HIV跨膜蛋白gp41和gp36原核表达及抗原性研究  

Expression of HIV trans-membrane protein gp41 and pronucleus gp36 and their antigenicity

作　　者：袁作为[1] 帅光泽[2] 张君[1] 胡接力[1] 郑建[1] 

机构地区：[1]蓖庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016 [2]成都大学校医院内科,成都610106

出　　处：《第三军医大学学报》2010年第21期2281-2285,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(30872419)~~

摘　　要：目的构建HIV跨膜蛋白gp41和gp36原核表达载体,诱导表达HIV抗原,为HIV快速诊断和蛋白疫苗研究提供材料基础。方法用PCR方法对HIV-1包膜蛋白gp41和HIV-2包膜蛋白gp36基因序列进行全长、截短扩增,分别或以融合表达方式克隆入原核表达载体pQE30、pET32a（＋）及pGST-MOLUC,重组质粒经异丙基硫代半乳糖苷（isopro-pyl-β-D-thiogalactopyranoside,IPTG）诱导表达,并经Ni-NAT或谷胱甘肽-Sepharose-4B层析柱亲和纯化HIV跨膜糖蛋白抗原,对获得的纯化抗原进行Western blot检测,利用HIV抗原制备ELISA检测试剂盒,并分析HIV临床样本。结果成功构建了HIV包膜蛋白的原核表达载体,利用亲和层析纯化得到了纯度较高的HIV包膜蛋白;Western blot分析结果证实表达纯化的HIV跨膜蛋白gp41（aa.538~718）、gp36（aa.533~764）以及gp41-gp36均能和HIV阳性血清反应;ELISA试剂盒检测临床样本显示,特异性达95%以上,灵敏度达100%。结论获得了有生物学活性的HIV截短包膜蛋白,并可用于HIV快速检测试剂盒的研发。Objective To construct the recombinant plasmids with HIV-1 gp41 and HIV-2 gp36 genes and induce the expression of HIV antigens for the rapid diagnosis of HIV and development of protein vaccine.Methods Full-length genes and/or fragment genes encoding HIV-1 trans-membrane protein gp41 and HIV-2 trans-membrane protein gp36 were amplified by PCR.Target genes were inserted into the expression vectors pQE30,pET32a（＋）and pGST-MOLUC,respectively.Recombinant vectors were transformed into E.coli BL21 and induced by IPTG.Expressed fusion proteins were purified by Ni2＋-affinity chromatography or GST-affinity chromatography.Antigenicity of purified proteins was identified by Western blotting and ELISA were prepared using the purified proteins.HIV samples were analyzed.Results The pronucleus vector of HIV trans-membrane protein was successfully constructed.HIV trans-membrane protein with a rather high purity was obtained by Ni2＋-affinity chromatography or GST-affinity chromatography.Western blotting showed that the expressed products including gp41（aa.538-718）,gp36（aa.533-764）and gp41-gp36 reacted to positive HIV in serum.HIV-1＋ or HIV-were detected with ELISA in HIV samples with a sensitivity of 100% and a specificity of over 95%.Conclusion The biological activity of HIV truncation cross membrane proteins can be used in research and development of HIV rapid diagnosis kits.

关 键 词：HIV-1包膜蛋白gp41 HIV-2包膜蛋白gp36 重组 抗原性 

分 类 号：R373.9[医药卫生—病原生物学] R392-33[医药卫生—基础医学]