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本研究在大肠杆菌BL21中融合表达人乳头瘤病毒16型E7蛋白,并初步评价其应用价值。采用PCR技术扩增出HPV16E7基因,将其克隆进原核表达载体pGEX6p-1,转化至大肠杆菌BL21,利用IPTG进行诱导表达。以纯化的融合蛋白作为检测抗原建立间接ELISA方法,用于检测重组李斯特菌(Lm1-2-E7)免疫小鼠后的E7血清抗体水平。在25℃,0.5mM IPTG诱导下,HPV16E7蛋白在大肠杆菌BL21中获得表达,融合蛋白以可溶性形式存在,Western blot结果显示其与HPV16E7单克隆抗体发生特异性反应。二次免疫后小鼠血清经间接ELISA结果表明E7特异性抗体滴度为1∶200。结果表明GST-E7融合蛋白具有较强的免疫活性。
In this study, human papillomavirus type 16 E7 protein was fused and expressed in E.coli BL21, and its application value was preliminary evaluated. HPV16E7 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX6p-1. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. The purified fusion protein was used as the detection antigen to establish an indirect ELISA method for detecting E7 serum antibody levels after immunization with recombinant Listeria monocytogenes (Lm1-2-E7). The HPV16E7 protein was expressed in E. coli BL21 under the condition of 25 ℃ and 0.5mM IPTG. The fusion protein existed in soluble form. Western blot results showed that HPV16E7 protein reacted specifically with HPV16E7 monoclonal antibody. The indirect ELISA results of mouse sera after secondary immunization showed that the E7 specific antibody titer was 1: 200. The results showed that GST-E7 fusion protein has strong immunogenicity.