人类乳头状瘤病毒E6相关蛋白的原核表达及纯化

Prokaryotic expression and purification of human papillomavirus E6-associated protein

目的原核表达及纯化带GST标签的人类乳头状瘤病毒E6相关蛋白E6AP,为研究泛素化修饰与多种疾病的机制关联提供实验基础。方法以人卵巢文库为模板,利用PCR技术扩增E6AP编码序列,将其插入pGEX KG GST载体中。利用DH5α感受态细胞鉴定重组质粒并大量克隆。质粒转入大肠杆菌Rossate菌株,小量诱导表达。利用GST融合蛋白纯化磁珠提取并纯化GST E6AP融合蛋白,SDS PAGE电泳和Western印迹检测纯化效率。结果PCR技术成功获取大小约为2559 bp的E6AP编码序列,与pGEX KG GST载体连接,双酶切鉴定及公司测序结果显示GST E6AP载体构建成功。转入Rossate菌株后诱导质粒小量表达,获取并提纯带有GST标签的E6AP重组蛋白。SDS PAGE电泳和Western印迹结果显示E6AP蛋白纯化成功。结论成功构建重组质粒并表达GST E6AP蛋白,获取纯化E6AP蛋白,为进一步研究泛素化在疾病发生发展中的地位和作用奠定坚实基础。

Objective To express human papillomavirus E6 assiociated protein(E6AP)with GST tag in prokaryotic cells and provide experimental reference for identification of mechanisms of ubiquitination and various diseases.Methods E6AP coding regions were amplified according to human ovary cDNA library by PCR and then inserted into the vector of pGEX KG GST in prokaryotic cells.The competent cells of DH5a were employed to identify the recombinant plasmids and clone them.The plasmids were introduced into E.coli Rossate.The expression of recombinant plasmids was induced mildly.GST beads were used to extract and purify recombinant proteins of GST E6AP.The effects of purification were tested by SDS PAGE and Western blotting analysis.Results E6AP coding regions were cloned by PCR with 2559 bp and inserted into plasmids of pGEX KG GST.The results of dual digestion and sequencing showed successful construction of vectors of GST E6AP.The expression of the recombinant plasmids was induced mildly in E.coli Rossate.The recombinant proteins of E6AP with GST tag were purified,which was verified by SDS PAGE and Western blotting analysis.Conclusion Recom binant plasmids of E6AP with GST tag are constructed.The prokaryotic expression protein of GST E6AP is also obtained and purified successfully.This study can help explore the relationships between ubiquitination and development of diseases.