HIV-1核心抗原p24基因的表达、纯化及抗原性分析

Expression,purification and analysis of HIV-1 core p24 antigenicity

目的在原核系统中表达全长HIV-1 p24抗原,并对其抗原性进行鉴定。方法利用PCR技术从HIV-1全基因质粒(BH-10)中扩增p24抗原基因,通过酶切消化后连接到表达载体pET22b上,用此连接产物转化大肠埃希菌BL21(DE3),经IPTG诱导,表达p24抗原。运用双酶切技术、SDS-PAGE电泳检测插入基因片段的正确性,并用W estern B lot(WB)及ELISA法对表达产物的抗原性进行检测。结果PCR产物和构建的重组质粒pET22b-p24经双酶切插入的外源基因片段均为690 bp,与预期p24抗原全基因片段大小一致。纯化蛋白SDS-PAGE电泳,可见1条相对分子量约26×103的外源表达蛋白带,与预期大小一致,未见杂蛋白带。WB结果显示重组蛋白与HIV-1阳性血清呈特异性反应,与健康人血清没有反应。ELISA检测p24抗原灵敏度为93.94%(62/66),特异性为93.33%(28/30)。结论构建了HIV-1 p24表达载体pET22b-p24,并在原核细胞中高效表达,其表达产物具有良好的抗原性,为研制HIV抗体确认试剂奠定了良好的基础。

Objective To express, purify and analyze antigenicity of HIV-1 core p24 antigen in prokaryotic system. Methods HIV-1 p24 gene amplified by PCR from plasmid（BH-10） was subcloned into vector pET-22b （ ＋ ） after enzymatic digestion. The recombinant plasmid was then transformed to E. coli host, BL21 （DE3）, and highly expressed after IPTG induction. Double enzyme digestion was used to confirm the correct insert in the recombinant plasmid. SDS-PAGE, Western blot and ELISA were used to analyze purity and antigenicity. Results PCR product and external gene section from the recombinant plasmid pET 22b-p24 showed the same size of 690 bp equal to p24 gene sequences. An external expressed protein band of Mr 26 × 10^3 was obtained after purified protein SDS-PAGE electrophoresis. Western blot showed recombinant protein had specific reaction with HIV-1 positive sera and no response with normal sera. Sensitivity and specificity of ELISA were 93.94% （62/66） and 93.33% （28/30） respectively. Conclusion The recombinant p24 antigen constructed and expressed in E. coli had good antigenicity and potential to develop HIV confirmation reagent.