牛支原体Vsp蛋白的原核表达及单克隆抗体的制备

Prokaryotic Expression of Vsp Protein and Preparation of Monoclonal Antibody against Mycoplasma bovis

为研发牛支原体的快速诊断试剂,利用大肠埃希菌原核表达系统表达重组CaIgV-Vsp蛋白,制备CaIgV-Vsp蛋白单克隆抗体。结果显示,成功构建了重组质粒pET-CaIgV-Vsp;SDS-PAGE电泳结果显示,融合蛋白为可溶性蛋白;ELISA与Western blot检测结果显示,重组CaIgV-Vsp蛋白与牛支原体阳性血清可发生特异性反应,具有较好的免疫原性;成功制备了4株稳定分泌CaIgV-Vsp蛋白单克隆抗体的杂交瘤细胞株,腹水ELISA效价分别为5.2×10^(5)、1.3×10^(5)、2.6×10^(5)、2.6×10^(5)。

In order to develop a rapid diagnostic reagent for Mycoplasma bovis,the monoclonal antibody against CaIgV‐Vsp protein was prepared after expressing recombinant CaIgV‐Vsp protein in the prokaryotic expression system of Escherichia coli.The results showed that the recombinant plasmid pET‐CaIgV‐Vsp was successfully constructed.SDS‐PAGE analysis showed that the fusion protein was soluble protein.The results of ELISA and Western blot showed that the recombinant CaIgV‐Vsp protein could react specifically with the positive serum of Mycoplasma bovis and had good immunogenicity.Four hybrioma cell lines secreting monoclonal antibodies against CaIgV‐Vsp protein were prepared successfully.The ELISA titers of ascites were 5.2×10^(5),1.3×10^(5),2.6×10^(5) and 2.6×10^(5),respectively.