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目的探讨对虾白斑综合症病毒极早期基因IE1原核表达载体的构建与表达。方法将IE1编码序列克隆入pGEX-4T-2原核表达载体,并转染大肠埃希菌BL21,异丙基-β-D硫代半乳糖苷(IPTG)诱导谷胱甘肽硫转移酶(GST-IE1)融合蛋白表达,亲和层析法纯化,并用聚丙烯酰胺变性凝胶电泳(SDS-PAGE)和蛋白印迹(western b lot)对融合蛋白进行鉴定。结果克隆到IE1基因序列长675 bp,编码224个氨基酸,理论分子量为25 kDa,等电点为4.87,所构建的重组体经PCR、双酶切及测序鉴定与目标基因相符,SDS-PAGE结果表明,目的基因在大肠埃希氏菌BL21中表达成功。结论克隆构建了对虾白斑综合症病毒极早期基因IE1,并在原核细胞中获得表达。
Objective To investigate the construction and expression of prokaryotic expression vector of very early gene of prawn white spot syndrome virus (IEV). Methods The IE1 coding sequence was cloned into pGEX-4T-2 prokaryotic expression vector and transfected into Escherichia coli BL21 and induced by glutathione S-transferase (GST) -IE1 fusion protein expression, purified by affinity chromatography, and the fusion protein was identified by polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Results The sequence of IE1 gene was 675 bp in length, encoding 224 amino acids. The theoretical molecular weight was 25 kDa and the isoelectric point was 4.87. The constructed recombinant was identified by PCR, double enzyme digestion and sequencing. The results of SDS-PAGE The results showed that the target gene was successfully expressed in Escherichia coli BL21. Conclusion Cloning and construction of a very early gene of White Spot Syndrome Virus IE1 and its expression in prokaryotic cells.