HPV 16L1基因的原核表达及表达条件的优化

Prokaryotic expression of HPV 16L1 and optimization of expression conditions

目的:构建HPV16L1基因的原核表达质粒,并优化其表达条件。方法:根据GeneBank中的HPV序列及pGEX-KG中的多克隆位点设计引物,以含有HPV全长基因片段重组质粒为模板,经PCR扩增出1 500 bp的DNA片段。将所得片段与pGEX-KG载体连接,转化JM109大肠杆菌,筛选阳性克隆。其扩增片段测序结果与原序列一致,表明原核表达载体pGEX-KG-HPV16L1已构建成功。提取pGEX-KG-HPV16L1质粒转化到BL21(DE3)表达菌株中,经IPTG诱导后收集菌体,进行SDS-PAGE,Western Blot鉴定。结果:在大肠杆菌中获得HPV16L1基因融合表达,融合蛋白的相对分子量为83kDa;表达的蛋白能与抗HPV16L1抗体发生特异性反应。结论:HPV16L1基因在大肠杆菌中获得高效表达,为HPV16L1疫苗的研制奠定了基础。

Objective： To construct the HPV16 L1 prokaryotic expression plasmid and to optimize its expression.Methods： A pair of primers was designed according to plasmid sequences of pGEX-KG and the HPV16L1 genes published by GeneBank.The DNA fragment of 1 500 bp was amplified by PCR from the HPV recombinant plasmid with HPV16L1 gene,then cloned into pGEX-KG and transformed into the host E.coli strain JM109.The pGEX-KG-HPV16L1 plasmid was taken and transformed into BL21（DE3） for expression.Induced by IPTG at 37℃,the expression product of HPV16L1 gene was identified by SDS-PAGE and Western blot.Results： HPV16L1 fusion protein was expressed successfully in the form of inclusion bodies.The molecular weight was 83 kD.Meanwhile,the optimum condition of HPV16L1 fusion protein expression was induced with 1.0 mmol·L-1 IPTG for 4 h.The fusion protein reacted specifically with antibodies against HPV16L1.Conclusion： The prokaryotic expression vector of HPV16L1 gene has been constructed and expressed in E.coli successfully.