甘蔗花叶病毒E株系外壳蛋白基因原核表达载体的构建与表达

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。

Construction of Plasmid Containing Coat Protein Gene of Sugarcane Mosaic Virus Strain E and Prokaryotic Expression of Coat Protein Gene

Objective: To obtain coat protein(CP) of strain E of sugarcane mosaic virus(ScMV-E) with 6×His tag by prokaryotic expression. Methods: The recombinant plasmid pNUSCP with target CP gene of strain E of ScMV-E and other genes was amplified by specific primers with the sites of double restriction enzyme digestion, BamH Ⅰ and Sal Ⅰ , respectively and the target CP gene with length of 942 bp was obtained. The CP gene fragment of ScMV-E was recovered by kit of Gel Purification. Then, CP gene of ScMV-E with two digested sites of BamH Ⅰ and Sal Ⅰ was subcloned into plasmid of pMD18-T simple vector. Positive recombinant plasmid was selected and tested by digestion with restriction endonucleases of BamH Ⅰ and Sal Ⅰ. The prokaryotic expression plasmid was constructed by inserting the CP gene into vector of pET-29a(+) and detected by sequencing. Positive clone was named as pET-29a-CP. Results: Plasmid DNA extracted from pET-29a-CP was used as foreign DNA and transformation of E. coli BL21(DE3) was conducted. According to SDS-PAGE analysis, positive transformant with specific expression of a 36 kD fusion protein corresponding to CP gene of ScMV-E achieved by IPTG induction was obtained. Conclusion: The target protein with 6×His tag will be propitious to purify and obtain pure CP of ScMV-E.