猪伪狂犬病病毒囊膜糖蛋白gD主要抗原表位区基因的克隆及原核表达

Cloning and Prokaryotic Expression of Major Domain of Pseudorabies Virus Envelop Glycoprotein gD in Escherichia coli

本试验通过PCR方法以猪伪狂犬病病毒SD株的基因组DNA为模板扩增得到了含gD主要抗原表位编码区的片段,将该PCR产物克隆到pGEM-T载体上,酶切后插入原核表达载体pET-32a的T7启动子下游。构建的重组质粒pET-gD经IPTG诱导,在大肠杆菌BL21(DE3)中获得了高效表达。SDS-PAGE结果显示,表达产物分子质量约为45.2ku,主要以包涵体形式存在。表达产物用His亲和层析柱纯化。Western blotting结果显示,该纯化蛋白能与猪伪狂犬病病毒抗体阳性血清发生特异性反应,表明该重组蛋白具有良好的抗原反应性,可以作为猪血清伪狂犬病病毒抗体诊断用抗原。

Use the genome DNA of pseudorabies（PRV） virus SD strain as template,the major epitope of PRV envelop glycoprotein gD was amplified by polymerase chain reaction（PCR）and cloned into pGEM-T vector,then inserted into the downstream of T7 promoter to construct an expression plasmid pET-32a.After induction by IPTG,the fusion protein was highly expressed in Escherichia coli BL21（DE3） in the form of inclusion bodies.The recombinant protein was purified with His-bind affinity chromatography.SDS-PAGE and Western blotting analyses revealed that the recombinant protein with the expected 45.2 ku could react with pig serum containing antibody against PRV.All results indicated that the recombinant fusion protein can be used as an antigen of diagnostic assay to detect the PRV antibody in pig serum.