山羊痘病毒RPO30蛋白的原核表达及其单克隆抗体的制备

Prokaryotic expression and monoclonal antibody preparation of goatpox virus RPO30 protein

为了研究山羊痘病毒RNA聚合酶亚基RPO30蛋白功能,构建了其原核表达质粒pET28a-RPO30和pGEX-6p-1-RPO30,并进行了诱导表达和纯化。将纯化的His-RPO30蛋白免疫BALB/c小鼠,并将免疫小鼠脾细胞与骨髓瘤细胞SP2/0融合,通过筛选得到了能够稳定分泌与RPO30蛋白特异性反应的单克隆抗体的杂交瘤细胞株7E5。对该单克隆抗体的轻链和重链类型进行测定,发现其重链为Ig G2b亚型,轻链为κ亚型。经测定,单克隆抗体7E5的腹水效价可达1∶128000。RPO30单克隆抗体的制备将为该蛋白功能的研究提供有力工具。

To study the function of goatpox virus RNA polymerase subunit RPO30,the prokaryotic expression plasmids p ET28 a-RPO30 and p GEX-6 p-1-RPO30 were constructed for further expression and purification.The purified His-RPO30 was then used to immunize BALB/c mice.Through cell fusion of SP2/0 and the immunized mice spleen cells,we got the hybridoma cell strain 7 E5 which could stably secrete monoclonal antibody and specially react with RPO30.The determination of monoclonal antibody 7 E5 heavy chain and light chain types indicated that the heavy chain was Ig G2 b subtype and the light chain wasκsubtype.The titer of the ascitic fluid could reach to 1∶128000.The preparation of RPO30 monoclonal antibody provides a powerful tool for its further functional study.