TAP CHl NGHIEN CU'U Y HQC
T A C H DONG, BI^U HIEN GEN M A HOA PROTEIN P24 CUA HIV-1
P H A N TYP CRF01_AE VA U'NG DUNG PROTEIN TAI T 6 HOT
P H A T HIEN K H A N G T H ^ K H A N G HIV TRONG HUY^T THANH BENH N H A N
m
Bach Thj Nhu' Quynh, Nguyin Thj Hoa, Ld Phu'ang Hdng, Nguyin Thj Phu-ang Trln Minh Dii'c, Nguyin Thanh Timg, Ddng Vdn Quyln, Dinh Duy Khang
Vidn Khoa hpc vd Cdng nghd Vidt Nam Khang nguyen p24 la mpt tmng sd ede khdng nguydn quan trpng Cmg dung tmng chin dodn HIV. Khdng nguyen nay cd thi dwac sw dpng di chi tpo kit chin dodn HIV trdn ea sd phdt hidn khdng thi khdng protein p24 tmng huyit thanh bpnh nhdn hodc sd dpng khdng nguydn p24 gdy miin dich cho ddng vdt thi nghipm thu nhpn khdng thi khdng p24 dung tmng chi tpo kit phdt hidn pmtein p24 di chin sdm HIV. Nghidn cuv dwac tiin hdnh nham: tdch ddng vd biiu hidn gen p24 tdphdn typ HIV-1 luv hdnh chu yiu d Vidt Nam thu nhdn pmtein p24 tdi td hap phuc vu chi tpo kit chin dodn. Kit qud nghidn cwu cho thiy gen p24 eua HIV-1 phdn typ CRF01_AE dwac biiu hidn thanh cdng tmng Escherichia coli chung BL21DE3 star Protein p24 tdi
td hap phdn ung ddc hipu vdi khdng thi khdng HIV tmng huyit thanh bdnh nhdn. Protein tai td hap biiu hidn tw gen p24 cua HIV-1 phdn typ CRF01_AE phdn ung ddc hidu vdi khdng thi tmng huyit thanh bpnh nhdn nhiem HIV-1 phdn typ CRF01_AE.
Tir khoa: Gen p24, HIV-1 phan typ CRF01_AE, protein p24 tai td hgp
I. OAT
V A NDt
Dai djch HIV (human immunodeficiency virus) ngay mpt lan rpng tren todn c l u vdi tdc dp 13.000 ca nhiem mdi mdi ngdy, dilu do cQng cd nghTa la cfr mdi phut cd 9,3 trudng hgp nhilm mdi vd 5.9 trudng hgp chit vi AIDS hodc cfr 6-7 gidy cd mft trudng hgp nhilm mdi vd cfr 10 gidy ed mpt ngudi chit vl AISD trdn t h i gidi [1]. Vift Nam la nude ed ty If nhiem HIV cao thfr 6 d Chdu A. Dk giam thilu sg lay lan ciia HlV/AlDS, vifc phdt hifn nhanh, sdm vd chfnh xdc ngudn bfnh d l cd bifn phap dg phdng va dilu tri Id vifc Idm hit sfre ein thilt. V l ly thuylt ngudi ta ed t h i phat hifn t i t ea cdc khang nguyen cua virus, nhung tren thgc te, do tfnh dac hifu va di Igc eua ede khdng t h i chu y l u hudng tdi cdc protein chfnh trong vd nhdn nhu p24, vl vdy khdng nguyen p24 ludn duge chpn d l thgc hifn chin dodn [3]. P24 tham gia vdo qua trinh "cdi do" sau khi virus xdm nhdp vao t l bdo ddng thdi cQng tham gia vdo qud trinh
sao chep nguge cua virus. Kiem tra khang the dac hieu cua HIV dugc coi Id dau chuan cua qua trinh lay nhilm [6]. Ddi vdi HlV-1, phan type luu hanh chu ylu tai Viet Nam Id phdn type CRF01_AE. Kit chan dodn tren ca sd cac protein tai td hgp bieu hien tfr phdn typ luu hanh d Vift Nam cd t h i se cho kit qua dac hifu va chinh xdc han. Muc tieu de tdi: Tdch ddng vd bilu hifn gen p24 thupc phdn typ CRF01_AE, thu nhdn protein p24 tdi td hgp phgc vg c h l tao kit chin dodn HIV d Viet Nam.
II. VAT LIEU VA PHUONG
PHAP 1. Vat lifuMiu mdu cua bfnh nhdn nhiem HIV dugc cung c l p bdi Vifn vf sinh phdng djch qudn dpi, vifn V f sinh djch te trung uang, bfnh vifn da khoa Quang Ninh vd Trung tdm cai nghifn Hal Phdng.
