Scytovirin在大肠杆菌中的可溶性表达

Soluble Expression in Escherichia coli and Purification of Recombinant Scytovirin

目的:获得Scytovirin(SVN)蛋白及其多克隆抗体。方法:按照NCBI上公布的SVN基因序列设计合成引物,合成SVN基因,构建pET32c-SVN原核表达重组质粒,经限制性酶切分析、DNA序列测定插入片段正确;将该重组质粒转化大肠杆菌BL21(DE3),IPTG诱导重组蛋白表达;用离子交换层析法及金属亲合层析法纯化蛋白,采用Tris-Tricine系统分析;以经过纯化的蛋白为抗原免疫白兔,制备SVN多克隆抗体。结果:对表达产物进行了分离纯化,SVN纯度达到91%;用纯化的样品制备了多克隆抗体,抗血清效价为1∶102400。结论:SVN在大肠杆菌表达系统中获得了高效可溶表达,并制备了其多克隆抗体,为进一步深入研究SVN提供了材料。

Objective： To obtain scytovirin（SVN） protein and its polyclonal antibody. Methods： The SVN sequence follow the NCBI was synthesized, the primers specific for SVN coding sequence was designed and synthesized. The SVN sequence was directly amplified using PCR and inserted into vector pET-32c to obtain the recombinant expression plasmid pET32c-SVN. The restriction enzyme analysis and DNA sequence direction confirmed that the insert fragment of clone pET32c-SVN was the SVN coding sequence. The recombinant DNA was transformed into the host cells E.coli BL21（DE3）, and induced with IPTG. The recombinant protein was purified use the ion exchange chromatography and metal affinity chromatography, then the rabbits were immuned to produce the polyclonal antibody. Results： Based on the experiments, purity of SVN reach 91%, and the titer of the antisera of the polyclonal antibody was 1：102 400. Conclusion： The soluble SVN was expressed effectively in the E.coli, which supplied a good material for further reseach.