中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2008年
4期
231-234
,共4页
张云智%靳虹%卢洪洲%程训佳%潘孝彰
張雲智%靳虹%盧洪洲%程訓佳%潘孝彰
장운지%근홍%로홍주%정훈가%반효창
HIV-1%基因产物,Vif%突变%多克隆抗体
HIV-1%基因產物,Vif%突變%多剋隆抗體
HIV-1%기인산물,Vif%돌변%다극륭항체
HIV-1%Gene products,Vif%Mutation%Polyclonal antibody
目的 分析上海HIV-1分离株病毒感染因子(Vif)蛋白编码基因突变特点,构建HIV-1vif基因原核表达质粒,并了解其免疫原性.方法 采用RT-PCR法扩增23例上海HIV-1分离株vif基因并测序,与HIV-1国际标准毒株比较;将vif编码基因克隆入原核表达载体pET32b(+),构建pET32b(+)-HIV-1/Vif重组质粒;将该质粒转化入细胞BL21D3(Star)表达,并纯化HIV-1 Vif蛋白,制备Vif蛋白鼠多克隆抗体,并以ELISA检测Vif蛋白及其鼠多克隆抗体的免疫原性.统计学处理采用t检验.结果 上海HIV-1分离株vif基因与国际标准毒株相比较,核苷酸突变率为(0.179±0.006)%,并具有多处相似的变异位点,上海HIV-1分离株Vif蛋白第151～240位氨基酸相对保守;成功构建HIV-1 vif基因原核表达质粒pET32b(+)-HIV-1/Vif,表达并纯化Vif蛋白,制备了Vif多克隆抗体;重组HIV-1 Vif蛋白与HIV感染者及健康人血清反应比较,差异无统计学意义(P＞0.05);鼠多克隆抗体与重组Vif蛋白反应与健康对照组血清比较差异有统计学意义(t=178.61,P<0.01).结论 上海HIV-1分离株vif基因与HIV-1国际标准毒株比较存在较高突变,成功地构建了HIV-1 vif基因原核表达载体pET32b(+)-HIV-1/Vif,制备了Vif多克隆抗体.
目的 分析上海HIV-1分離株病毒感染因子(Vif)蛋白編碼基因突變特點,構建HIV-1vif基因原覈錶達質粒,併瞭解其免疫原性.方法 採用RT-PCR法擴增23例上海HIV-1分離株vif基因併測序,與HIV-1國際標準毒株比較;將vif編碼基因剋隆入原覈錶達載體pET32b(+),構建pET32b(+)-HIV-1/Vif重組質粒;將該質粒轉化入細胞BL21D3(Star)錶達,併純化HIV-1 Vif蛋白,製備Vif蛋白鼠多剋隆抗體,併以ELISA檢測Vif蛋白及其鼠多剋隆抗體的免疫原性.統計學處理採用t檢驗.結果 上海HIV-1分離株vif基因與國際標準毒株相比較,覈苷痠突變率為(0.179±0.006)%,併具有多處相似的變異位點,上海HIV-1分離株Vif蛋白第151～240位氨基痠相對保守;成功構建HIV-1 vif基因原覈錶達質粒pET32b(+)-HIV-1/Vif,錶達併純化Vif蛋白,製備瞭Vif多剋隆抗體;重組HIV-1 Vif蛋白與HIV感染者及健康人血清反應比較,差異無統計學意義(P＞0.05);鼠多剋隆抗體與重組Vif蛋白反應與健康對照組血清比較差異有統計學意義(t=178.61,P<0.01).結論 上海HIV-1分離株vif基因與HIV-1國際標準毒株比較存在較高突變,成功地構建瞭HIV-1 vif基因原覈錶達載體pET32b(+)-HIV-1/Vif,製備瞭Vif多剋隆抗體.
목적 분석상해HIV-1분리주병독감염인자(Vif)단백편마기인돌변특점,구건HIV-1vif기인원핵표체질립,병료해기면역원성.방법 채용RT-PCR법확증23례상해HIV-1분리주vif기인병측서,여HIV-1국제표준독주비교;장vif편마기인극륭입원핵표체재체pET32b(+),구건pET32b(+)-HIV-1/Vif중조질립;장해질립전화입세포BL21D3(Star)표체,병순화HIV-1 Vif단백,제비Vif단백서다극륭항체,병이ELISA검측Vif단백급기서다극륭항체적면역원성.통계학처리채용t검험.결과 상해HIV-1분리주vif기인여국제표준독주상비교,핵감산돌변솔위(0.179±0.006)%,병구유다처상사적변이위점,상해HIV-1분리주Vif단백제151～240위안기산상대보수;성공구건HIV-1 vif기인원핵표체질립pET32b(+)-HIV-1/Vif,표체병순화Vif단백,제비료Vif다극륭항체;중조HIV-1 Vif단백여HIV감염자급건강인혈청반응비교,차이무통계학의의(P＞0.05);서다극륭항체여중조Vif단백반응여건강대조조혈청비교차이유통계학의의(t=178.61,P<0.01).결론 상해HIV-1분리주vif기인여HIV-1국제표준독주비교존재교고돌변,성공지구건료HIV-1 vif기인원핵표체재체pET32b(+)-HIV-1/Vif,제비료Vif다극륭항체.
Objectives To analyze the characteristic of HIV-1 viral infectivity factor (Vif) gene variants isolated from Shanghai. To construct the prokaryotic expression vector of HIV-1 vif gene and understand its immunogenieity. Methods HIV-1 vii genes were amplified and sequenced from 23 serum samples of HIV-1 infected patients in Shanghai and then compared with the international standard HIV-1 strain. Subsequently, these amplified Vif fragments were sub cloned into pETS2b(+)expression vector. The recombinant prokaryotie plasmids pETS2b (+)-HIV-1/Vif were then transferred into BL21DS(Star)cells for expressing and purifying HIV-1 Vif protein. HIV-1 Viff rat polyclonal antibody was then preparing by injecting the purified Vif proteins into the mice. ELISA was used to determine the purity of Vif proteins and the immunogenicity of its polyclonal antibodies. Results The nucleotide acid mutation rate of HIV-1 vif gene in Shanghai AIDS patient was (0. 179±0. 006)% compared with the international standard HIV-1 strain. Some similar mutations in vif gene were found in HIV-1 strains isolated from Shanghai while the amino acid sequence between 151 and 240 in Vif protein was conserved. The construction of HIV-1 Vif prokaryotic expression plamids and the preparation of Vif polyclonal antibodies were successfully done in this study. The reaction between recombinant HIV-1 Vif protein and the serum from HIV-1 infected patient was not significantly different from that between the recombinant protein and healthy control serum(P＞0.05). HIV-1 Vif polyclonal antibody reacted differently with recombined HIV-1 Vif protein compared with healthy control serum samples (t=178.61, P＜0.01). Conclusions The Vif gene mutation rate is high in HIV-1 strains isolated from Shanghai compared with international standard HIV-1 strain. The prokaryotic expression plasmids of HIV-1 vif antigen are successfully constructed and Vif polyclonal antibodies are prepared well.
