摘要目的 在真核系统中表达全长人类免疫缺陷病毒1(human immunodeficiency virus 1,HIV-1)p24抗原,鉴定其抗原性,探讨其在HIV早期诊断中的价值.方法 利用PCR技术分别扩增p24基因和DRVI信号肽基因,通过融合PCR在p24基因前添加来自gp140质粒的DRVI信号肽序列,并分别将其克隆至pDRVI1.0载体,获得两种重组质粒pDRVI-p24和pDRVI-p24s.将重组质粒转染293F细胞,通过SDS-PAGE检测p24蛋白的表达与分泌,利用Ni-NTA金属螯合层析法和分子筛纯化p24s蛋白,并利用免疫印迹法检测该蛋白质的抗原特异性.通过间接ELISA法进行HIV-1阳性小鼠和HIV-1阳性感染者血清的检测.结果 成功构建了高效表达HIV-1 p24蛋白的真核表达系统,细胞上清中可检测到明显的p24s蛋白分泌.经HIV-1阳性和阴性小鼠,以及经临床验证的30份HIV-1阳性感染者和50份HIV阴性血清标本鉴定,重组蛋白具有较高的检出特异性和敏感性.结论 成功构建了HIV-1 p24抗原真核表达载体,表达纯化的p24s抗原具有较好的抗原性,初步建立了检测HIV-1的间接ELISA法,并证明该检测法具有较高的特异性和灵敏度,具有良好的应用前景.

abstractsObjective To express HIV-1 capsid p24 antigen in an eukaryotic expression system and to evaluate its antigenicity and potential in the early diagnosis of HIV. Methods The full-length gene of HIV-1 p24 and the signal peptide DRVI gene were amplified by PCR. The signal peptide DRVI preceding the p24 gene was introduced using fusion PCR, and cloned into vector pDRVI1. 0. Two recombinant plas-mids pDRVI-p24 and pDRVI-p24s were constructed and transfected into 293F cells. Expression and secre-tion of p24 protein were detected by SDS-PAGE, Ni-NTA column chromatography and molecular sieve were used to purify p24s protein. The purified protein was identified by Western blot and indirect ELISA using hu-man/mouse HIV-1-positive serum samples. Results The eukaryotic expression system for HIV-1 p24 anti-gen was successfully established with high efficiency. The target protein of interest with the signal peptide DRVI was obviously detected in the supernatants of cell culture. The recombinant protein had good specifici-ty and sensitivity based on the results of serological tests using serum samples of five HIV-1-positive and five HIV-negative mice , 30 HIV-1-positive patients and 50 HIV-1-negative healthy individuals . Conclusions The eukaryotic expression system for HIV-1 p24 antigen was successfully established. The purified HIV-1 p24s antigen had good antigenicity. An indirect ELISA assay with good specificity and sensitivity for the de-tection of HIV-1 was preliminarily constructed and showed great potential for application.