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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 construct HPV11E6 and human IFNα2b fusion gene expression vector and lay the foundation for further prokaryotic expression. Methods ccatggct was added before the start codon of IFNα2b and its stop codon was replaced by a 45 base sequence encoding (GGSGS) 3. The modified human IFNα2b gene was artificially synthesized and then inserted into NcoI of plasmid pET32a And BamH Ⅰ restriction sites; PCR amplified HPV11E6 gene fragment. The plasmid pET32a containing IFNα2b gene fragment and HPV11E6 containing BamHⅠ and EcoRⅠ restriction sites were digested with BamHⅠ and EcoRⅠ at the same time. The digested product was purified and ligated. The ligated product was transformed into E. coli DH5α competent cells randomly and randomly Positive clones were picked and digested with restriction endonucleases, and the positive clones selected were sent to the company for sequencing. Results The sequencing results showed that HPV11E6 and human IFNα2b were successfully ligated into the NcoⅠ and EcoRⅠ restriction sites of pET32a through the connecting peptide (GGSGS) 3, and the sequence of the gene was completely consistent with the design. Conclusion The successful construction of HPV11E6 and human IFNα2b fusion gene expression vector laid the foundation for further prokaryotic expression.