非洲猪瘟病毒主要抗原p54-1的原核表达及多克隆抗体的制备与鉴定

Prokaryotic Expression of African Swine Fever Virus p54-1 and Preparation and Characterization of Its Polyclonal Antibody

为进一步深入研究非洲猪瘟病毒(African swine fever virus,ASFV)p54蛋白的主要抗原表位及抗原性质,本试验根据GenBank中p54基因序列(登录号:NC_001659.2)设计表达区域特异性引物,PCR扩增后连接表达载体pGEX6p-1构建pGEX6p-1-p54-1原核表达质粒;将该质粒转化大肠杆菌BL21感受态细胞,经IPTG诱导,SDS-PAGE鉴定融合蛋白的表达,切除GST标签后采用阴离子柱纯化目的蛋白并鉴定;将纯化蛋白与佐剂混合乳化后作为免疫原,免疫小鼠制备p54-1蛋白多克隆抗体;采用ELISA和Western blotting检测抗体的效价和反应特性。结果显示,重组p54-1融合蛋白以可溶形式表达,切除标签后的纯化蛋白能够与ASFV阳性血清发生反应,而与PRRSV和PCV3不发生反应。利用该蛋白免疫获得的多克隆抗体经ELISA检测其抗体效价为1∶128 000。Western blotting结果显示,制备的多克隆抗体能够识别ASFV p54蛋白。表明本研究成功获得了较高纯度的p54-1蛋白,制备的p54-1多克隆抗体具有较高反应性和特异性,为后续非洲猪瘟双抗夹心ELISA检测方法的建立提供依据。

In order to further study the major epitopes and antigen properties of African swine fever virus(ASFV)p54-1 protein,specific primers were designed according to p 54 gene sequence retrieved from GenBank(accession No.:NC_001659.2),the target sequence of encoding p54-1 protein was amplified by PCR.Then it was ligated into pGEX6p-1 vector and constructed prokaryotic expression plasmid(pGEX6p-1-p54-1).The plasmid was transfected into E.coli BL21,and the expression of recombinant protein was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The fusion protein cleaved the GST-tag using thrombin,and then purified by anion exchange column.The protein was identified by ELISA and emulsify with adjuvant,the prepared immunogen was inoculated into mouse to prepare of p54-1 protein specific polyclonal antibody.The immune-activity and titers of the prepared polyclonal antibody were determined by ELISA and Western blotting.The results showed that the expressed recombinant protein p54-1 existed in a soluble form.The p54-1 protein cleaved GST-tag could react with the positive serum of ASFV,but no with negative serum,PRRSV and PCV3.The prepared polyclonal antibody titer was 1∶128 000.Western blotting result demonstrated that the prepared polyclonal antibody could recognize the p54 protein.In conclusion,the high purified expressed protein of ASFV p54-1 had been successfully prepared and p54-1 specific polyclonal antibody showed wonderful immunocompetence and specificity,providing foundation for the development of sandwich ELISA detection method of ASF.