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目的:用原核表达的方法获取大量带6个His标记的甘蔗花叶病毒E株系(ScMV-E)外壳蛋白(CP)。方法:用带有BamHⅠ和SalⅠ酶切位点的特异引物,以带有多个基因的重组质粒pNUSCP为模板,扩增出片段长度为942bp的ScMV-E外壳蛋白基因,亚克隆到pMD18-T载体上,转化E.coliDH5α,经双酶切检测获得阳性克隆。BamHⅠ和SalⅠ双酶切阳性克隆质粒,回收目的片段ScMV-E的CP基因。把目的片段插入表达载体pET29a(+),转化E.coliBL21(DE3),测序。结果:阳性质粒pET29a-CP在E.coliBL21(DE3)中得到大量特异表达。SDS-PAGE分析表明,该蛋白的相对分子质量约36000,与预测一致。结论:以上方法可以得到带6个His标记的目的蛋白,有利于纯化并获取高纯度的ScMV-E的外壳蛋白。
OBJECTIVE: To obtain a large number of ScMV-E coat protein (CP) containing 6 His-tagged sugarcane mosaic virus (E) strains by prokaryotic expression. METHODS: The ScMV-E coat protein gene of 942bp in length was amplified by using a specific primer pBluescript BamHⅠ and SalⅠ and the recombinant plasmid pNUSCP with multiple genes as a template, and subcloned into pMD18-T On the vector, transformed E. coliDH5α, double digestion detected positive clones. The positive clones were digested with BamH I and Sal I to recover the CP gene of ScMV-E. The target fragment was inserted into the expression vector pET29a (+) and transformed into E. coli BL21 (DE3) for sequencing. Results: The positive plasmid pET29a-CP was highly expressed in E.coli BL21 (DE3). SDS-PAGE analysis showed that the relative molecular mass of the protein was about 36000, which was consistent with the prediction. Conclusion: The above method can be obtained with 6 His-tagged target protein, which facilitates the purification and acquisition of high purity ScMV-E coat protein.