深圳地方株人乳头瘤病毒18型E6E7基因的克隆、序列分析及E6/E7融合基因的构建

[摘要] 目的 克隆人乳头瘤病毒18型E6E7基因序列，构建E6/E7融合基因，为HPV治疗性疫苗的研制打下基础。方法 采用PCR 技术扩增1例HPV18阳性患者组织中HPV18 E6E7基因。将PCR产物回收，插入pSC-A质粒载体构建pSC-A/HPV18 E6E7重组质粒并进行核酸限制性内切酶鉴定及DNA测序分析；通过点突变方法使HPV18 E6、E7基因的开放读码框(ORF)融合以简化后续的基因表达问题。结果 成功构建了pSC-A/HPV18E6E7重组质粒，测序分析表明克隆的HPV18 E6E7基因与GenBank收录的德国株(AY262282)完全一致，点突变成功使E6基因的终止密码子突变，使HPV18 E6、E7基因融合。结论 本研究获得了正确的HPV18 E6E7基因，为其重组表达及其相关研究奠定了良好基础。

Cloning and Sequencing of Human Papillomavirus 18 E6E7 Gene from a patient in Shenzhen City and the Construction of the HPV18 E6/E7 Fusion Gene

Construction of the HPV18 E6/E7 Fusion Gene Deng Jingui, Li Tiyuan. Clinical Medical Research Center, Jinan University 2nd Clinical Medical College (Shenzhen People’s Hospital), (Shenzhen, 518020)[Abstract] Objective To clone the HPV18 E6E7 gene and construct the E6/E7 fusion gene for the construction of HPV therapeutic vaccine. Methods The HPV18 E6 and E7 genes were amplified by PCR. The PCR products were recovered and cloned into pSC-A vector to construct the pSC-A/HPV18 E6E7 recombinant plasmid and was sequenced. To simplify the problem of express, the E6 and E7 coding sequences were fused together by site-directed mutagenesis. Results The recombinant plasmid pSC-A/HPV18 E6E7 was constructed successfully. DNA analysis showed the sequence of HPV18 E6E7 was the same as what was published by GeneBank(AY262282) and the E6、E7 open reading frame were fused together. Conclusion A confirmed HPV18 E6E7 genes has been obtained, providing a good foundation for recombination, expression and related study.