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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 prokaryotic expression plasmid of HPV16 L1 gene and optimize its expression conditions. Methods: According to the HPV sequences in GeneBank and the multiple cloning sites in pGEX-KG, primers were designed and a 1500 bp DNA fragment was amplified by PCR using the recombinant plasmid containing the full-length HPV gene fragment as a template. The resulting fragment was ligated with pGEX-KG vector and transformed into E. coli JM109 to screen positive clones. The amplified fragment was sequenced consistent with the original sequence, prokaryotic expression vector pGEX-KG-HPV16L1 has been constructed successfully. The pGEX-KG-HPV16L1 plasmid was transformed into BL21 (DE3) expression strain. After induced by IPTG, the cells were collected and identified by SDS-PAGE and Western Blot. Results: The fusion protein of HPV16 L1 was expressed in E. coli. The relative molecular weight of fusion protein was 83 kDa. The expressed protein could specifically react with anti-HPV16 L1 antibody. Conclusion: HPV16L1 gene is highly expressed in E. coli, which lays the foundation for the development of HPV16L1 vaccine.