猪伪狂犬病毒囊膜糖蛋白gB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 gB in Essherichia Coli

参照GenBank发表的猪伪狂犬病毒囊膜糖蛋白gB主要抗原表位的编码区基因序列,设计一对引物,通过PCR扩增后,将约为600bp的目的片段克隆到pGEM-T载体上,酶切后插入原核表达载体pET-32(a)的T7启动子下游,构建的重组质粒pET-gB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,表达产物分子量约为42.4KDa,主要以包涵体形式存在。BandScan分析表明,表达量约占菌体蛋白的60.5%。利用His亲和层析方法得到了纯化的表达产物。Western blotting结果显示,重组蛋白能与阳性血清发生特异性反应,具有较好的抗原反应原性,可以作为检测用抗原。

According to the gene sequences of pseudorabies virus envelope glycoprotein gB published in genbank, a pair of primers was designed. The major epitope of pseudorabies virus envelop glyeoprotein gB was amplified by polymerase chain reaction （PCR） and cloned into pGEM-T vector, then inserted into the downstream of T7 promoter to construct a recombinant plasmid pET-gB. After induction by IPTG, the fusion protein was highly expressed in Escherichia Coli BL21 （ED3） in the form of inclusion bodies. BandScan analysis showed that the ratio of the protein is 60.5% of the total protein.The recombinant protein was purified with His-Bind affinity chromatography. SDS-PAGE and Western blotting analyses revealed that the recombinant protein with the expected 42.4KDa could react with pig serum containing antibody against PRV. All result indicated that the recombinant fusion protein can be used as an antigen of diagnostic assay to detect the PRV antibody in swine serum.