人腺病毒六邻体高变区蛋白的亚克隆表达及纯化

Molecular cloning,expression and purification of hyper-variable region in the hexon protein of human adenovirus

目的克隆表达人腺病六邻体高变区片段,了解其结构和功能,为研发腺病毒基因工程重组疫苗奠定基础。方法根据生物信息学软件预测的腺病毒六邻体的高变区域,选择目标基因,设计其对应的引物,并以团队保存的人腺病毒六邻体核酸序列为模板,聚合酶链反应扩增目标基因,定向插入载体pQE32中构建工程质粒。通过酶切和基因测序鉴定阳性工程质粒,并诱导其在大肠杆菌M15中高效表达带有组氨酸标记的靶蛋白,应用镍树脂亲和层析的方法分离纯化靶蛋白。结果构建了含有人腺病毒六邻体高变区片段的重组质粒,同时探索出其在大肠杆菌M15中高效表达的条件,纯化目标蛋白,其分子量约为188000,与应用生物信息学分析预测的结果相同。结论成功地克隆、表达和纯化了人腺病毒六邻体高变区片段。

Objective To clone and express the hyper-variable region in the hexon protein of human adenovirus,understand its structure and function,and lay a foundation for a genetic engineering vaccine candidate of human adenovirus.Methods We selected the target gene which was the hypervariable region of hexon,according to the model of human adenovirus hexon predicted by bioinformatics software.The corresponding primers were designed.The hexon,preserved by our team,was as a template to amplify the desired gene by PCR.The recombinant plasmid was constructed by directly cloning into the vector pQE32.The positive engineering vector,confirmed by the cleavage of endonuclease and nucleic acid sequence,was transformed into E.coli.M15 to express the His-tagged protein.Then the target protein was purified by Ni-Resin column affinity chromatography.Results The recombinant plasmid containing the hyper-variable region fragment in the hexon protein of human adenovirus was constructed.The conditions for the target gene expressing protein efficiently in E.coli M15 were found.We purified the target protein with a molecular weight of 188 000,which was consistent with the prediction by bioinformatics.Conclusion The fragment of hexon protein hyper-variable region is cloned,expressed and purified successfully.