2. Phu'ang phdp
Mau todn phin cua bfnh nhan nhilm HIV dugc filn hdnh tdch chiit DNA tdng sd. DNA
TAP CHl NGHllN CO'U Y H Q C / -
tdng sd dugc sfr dgng ldm khudn d l khulch dgi gen p24 bdng phuang phdp PCR Idng.
Xdc djnh trinh tg gen dugc filn hdnh theo phuang phdp cua Sanger trdn mdy phdn tieh trinh tg ABl PR1SM®3100 Genetic Analyzer vd phin m i m xfr ly dfr lifu PC/GENE. Gen p24 duge g i n vdo hf vecta bilu hifn pET32a(+) thdng qua hai vj tri cua enzym gidi hgn BamHl vd Xhol d l bilu hifn ra protein tdi td hgp dgng lidn kit Trx-p24-6Hls. Vecta tdi td hgp dugc biln ngp vd bilu hifn trong t l bdo
£ CO//Chung BL21-DE3 star. Protein p24 tdi td hgp dugc finh c h l bing eft ProBond Nickel- Chelafing Resin sau dd kilm tra bing phin frng western blot vdi khdng t h i 1 Id huylt thanh bfnh nhdn duang tinh HIV dugc pha lodng 500 lln vd khdng t h i 2 Id cfng hgp khdng IgG ngudi g i n HRP
III. K^T QUA
1. Tach ddng va xdc djnh trinh t g gen ma hod protein p24
Dogn gen md hda protein p24 dd dugc nhan len mft cdch ddc hifu, kfch thude eiia san phim PCR dung theo dg dodn 11 thuylt Id khoang 600bp.
Sau khi khulch dgi, gen p24 d[jegc tdch ddng vdo vector pCR2.1 vd xdc djnh trinh tg.
Gen p24 cua HIV phdn typ CRF01_AE cd chilu ddi 588 bp dd disgc ddng ky trong ngdn hdng gen vdi sd ddng ky FM162180.
4-588
Dudng chgy 1: Thang DNA chuln.
Dudng chgy 2, 3: Sdn phim PCR vdng 2 2. Bilu hifn vd tinh sgch protein p24 Hf vector bilu hifn mang gen p24 sau khi thilt k l thdnh cdng duge dft tdn Id pQH09-p24.
Plasmid tdi td hgp pQH09-p24 dugc biln ngp vdo trong t l bdo £ coli BL21 DE3 Star, elm frng bing 1 mM IPTG, lie 200 vdng/phut d 3r'C trong 3 gid. K i t q u i bilu hifn duge t h i hifn d Inh difn di trdn gel polyacrylamid 12,5%.
M 116 66,2 45,0 35,0 25,0 18,0
p24
' • W K
Hinh 1. Kit qua difn di san phim PCR tren gel agarose 1 %
Hinh 2. Kit qua bilu hifn Protein tai td hgp M: thang protein chuln
1: M i u khdng cam frng vdi IPTG.
2: M i u e l m frng vdi IPTG
Bdng protein d f m xult hifn d vj tri khoang 40 kDa dung vdi dg todn theo ly thuylt, chlnh Id protein tdi td hgp p24. Protein tdi td hgp dvegc tinh c h l bing eft sdc ky di Igc Ni^*- resin. Kit qud finh c h l duge ndu trong hlnh 3.
3. Phdt hifn khdng t h i trong huylt thanh bfnh nhdn
Protein tdi td hgp sau khi finh c h l dugc frng dgng d l phdt hifn khdng t h i trong hgylt thanh bfnh nhdn. Ky thudt Western blot dd duge thgc hifn d l ddnh gid khd ndng phan frng eiia p24 tdi td hgp vdi khdng t h i trong 30 m i u huylt thanh .eiia bfnh nhdn nhilm HIV thufc hai phdn typ B vd CRF01_AE.^udng chgy IA, 13B: Thang protein chuin; Dudng chgy 2A, 3A, 4A, 6A, 8A, 4B, 6B, 8B, 9B, 11B, 12B: Phan frng gifra khdng nguydn p24
8
1
TAP CHl NGHIEN CLPU Y HQC vdi huylt thanh bfnh nhdn nhilm HIV thufc
phdn typ CRF01_AE; Dudng chgy: 7A. 9A, 5B, 7B: Phan frng gifra khdng nguydn p24 vdi huylt thanh bfnh nhdn nhilm HIV thufc phdn type B; Dudng chgy 5A: Phan frng gifra khdng nguydn Thioredoxin vdi vdi huylt thanh bfnh
kDa 66,2 45,0 35,0 25,0 18,0
nhdn nhilm HIV thufc phdn typ AE; Dudng chgy 10B: Phin frng gifra khdng nguydn Thioredoxin vdi vdi huylt thanh bfnh nhdn nhilm HIV thufc phdn typ B; Dudng chgy 10A, IB, 2B, 3B: Phin frng gifra khdng nguydn p24 vdi huylt thanh dm finh (xem hlnh 4).
