【摘要】 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。更多还原

【Abstract】 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.更多还原