牛病毒性腹泻病毒E0基因重组表达质粒pMG36e-E0的构建与免疫原性分析

利用SacⅠ、HindⅢ限制性内切酶分别对pMG36e与pMD19-T-E0进行双酶切,将目的基因E0整合入穿梭表达载体pMG36e,构建牛病毒性腹泻病毒E0基因重组表达质粒pMG36e-E0。提取重组质粒pMG36e-E0,进行双酶切鉴定及PCR鉴定。将阳性重组质粒pMG36e-E0转化到大肠杆菌DH5α中进行表达,对表达产物进行SDSPAGE和Western-blotting鉴定。用表达的E0蛋白二次免疫家兔,间接ELISA法检测其血清抗体水平。结果,重组质粒经双酶切得到大小分别约为3 600、691bp的2个片段,PCR扩增出691bp的片段,双酶切与PCR鉴定结果表明目的基因E0正确插入到载体pMG36e中。SDS-PAGE电泳结果表明E0基因在大肠杆菌获得了表达,表达产物相对分子质量大小约为27 000。Western-blotting分析结果证明表达产物能被BVDV阳性血清所识别。ELISA检测结果证明表达的E0融合蛋白能刺激动物产生抗体,具有天然蛋白的免疫原性。

Construction and prokaryotic expression of recombinant plasmid pMG36e-E0 of bovine viral diarrhea virus and immunogenicity of the recombinant protein

The pMG36e vector and pMD19-T-E0 were digested by restriction endonuclease SacI and HindⅢ ,respectively. The E0 gene was subcloned into the expression vector pMG36e to constructed recombinant plasmid pMG36e-E0, and then transformed into E. coli DHSa strain. The constructed recombinant plasmids pMG36e-E0 were extracted and identificated by Sac I/Hind Ⅲ double enzyme digestion and PCR. The expression of E0 fusion protein was confirmed by SDSPAGE and Western-blotting. The healthy male rabbits were immunized with recombinant E0 pro- tein,and its antibody levels against BVDV in serum were detected by an indirect ELISA. The Sac I/Hind Ⅲ enzyme digestion of the recombinant plasmids obtained a target band of 690bp and a band of 3 600 bp in size corresponding pMG36e vector genome. PCR amplification of E0 genes u- sing recombinant plasmids as a template resulted in single band of 690 bp in size. The results showed that the constructed recombinant plasmid pMG36e-E0 was positive plasmid by enzyme cleavage and PCR. The E0 fusion protein band of about 27 000 was identificated by SDS-PAGE and Western blotting. The recombinant plasmid pMG36e-E0 was successfully constructed and a- ble to express in E. coli DH5a. Western blotting analysis indicated that the recombinant protein could specifically react to antibodies against BVDV. The results of studies on immunogenicity by indirec, ELISA indicated that the expressed E0 protein could induce animals to produce specific andbodies against BVDV