B组轮状病毒WH-2株vp7基因的原核表达与抗体的制备

Prokaryotic expression of vp7 gene from human group B rotavirus and generation of antibody

目的:在大肠杆菌中表达人B组轮状病毒WH-2株VP7蛋白并制备其兔抗血清。方法:根据B组轮状病毒(GBRV)WH-2株vp7基因的全序列设计引物,用PCR的方法扩增得到vp7基因的编码区。将其克隆到原核GST融合表达载体pGEX-KG内,转化大肠杆菌E.coliDH5α,IPTG诱导表达人B组轮状病毒WH-2株VP7蛋白,经SDS-PAGE分离纯化表达的蛋白免疫新西兰兔,制备人B组轮状病毒WH-2株VP7蛋白抗血清。结果:经鉴定确认,vp7基因以正确的方式插入到载体中,此重组表达载体经IPTG诱导后,可表达相对分子量为53.4 kDa的GST-VP7融合蛋白。制备的抗血清经同样诱导表达的表达载体pGEX-KG表达产物吸收后1:500倍稀释后用Western Blot分析,与53.4 kDa的GST-VP7融合蛋白获得特异性显色信号。结论:人B组轮状病毒WH-2株VP7蛋白成功在大肠杆菌中GST融合表达,所表达的蛋白和制备的抗体不但为研究结构与功能提供了物质基础,也为GBRV所引起的疾病预防、诊断和治疗等流行病学研究和临床诊断奠定了基础,具有重要实际应用价值。

Objective：To express structural protein VP7 of human group B rotavirus in E. coli and prepare rabbit antibody against the VP7. Methods：A pair of primers were designed according to the vp7 gene of human group B rotavirus stain WH - 2. The ORF of vp7 gene was amplified by PCR using the primer pair from the cloning plasmid pUCmT - vp7 of human Group B rotavirus stain WH - 2. The GST fusion expression vector of the VP7 pGEX - KG - VP7 was constructed by cloning the PCR product of vp7 gene ORF into the pGEX - KG and was transformed into E. coli DHSct. The transformed E. coli DHSct was induced with IPTG to express GST - VP7 fusion protein. The purified fusion protein was then used to immunize the new Zealand rabbits to produce the polyclonal antibody against the VP7. Results： The sequence of VP7 in the recombinant pGEX - KG - VP7 was confirmed by restriction endonuclease digestion and sequence analysis. SDS - PAGE analysis showed that fusion protein GST - VP7 was expressed in E. coli with a molecular weight of 53.4 kDa. Western Blot analysis using the polyclonal antibody derived from the rabbits indicates that these antibodies could react with the target protein. Conclusion：The preparation of the polyclonal antibody against VP7 lay a foundation for the further study of VP7 expression at protein levels and its function.