非洲猪瘟病毒I215L蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of African swine fever virus I215L protein

本研究聚焦于非洲猪瘟病毒(ASFV)I215L基因编码的泛素结合酶(UBCv1),对其进行原核系统表达并制备了多克隆抗体。以ASFV CN/GS/2018株为模板,成功扩增I215L基因并插入原核表达载体,形成重组质粒pET28a-I215L。经大量诱导表达后,利用切胶方法成功纯化出重组蛋白。将提纯后的I215L蛋白与佐剂混合对动物进行多次免疫,获得鼠/兔抗I215L蛋白的多克隆抗体。采用酶联免疫吸附试验(ELISA)测定抗体效价,鼠抗I215L多抗效价达到1∶64000以上,兔抗I215L多抗效价达到1∶512000;间接免疫荧光(IFA)和免疫印迹(Western-blot)结果显示,该多抗具有特异性识别ASFV感染后I215L蛋白的能力,并且显示出良好的效果。本研究在体外表达并纯化了ASFV I215L蛋白,成功获得鼠/兔抗I215L蛋白的多克隆抗体,这为后期ASFV I215L蛋白的结构解析、功能研究及病毒相关致病机制的探索奠定了基础。

This study focused on the ubiquitin-conjugating enzyme(UBCv1)encoded by the African swine fever virus I215 L gene,expressed its prokaryotic system and prepared polyclonal antibodies.According to the sequence of the ASFV-CN/GS/2018 strain isolated by the team,the I215L gene was introduced into the prokaryotic expression vector to form a recombinant plasmid(p ET-28a-I215L),which was introduced into E.coli BL21(DE3)competent cells.After a large number of induction and expression,the recombinant protein was purified by gel cutting method.The purified I215L protein was mixed with adjuvant to immunize animals for many times to obtain mouse/rabbit polyclonal antibody against I215L protein.The antibody titer was determined by enzyme-linked immunosorbent assay(ELISA).The mouse anti-I215 L polyclonal antibody titer reached more than 1∶64000,and the rabbit anti-I215 L polyclonal antibody titer reached 1∶512000.The results of indirect immunofluorescence(IFA)and Western-blot showed that the polyclonal antibody could specifically recognize the expression of I215L protein after PAMs infected with ASFV,and the effect was good.In this study,the African swine fever virus I215L protein was expressed and purified in vitro,and the polyclonal antibody of mouse/rabbit anti-I215L protein was successfully obtained,which laid a foundation for the structural analysis,functional study and virus-related pathogenesis of ASFV I215L protein.