柯萨奇病毒A组16型VP1蛋白的原核表达及纯化

Okaryotic Expression of VP1 Protein of Coxsackie Virus Group A 16 Strain and Purification

[目的]原核表达并纯化柯萨奇病毒A组16型(CVA16) VP1蛋白。[方法]根据Gen Bank CVA16 VP1基因序列,合成VP1全基因,连接到载体p ET30a,构建原核表达质粒p ET30a-CVA16-VP1。将测序正确的重组质粒转化至大肠杆菌BL21(DE3),IPTG诱导蛋白表达,Ni-NTA亲和层析纯化,SDS-PAGE和Western Blot鉴定重组蛋白。[结果]成功构建重组质粒p ET30a-CVA16-VP1,表达蛋白经SDS-PAGE显示分子量约为38.7 k Da,目的蛋白以包涵体形式存在,蛋白经复性、纯化,纯度达90%以上;经Western Blot检测,证实表达的蛋白为VP1蛋白。[结论]成功获得了高纯度的VP1蛋白,为此抗原用于临床诊断打下了基础。

[Objective]To express and purify the VP1 protein of coxsackie virus group A 16 strain （CVA16） in E.coli. [Method]The CVA16 VP1 gene was manually synthesized according to the GenBank VP1 sequence,and then cloned into pET30a to construct the expressional plasmid pET30a- CVA16-VP1. The plasmid pET30a- CVA16-VP1 confirmed through sequencing was transformed into E.coli BL21 （DE3） for inducing VP1 expression by IPTG and purification by Ni-NTA affinity chromatography.The purified protein was identified by SDS-PAGE and Western Blot.[Result]The recombinant plasmid pET30a- CVA16-VP1 was obtained.The relative molecular mass of the protein was 38.7 kDa by SDS-PAGE analysis.The target protein existed as inclusion bodies.The purity of the purified protein was above 90%.Western Blot proved that the expressed product is VP1 protein.[Conclusion]The recombinant CVA16 VP1 protein was successfully expressed in E.coli, which laid a solid foundation for the clinical diagnosis of MTB infection.