猪瘟病毒E2蛋白部分抗原区基因在原核系统的分泌表达与鉴定

基于猪瘟病毒主要保护性抗原E2囊膜糖蛋白有两个相对独立的抗原结构单位B/C抗原区和A/D抗原区,根据GeneBank中猪瘟病毒B/C基因核苷酸序列设计合成一对引物,经RT-PCR扩增猪瘟病毒B/C基因。将扩增所得猪瘟病毒B/C基因克隆于PMD18-T载体,然后定向亚克隆猪瘟病毒B/C基因至原核表达载体PET30(a)的多克隆位点中,经酶切鉴定后,将含有B/C基因的重组质粒命名为PETB/C。用PETB/C转化受体菌BL21,挑取单个菌落,用诱导剂IPTG以不同浓度进行诱导,并在不同诱导时间收集样品。经SDS-PAGE电泳分析证实B/C基因获得了表达,并在终浓度为1mM的IPTG,诱导6h其表达达到高峰,经Western-blot分析证实表达的重组B/C蛋白具有反应活性,大小约为9KD。用表达产物作ELISA,结果表明表达产物与猪瘟病毒抗血清可发生明显的反应,而与其它8种疫病阳性血清不发生反应。正常菌体蛋白与猪瘟病毒阳性血清也不发生反应。通过对58份送检血清样品的检测结果显示其与Dot-ELISA的相符率高达95%以上。试验证明利用原核表达系统制备的重组蛋白作为诊断用抗原,为研究亚单位疫苗或诊断抗原打下坚实基础。

Secretion Expression of the Gene Encoding Classical Swine Fever Virus E2 B/C Antigenic Domain in E.Coli and Identification of the Protein

Since the envelope glycoprotein E2 of classical swine fever virus(CSFV)has two structural antigenic units B/C and A/D,and according to the sequence of B/C gene published in GeneBank,a pair of primers were designed and synthesized,and CSFV was amplified and cloned into PMD18-T plasmid.The CSFV gene then was subcloned into prokaryotic expression plasmid PET30(a).The recombinant plasmid carried B/C gene was designated PETB/C.By transformation of BL21 with PETB/C,the recombinant strain BL21(PETB/C) was obtained.The expressed fusion protein was identified by SDS-PAGE in E coli BL21 after induction with IPTG.And the biological activities were confirmed by western-blot and ELISA test with polyclonal antibodies.The recombinant B/C protein was about 9KD in size.In the ELISA test,the serum samples from the swine infected with 8 infectious pathogens other than CSFV were negative.And there were no reactions between CSFV positive serum and BL21 E.coli.The total of 58 serum samples were evaluated by ELISA and Dot-ELISA test,the correspondence between ELISA and Dot-ELISA was about 95%.The results showed that the recombinant protein was more specific,economic,and easier to perform for serologically diagnosis of CSFV infection in swine.The findings provided the base to develop sub-unit vaccine and diagnostic antigen.