M 1 2
.p24
Hinh 3. Tinh c h l protein tdi td hgp p24 bdng cdt Ni-Resin
Dudng chgy M: Chi thj phdn tfr protein; Dudng chgy 1-3: san pham finh sgch d cdc phdn doan khae nhau.
kDa 72- 55- 43- 34- 26- 17-
1 2 3
m
4 5 6
A
7 8 9 10 1 2 3 4
'
5 6 7 8
B
9 10 11 12 13
Hinh 4. K i t qua kilm tra phan u'ng ciia protein p24 tai td hgp vd-i khang t h i trong huylt thanh bfnh nhan bdng ky thuat Western blot
IV. BANLUAN
Wirachsllp P vd cfng sg [9], dd nghidn cfru xdc djnh phdn typ HlV-1 vdi khoing 10.000 miu HIV duang tfnh d Thdi Lan. Kit qua cho thiy, phdn typ CRF01_AE chilm da sd vdi 97,3 %. Phan typ B ehi chilm 2,7%. Khdng fim thiy cac phdn typ khde. Vfy sg luu hdnh eua ede phdn typ HlV-1 d Vift Nam ra sao? Trong sd 240 m i u mdu duang finh vdi HIV thu nhfn
tfr 13 tinh thdnh trong c l nudc ehung tdi dd xdc djnh dugc phdn typ HlV-1 luu hdnh chu y l u d Vift Nam cQng Id phdn typ CRF01_AE vdi 96,7% vd chi cd 3,3% Id phdn typ B. Chung tdi cQng khdng fim thiy cdc phdn typ khde [8].
Nhu vdy kit qua ban d i u cho thiy sg luu hdnh cua phdn typ HIV-1 d Vift Nam g i n gidng vdi Thdi Lan. Cdu hdi duge ddt ra Id cdc khdng nguydn tdi td hgp bilu hifn tfr cdc gen ma hda cho cde khdng nguyen quan trong eua
TAP CHl NGHllN CU'U Y HQC
HIV-1 nhu p24, gp41, gp120 eua phdn typ CRF01_AE lifu cd phan frng tdt han vdi khdng t h i trong huylt thanh bfnh nhdn nhilm cung phdn typ hay khdng? Dk trd Idi eho cdu hdi ndy, chQng tdi dd nghidn cfru tdch ddng, xdc djnh trinh tg vd bilu hifn cdc gen p24, gp41, gp120 eua phdn typ CRF01_AE luu hdnh d Vift Nam. Bdi bdo ndy cdng bd kit q u i nghidn cfru tdch ddng, xdc djnh trinh tg, bilu hifn gen p24 vd ddnh gid kha ndng phin frng vdi khdng t h i trong huylt thanh bfnh nhdn nhilm phdn typ HIV-1 CRF01_AE vd phdn typ HIV-1 B. Kit q u i xdc djnh trinh tg cho thiy df tuang ddng gifra trinh tg gen p24 phdn typ CRF01_AE vdi trinh tg gen p24 cua ede Chung HIV-1 phdn typ CRF01_AE luu hdnh d Trung Qudc vd Thdi Lan, dd cdng bd trong ngdn hdng gen qudc t l dat 98-99. So sdnh trinh tg gen p24 phdn typ CRF01_AE vdi trinh tu gen p24 cua phdn typ B do chung tdi tdch ddng vd xdc djnh trinh tg trudc ddy [7], dp tuang ddng ve nucleofid gifra hai phdn typ nay chi dat 89% vd df tuang ddng v l trinh tg amino acid chT dgt 91%. Nhu vfy lifu khdng t h i khdng protein p24 eua phdn typ ndy cd phan frng tdt vdi protein p24 eua phdn typ khde hay khdng ? Khdng nguydn p24 tdi td hgp bilu hifn tfr gen p24 cua phdn typ CRF01_AE dd dugc finh c h l vd cho phan frng vdi khdng t h i trong huylt thanh bfnh nhdn nhilm HlV-1 phdn typ CRF01_AE vd B. Kit qua ban d i u cho thiy khdng nguydn p24 tdi td hgp phan frng ddc hifu vd mgnh vdi khdng t h i trong huylt thanh bfnh nhdn nhilm HlV-1 phdn typ CRF01_AE, tuy nhidn phdn frng y l u hofc khdng phan frng vdi khdng t h i trong huylt thanh bfnh nhdn nhiem HlV-1 phdn typ B. Nhu vfy, vife su dung khdng nguydn tdi td hgp bilu hifn tfr gen cua phdn typ luu hdnh cd khd ndng phin frng tdt han vdi khdng t h i do phdn typ ndy tgo ra trong huylt thanh bfnh nhdn. Dilu dd ed nghTa ring kit chin dodn vdi cdc protein tdi td hgp ndy ed kha ndng cho d f
nhgy vd d f dfc hifu eao han khi sfr dgng protein tdi td hgp bilu hifn tfr phdn typ khde.
