非洲猪瘟病毒I177L蛋白的原核表达及其多克隆抗体的制备

Prokaryotic Expression of I177L Protein of African Swine Fever Virus and Preparation of Its Polyclonal Antibodies

为了制备抗非洲猪瘟病毒I177L蛋白的多克隆抗体,根据HLJ/2018株的I177L序列,设计引物,扩增带有His标签序列的I177L序列,并将其插入pMAL-c5x原核表达载体中,转化至Rosetta(DE3)感受态细胞中,经IPTG诱导表达和切胶纯化后,用Xa蛋白酶因子去除重组蛋白中的MBP标签。将处理后的I177L蛋白免疫新西兰大白兔,制备多克隆抗体。结果显示,在37℃、0.6 mmol/L IPTG条件下,诱导5 h后,重组MBP-I177L蛋白表达量最高,蛋白分子质量大小约为67 ku,与预期大小相符;重组蛋白以可溶性表达为主;多克隆抗体经Western blot、间接ELISA及IFA试验结果显示,抗体效价为1∶128000,并且该抗体能特异性识别I177L蛋白。

To prepare the anti-I177L polyclonal antibody,the gene of I177L,contains the His tag sequence,was amplified and inserted into the pMal-c5 x prokaryotic expression vector.Then,the vector was transfected into the E.coli Rossetta(DE3)competent cells.The recombinant protein was induced by IPTG,purified by gel purification and temoved the MBP tag by Xa protease;Next,the New Zealand white rabbits were immunized with protein to prepare polyclonal antibodies.The results showed that under the condition of 0.6 mM IPTG at 37℃,after 5 h induction,the recombinant I177L protein expression was the highest,the protein was soluble and the size,67 ku,was met the prediction.The prepared polyclonal antibody was tested by Western-blot,indirect ELISA and IFA tests.The result showed that the titer of polyclonal antibody was up to 1:128000.It also could recognize the ASFV I177L protein.