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目的在原核系统中表达全长HIV-1 p24抗原,对其抗原性进行鉴定,探讨其对HIV早期诊断的价值。方法利用PCR技术从含有HIV-1 gag基因质粒PTM-1中扩增p24蛋白基因,通过酶切消化后连接到表达载体pET28a上,转化大肠埃希菌BL21(DE3),经IPTG诱导,表达p24抗原。通过ELISA和Western blotting(WB)法检测表达产物的免疫反应活性及特异性。结果PCR产物和构建的重组质粒pET28a-p24经双酶切均显示约690 bpDNA片段,与预期p24抗原全基因片段大小一致。SDS-PAGE电泳可见纯化蛋白为1条相对分子量约26×103Mr的外源表达蛋白带,与预期大小一致。ELISA及WB实验证实表达的p24抗原具有较好的活性及特异性。时间分辨荧光免疫双抗体夹心法显示与市售p24抗原线性一致。结论构建了HIV-1 p24/pET28a原核高效表达系统,表达产物与市售的p24抗原具有相似的抗原性。
Objective To express full-length HIV-1 p24 antigen in prokaryotic system and identify its antigenicity to explore its value in the early diagnosis of HIV. Methods The p24 gene was amplified by PCR from PTM-1 containing gag gene of HIV-1, digested by restriction enzyme and ligated into expression vector pET28a. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG to express p24 antigen. The immunoreactivity and specificity of the expressed product were detected by ELISA and Western blotting (WB). Results The PCR product and the constructed recombinant plasmid pET28a-p24 were both double digested and showed about 690 bp DNA fragment, which was consistent with the expected size of p24 antigen gene fragment. SDS-PAGE electrophoresis showed that the purified protein was an exogenous protein with a relative molecular mass of about 26 × 103Mr, consistent with the expected size. ELISA and WB experiments confirmed the expression of p24 antigen has good activity and specificity. Time-resolved fluorescent immuno-antibody sandwich showed linearity with the commercially available p24 antigen. Conclusion The prokaryotic expression system of HIV-1 p24 / pET28a was constructed, and its expression product has similar antigenicity with the commercially available p24 antigen.