大鲵虹彩病毒主要衣壳蛋白的原核表达及多克隆抗体制备

摘要

According to major capsid protein (MCP) gene of chinese giant salamander iridovirus (GSIV),primers were de-signed,and MCP gene coding frame full-length sequence (1392 bp in length) was amplified by PCR and then cloned into prokaryotic expression vector pET-32a,to construct the recombinant expression vector pET-32a-MCP. The recombinant expres-sion vector pET-32a-MCP was successfully expressed in E.coli BL21 as a fused recombinant protein with a size of 70 ku e-qual to the expected size, and presented mainly in inclusion body. The optimum induction condition for expression of the re-combinant protein was 0.5 mmol/L IPTG,37 ℃,6 h. The purified recombinant proteins were used to immune New Zealand white rabbits to produce polyclonal antibody,the titers of polyclonal antibodies was above 1:50000 by ELISA. Western blot test demonstrated that the recombinant protein was recognized by the polyclonal antibody. An indirect immunofluorescent assay also showed that the polyclonal antibody was able to interact specifically with the pathological EPC cells (GICB) induced by GSIV infection. The conclusions made a preparations for the establishment of GSIV immunodiagnosis and the study of GSIV MCP gene coding protein functions.%根据大鲵虹彩病毒(Chinese giant salamander iridovirus,GSIV)主要衣壳蛋白(Major Capsid Protein,MCP)基因设计引物,PCR扩增得到MCP基因编码框全长序列1392 bp,将其克隆到原核表达载体pET-32a中,构建了重组原核表达载体pET-32a-MCP,并在大肠杆菌BL21中得到了表达,融合表达的重组蛋白分子量约为70 ku,与预期大小一致,主要以包涵体的形式存在.对IPTG浓度,诱导温度等诱导表达条件进行优化,确定0.5 mmol/L的IPTG于37℃的条件下诱导6 h重组蛋白的表达量最佳.纯化GSIV-MCP重组蛋白免疫新西兰大白兔,制备了GSIV-MCP多克隆抗体,ELISA检测抗体效价大于1:50000.Western blot检测显示该抗体可以特异性识别重组蛋白.间接荧光免疫结果表明,该多克隆抗体可与由GSIV感染引起细胞病变的EPC细胞(GICB)发生特异性的结合.研究为建立GSIV免疫诊断方法以及为研究GSIV MCP基因编码蛋白的功能奠定了前期基础.