非洲猪瘟病毒UBCv1的原核表达及多抗制备

Prokaryotic expression and polyclonal antibody preparation of ubiquitin-conjugating enzyme from African swine fever virus

【目的】为研究非洲猪瘟病毒(ASFV)泛素结合酶(UBCv1)的生物学功能及致病机制奠定基础。【方法】以p3XFLAG-CMV-7.1-I215L质粒为模板,利用PCR方法扩增非洲猪瘟病毒I215L基因,将其克隆至原核表达载体pET-28a,构建pET-28a-I215L重组质粒,并将其转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达重组蛋白。重组蛋白经His柱层析纯化后免疫BALB/c小鼠制备多克隆抗体,采用ELISA方法检测抗体效价,Western blot检测抗体特异性。【结果】成功构建了重组质粒pET-28a-I215L,重组菌经IPTG诱导后能够可溶性表达重组蛋白,产物约28 kDa,与预测大小一致。用纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体,ELISA测定抗体效价为1∶7290000;Western blot结果显示抗体具有较好的特异性。【结论】成功表达纯化了ASFV的泛素结合酶并制备其多克隆抗体,为后期解析ASFV泛素结合酶的结构、功能及病毒相关致病机制提供了重要的生物材料。

[Objective]This study aims to provide a basis for investigation of the biological function and pathogenic mechanism of African swine fever virus UBCv1.[Methods]Using the p3XFLAG-CMV-7.1-I215L plasmid as a template,the I215L gene of ASFV was amplified by PCR and cloned into the prokaryotic expression vector pET-28a.The recombinant plasmid pET-28a-I215L was transformed into E.coli BL21(DE3)competent cells to express the recombinant protein by IPTG induction.The purified fusion protein was used as antigens to immunize BALB/c mice to prepare polyclonal antibody.The obtained polyclonal antibody was titrated by ELISA,and the specificity was identified by Western blot.[Results]The pET-28a-I215L recombinant plasmid was successfully constructed,and the soluble expression was obtained after induction.The product size was about 28 kDa,which was consistent with the expected protein size.Polyclonal antibodies were prepared by immunization of BALB/c mice using the recombinant protein,and the antibody titer was 1∶7290000 by ELISA.Western blot results showed that the antibody had good specificity.[Conclusion]In this study,African swine fever virus UBCv1 was successfully expressed and purified,and the polyclonal antibody against it was prepared,which provided important biological materials for the study of the structure,function and virus-related pathogenic mechanism of African swine fever virus UBCv1.