dng thuc chng virus ganciclovir (GCV), to thnh phc hp ganciclovir-phosphat c ch hot ng ca enzym DNA polymerase qu trnh ti bn gen ca t bo b ngng ,s phn chia t bo b gin on t bo co li v cht.

3. VACCIN DNA TRONG IU TR IIIV/AIDS


-Vaccin DNA c xem l mt chin lc hiu qu ca liu php gen phng chng HIV/AIDS. -Vaccin DNA khc vi cc loi vaccin khc ch vaccin DNA l phc hp cc gen (DNA), khi a vo t bo cc gen c ti bn, phin m v dch m trong t bo to nn cc loi protein liu php tng ng. Protein liu php c (c dng ngn cn s xm nhp ca HIV, hoc c ch s pht trin v nhn ln ca HIV trong t bo. Cc loi vaccin khc (vaccin vi khun, virus gim c t; vaccin mnh peptid, vaccin ti t hp...) u l cc protein khng nguyn, khi a protein khng nguyn vo c th gy p ng min dch t bo, kch thch cc loi t bo tng ng vi khng nguyn sn sinh cc khng th c hiu.

S VAI TR CA VACXIN DNA TRONG LIU PHP GEN PHNG CHNG HIV/AIDS

-Hin nay vaccin DNA HIV-1 v ang c th nghim phng chng HIV/AIDS trn chui c hiu qu cao. Bn cht ca vaccin DNA HIV-1 l mt vect liu php gen, c ci tin t b gen HIV-I. Mt s loi vaccin DNA, c th nghim phng chng HIV/AIDS c to thnh t cc plasmid ti t hp, gm plasmid pTXGE v cc gen c hiu cho h thng min dch ngi nh gen IL-2, gen IFN-y v gen GM- CSF. Plasmid ti l hp pTXGE c to thnh t plasmid pTX (4,5 -5 kb) ci thm b gen H1V-1 bin i ( xo b mt s vng gen ca HIV nh gen pol, cc gen vpu, vpr, tat b xa mt vi vng nh).

-Th nghim th nghim vaccin DNA phng chng HIV/AIDS trn chut thnh cng, m ra mt trin vng mi trong iu tr HIV/AIDS cho ngi bng vaccin DNA.

CM N S QUAN TM THEO
DI CA THY V CC BN

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