非洲猪瘟p62蛋白的原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibodies Production of African Swine Fever Virus p62 Protein

为了获得非洲猪瘟(african swine fever virus,ASFV)p62蛋白进行血清学诊断技术研究。根据GenBank ASFV参考序列合成CP530R基因(编码p62蛋白),插入pET-32a(+)载体,转化到大肠杆菌BL21(DE3)感受态细胞,SDS-PAGE及Western blot鉴定。采用Ni柱纯化p62蛋白,然后免疫Balb/c雌鼠,制备鼠抗ASFV p62蛋白的多克隆抗体,ELISA检测多抗效价。结果表明,成功构建了pET32a-p62质粒,表达蛋白为56.1 kDa,以包涵体表达为主。Western blot显示该蛋白能与ASFV阳性血清结合。Ni柱纯化获得p62蛋白浓度为2.9 mg/mL。用该蛋白免疫小鼠,三免后抗体效价达1∶256000。表明表达蛋白具有良好抗原性,制备的p62多克隆抗体具有较高的反应性和特异性,为进一步研究p62蛋白的特性和诊断奠定基础。

This study aims to obtain the p62 protein of African swine fever virus(ASFV)for further development of serological diagnostics.According to the ASFV reference sequence in the Genbank,CP530R sequence was synthesized and inserted into the pET-32a(+)vector,subsequently transformed into E.coli BL21(DE3)competent cell.After inducing expression by IPTG,it was identified through SDS-PAGE and Western blot.The synthesized p62 protein was purified by Ni column and immunized Balb/C female mice to The synthesized p62 protein was purified by Ni column and immunized Balb/c female mice to obtain polyclonal antibody against ASFV p62 protein.The titer of polyclonal antibody was detected by ELISA.The results suggested that the plasmid pET32a-p62 was successfully constructed.SDS-PAGE showed that the recombinant protein with 56.1 kDa was expressed as inclusion body.Western blot showed that the protein could bind to ASFV antiserum.The p62 protein was purified by Ni column,and its concentration was determined to be 2.9 mg/mL.Polycolnal antibodies were produced by immunizing mice with the purified protein.The titer of antibodies reached 1∶256,000 after three rounds of immunization.The results suggested that the purified p62 protein had good antigenicity,and the polyclonal antibodies showed high reactivity and specificity,therefore laying a foundation for further study on the structure and function of p62 protein,and also on serological diagnosis of ASFV.