非洲猪瘟病毒p72蛋白的原核表达及其单克隆抗体的制备

African Swine Fever Virus p72 Protein Expression and Production of Monoclonal Antibodies

非洲猪瘟病毒(African swine fever virus, ASFV)B646L基因编码的结构蛋白p72是高度保守的抗原,常用作ASFV检测方法的建立。论文将截短的B646L基因连接在pET28b载体,重组质粒命名为pET28b-B646L。将重组质粒转化至大肠埃希氏菌BL21(DE3)感受态细胞,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,纯化p72蛋白并免疫Balb/c小鼠制备单克隆抗体。Western blot和IFA验证结果表明,成功获得1株IgG1亚型的ASFV p72特异性单克隆抗体,可为建立ASFV血清学检测方法提供参考。

P72,a highly conserved structural protein encoded by African swine fever virus(ASFV)B646L gene,is usually used as antigen for ASFV detection method development.In this study,we constructed recombinant plasmid named as pET28b-B646L by ligating truncated B646L gene and the pET28b vector.Then,the pET28b-B646L recombinant plasmid was transformed into E.coli BL21(DE3)competent cells.The truncated p72 protein was purified and identified by SDS-PAGE and Western blot assays after induction by isopropyl-β-D-thiopental(IPTG),and was used to immunize Balb/c mice to produce monoclonal antibodies.The results showed that we successfully obtained a IgGl subtype of p72 specific monoclonal antibody,which is confirmed by Western blot and IFA assays.Thus,this study provides the basis for establishing an ASFV-associated serum detection method.