人乳头瘤病毒11型E6基因与人IFN-α2b融合基因原核表达质粒的构建与鉴定

Constrution and Selection of Proeukaryotic Expression Plasmid of Human Papillomavirus Type 11 E6 and Human IFNα-2b Fusion Gene

目的构建HPV11E6与人IFNα2b融合基因表达载体,为进一步原核表达奠定基础。方法在IFNα2b的起始密码子之前加入ccatggct,同时以编码(GGSGS)3的45个碱基序列取代其终止密码子,将改造后的人IFNα2b基因人工合成,然后将其装入质粒pET32a的NcoⅠ和BamHⅠ酶切位点之间;PCR扩增HPV11E6基因片段。将含有IFNα2b基因片段的质粒pET32a和含有BamHⅠ,EcoRⅠ酶切位点的HPV11E6同时用BamHⅠ,EcoRⅠ酶切,将酶切产物纯化回收后做连接反应,连接产物常规转化大肠杆菌DH5α感受态细胞,随机挑选阳性克隆并提取质粒酶切鉴定,同时将挑选的阳性克隆菌液送公司测序。结果测序结果表明成功的将HPV11E6和人IFNα2b通过连接肽(GGSGS)3连接并装入pET32a的NcoⅠ和EcoRⅠ酶切位点之间,且基因序列与设计完全一致。结论HPV11E6与人IFNα2b融合基因表达载体的成功构建,为进一步原核表达奠定了基础。

Objective To construt the expression plasmid of HPV11-E6/human IFNα-2b fusion gene and give the way for further proeukaryotic expression.Methods The changed human IFNα-2b gene fragment was synthesized artifitially and was inserted into pET-32a plasmid between NcoⅠand BamHⅠsite;HPV11-E6 gene fragment was amplified by PCR.The changed human IFNα-2b and HPV11-E6 gene fragment were digested by BamHⅠand EcoRⅠenzyme,digested product was linked by T4DNA ligase,DH5αwas transformed with the linked product,positive clones were identified after BamHⅠand EcoRⅠdigestion, and DNA sequencing was carried out.Results The DNA sequencing result showed that HPV11-E6/human IFNα-2b fusion gene was successfully inserted into the pET-32a plasmid between NcoⅠand EcoRⅠsite.Conclusion The successful construction of proeukaryotic expression plasmid of HPV11-E6/human IFNα-2b fusion gene enables the further proeukaryotic expression.