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目的构建人乳头瘤病毒l6型(HPV16)E6-E7融合蛋白真核表达载体,为研究其基因疫苗免疫活性奠定实验基础。方法 PCR扩增HPV16 E6-E7基因片段,将其连接到真核表达载体pcDNA3.1(+),构建真核表达载体pcDNA3.1(+)/HPV16 E6-E7,双酶切及测序鉴定。将质粒转染HeLa细胞,RT-PCR鉴定E6-E7基因在HeLa细胞中的表达。提取质粒免疫小鼠,利用免疫组化方法检测在其肌肉组织中的表达。结果成功构建了真核表达载体pcDNA3.1(+)/HPV16 E6-E7;在转染pcDNA3.1(+)/HPV16 E6-E7的细胞中检测到HPV16 E6-E7基因。在免疫该质粒的小鼠肌肉组织中可以检测到该质粒的蛋白表达。结论成功的构建的了真核表达载体pcDNA3.1(+)/HPV16 E6-E7,该载体能在HeLa细胞内以及小鼠骨骼肌细胞内有效表达。
Objective To construct eukaryotic expression vector of human papillomavirus type 16 (HPV16) E6-E7 fusion protein and lay the experimental foundation for the study of its immunogenicity of gene vaccine. Methods The HPV16 E6-E7 gene fragment was amplified by PCR and ligated into the eukaryotic expression vector pcDNA3.1 (+). The eukaryotic expression vector pcDNA3.1 (+) / HPV16 E6-E7 was constructed and double digested and sequenced. The plasmid was transfected into HeLa cells, and the expression of E6-E7 gene in HeLa cells was identified by RT-PCR. The plasmid was extracted to immunize mice, and the expression in muscle was detected by immunohistochemistry. Results The eukaryotic expression vector pcDNA3.1 (+) / HPV16 E6-E7 was successfully constructed. The HPV16 E6-E7 gene was detected in the cells transfected with pcDNA3.1 (+) / HPV16 E6-E7. The protein expression of this plasmid can be detected in the muscle tissue of mice immunized with this plasmid. Conclusion The eukaryotic expression vector pcDNA3.1 (+) / HPV16 E6-E7 was successfully constructed, which can be efficiently expressed in HeLa cells and in mouse skeletal muscle cells.