传染性鼻气管炎病毒gE基因的表达与免疫原性

Expression and Immunogenicity of Gene g E of Infectious Bovine Rhinotracheitis Virus

[目的]对牛传染性鼻气管炎病毒(IBRV)gE基因表位进行原核表达,并对表达产物进行免疫原性鉴定。[方法]利用PCR扩增出gE基因2段表位(gE-A、gE-B),并构建原核表达重组质粒pET-32a-gE。将其转化BL21(DE3)表达菌中,IPTG诱导表达。表达的gE重组蛋白经亲和层析纯化后,进行Westernblot分析。[结果]重组表达质粒pET-32a-gE经过PCR、双酶切及测序证明成功构建重组质粒。SDS-PAGE结果表明,gE蛋白在大肠埃希菌中高效表达,表达的重组蛋白相对分子量约52ku,与预期蛋白分子量一致。纯化后的gE重组蛋白浓度为0.254mg/mL。免疫印迹结果表明,纯化后的gE重组蛋白能与IBR标准阳性血清发生特异性反应,说明其免疫原性良好。[结论]成功表达了gE基因表位蛋白,该蛋白具有良好的免疫原性,可作为检测IBRV的gE抗体的候选抗原。

[Objective] To express gene gE of infectious bovine rhinotracheitis virus,and to identify the immunogenicity of expression products.[Method] Two epitopes （gE-A,gE-B） of gene gE were amplified by PCR amplification.Prokaryotic expression recombinant plasmid pET-32a-gE was constructed,and transformed to BL21 （DE3）expression fungus.After induced by IPTG,the expressed recombinant protein gE was purified by nickel ion affinity chromatography and analyzed by Western blot.[Result] The recombinant plasmid was confirmed by PCR,restriction analysis and sequencing.SDS-PAGE showed that gene gE showed high-level expression in Escherichia coli,with the relative molecular weight of expressed recombinant protein being about 52 ku,which was consistent with the predicted molecular weight.The concentration of the purified recombinant protein was 0.254 mg/mL for gE.The fusion protein specifically reacted with IBRV polyclonal antibody from mice,indicating good immunogenicity.[Conclusion] Gene gE epitope protein is successfully expressed.This protein has good immunogenicity and can be used as the candidate antigen for prevention of IBR.