鲤春病毒血症病毒核蛋白原核表达及单克隆抗体制备

Prokaryotic expression and preparation of monoclonal antibody against nucleoprotein of spring viremia of carp virus

以含有鲤春病毒血症病毒核蛋白基因的质粒N-RFP为模板,通过PCR方法扩增N基因编码区全长,大小为1 254bp。将该基因连接到pGEX-KG载体上,构建了SVCV-N-KG重组质粒。将重组质粒转化到BL21菌株中进行诱导表达,采用SDS聚丙烯酰氨凝胶电泳分析鲤春病毒血症病毒N蛋白的表达情况。以纯化后的N蛋白为抗原免疫BALB/c小鼠,ELISA测定免疫小鼠的血清效价。将血清效价较高的小鼠脾细胞和骨髓瘤细胞融合,4次亚克隆后,通过ELISA及染色体数目分析筛选出1株杂交瘤细胞,命名为N-2。将杂交瘤细胞注射到小鼠腹腔制备单克隆抗体。经间接免疫荧光和免疫印迹实验证明该抗体可以特异性识别鲤春病毒血症病毒N蛋白。进一步分析表明,该抗体识别的靶位点位于N蛋白第227~336位氨基酸之间。

The full-length coding sequence of the nueleoprotein(N)gene in spring virema of carp virus(SVCV)was amplified by PCR method using N-RFP plasmid as the template.The PCR production was cloned into the prokaryotic expression vector pGEX-KG.The fusion protein SVCV-N-KG was analyzed by SDS-PAGE.The BALB/c mice was immuned by purified SVCV-N-KG protein and the serum titer was determined by ELISA.One monoclonal antibody(McAb N-2)against N protein were generated and selected by ELISA and chromosome number analysis after four times of subcloning selection.McAb N-2was characterized by IFA(indirect immunofluorescent assay)and Western blot assay.Further analysis showed that McAb N-2targeted the 227-336 amino acid region of N protein.This study laid a foundation for the study of pathological mechanisms and clinical diagnosis of SVCV.