V. K^T LUAN
Dd bilu hifn thdnh cdng gen md hPa protein p24 cua virus HIV-1 phdn typ CRF01_AE luu hdnh d Vift Nam. Kit q u i ban d i u eho thiy khdng nguydn p24 tdi td hgp phin frng dfc hifu vd manh vdi khdng th^
trong huylt thanh bfnh nhdn nhilm HIV-1 phdn typ CRF01_AE, tuy nhidn phan frng ylu hofc khdng phan frng vdi khdng t h i trong huylt thanh bfnh nhdn nhiem HIV-1 phdn typ B. Cdc khdng nguydn quan trpng khde nhu- gp120 vd gp41 cua HlV-1 phdn typ CRF01_AE cQng dd dugc ehung tdi nghidn cfru bilu hifn thdnh cdng vd phan frng tdt vdi khdng t h i trong huylt thanh bfnh nhdn nhilm HIV-1 phdn typ CRF01_AE.
TAI LIEU THAM KHAO
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html/pub/publieations/ire-pub06/je943- epiupdate2009_en_pdf.htm
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27: 288-295.
3. CDC (1989). (Centers for Disease Control and Prevention). Interpretation and Use of the Western-Blot Assay for Serodiagnosis of Human Immunodeficiency Virus Type 1 Infection, MMWR; 38: 1-7. http://
amedeo.eom/litphp?ld= 2501638
4. Ghosh, P K. and Sharma, N. C (2000).
HIV antibody deteefion methods: current approaches, J . Sci. Ind. Res., 59, 919-931.
5. Nguyin Hodi Giang, Pham Hdng Thing vd cs (2005). Phdn tieh trinh tg gen gag, gdp phin djnh logi HIV eiia cde bfnh nhdn mft sd tfnh phfa Ble Vift Nam, TC Y hpc 10
TAP CHl NGHllN CO'U Y ^QC dg phdng, 5: 96-100.
6. Hoffman S, Exner TM, Leu CS, Ehrhardt AA, Stein Z (2003) Female- condom use in a gender-specific family planning cllnie trial. Am J Public Health. 93 (11): 1897-1903.
7. Ddng Van Quyln, Hoang Anh, Bgch Thj Nhtf Quynh va cs (2008). Tdch ddng, bilu hifn, finh sgch gene p24/B tfr ehung HIV dang luu hdnh d Vift Nam vd nghidn cfru phan frng eua protein tdi td hgp vdi khdng t h i khdng HIV trong huylt thanh bfnh nhdn Tap
chl Cdng nghf Smh hpc, 6 (1): 27-34.
8. Bgch Thj Nhu' Quynh, Le Phu'ang Hdng, Hodng Thj Thanh Huyin vd cs (2009). Xdc djnh phdn typ HIV luu hdnh tgi mft sd dja phuang d Vift Nam, Tap chl Y hpc Vift Nam, 360 (2): 41-48.
9. Wirachsllp P, Kantakamalakul W, Foongladda S, Chuenchitra T et al (2007).
Surveillance of subtype and genetic variafion of the eireulafing strains of HlV-1 in Thailand, Southeast Asian J Trop Med Public Health, 38 (5):814-27
Summary
CLONING AND EXPRESSION OF THE GENE CODING FOR P24 PROTEIN FROM HIV-1 SUBTYPE CRF01_AE AND USING
RECOMBINANT PROTEIN FOR DETECTION OF HIV ANTIBODIES IN THE PATIENT'S SERA
P24 antigen is one of the important antigens to apply for HIV diagnosis. We can use this anfigen for HIV kit development based on the p24 anfibody in the patient's serum deteefion or using p24 protein for immunization to produce antibody against 24 to develop the kit for eariy diagnosis of HIV infecfion. Cloning and expression of the gene coding for p24 protein from the HlV-1 subtype CRF01_AE circulating prevalently in Vietnam to obtain recombinant protein for diagnostic kit development The results show that the p24 gene of HlV-1 subtype CRF01_AE was expressed successfully in Escherichia coli BL21DE3 star with pET32a(+) expression vector.
Recombinant p24 protein reacts specifically with anfibodies in the sera of HIV infected pafients. In conclusion, recombinant protein expressed from the p24 gene of HlV-1 subtype CRF01_AE reacts specifically with anfibodies in the sera ofthe pafients infected HIV-1 subtype CRF01_AE.
Key words: p24 gene, p24 antigen, p24 protein