猪轮状病毒VP4、VP6蛋白原核表达及多克隆抗体的制备

Prokaryotic expression of VP4 and VP6 proteins of porcine rotavirus and preparation of polyclonal antibodies

猪轮状病毒(porcine rotavirus,PoRV)属于呼肠孤病毒科,轮状病毒属,是引起幼年仔猪发生腹泻的主要病原之一,常与其他病原发生混合感染。PoRV结构蛋白VP4、VP6分别组成病毒的纤突及内衣壳,在病毒侵入、复制以及维持病毒粒子形态中起到重要作用。为研究PoRV VP4和VP6蛋白相关特性,分别扩增VP4开放阅读框中抗原性较好的两部分:58~1080 bp基因片段(1023 bp)、1657~2328 bp基因片段(672 bp),以及VP6完整开放阅读框(1191 bp),成功构建重组表达质粒pET32a-VP4-672、pET32a-VP4-1023和pET32a-VP6,经测序正确后转化BL21感受态细胞中,经IPTG诱导,重组蛋白均以包涵体形式表达,融合蛋白大小分别约为44,58,64 kDa,与预期大小相符。将纯化后的蛋白免疫新西兰大白兔,制备兔源多克隆抗体,间接ELISA方法测定多抗效价均在1∶10^(6)以上,免疫印迹(Western blot)和间接免疫荧光(IFA)检测结果显示多抗均可与病毒蛋白产生良好的抗原抗体反应。本研究结果可为广西地区PoRV抗原抗体检测方法的建立以及疫苗效果评估提供参考,也为病毒结构蛋白功能研究奠定基础。

Porcine rotavirus(PoRV)belongs to Rotavirus genus,reoviridae,and is one of the main pathogens causing diarrhea in young piglets.It is often co-infected with other pathogens.The structural proteins VP4 and VP6 of PoRV form fibrin and underwear shell of the virus,respectively,and play an important role in the invasion,replication and maintenance of virus particles.In order to study the related characteristics of PoRV VP4 and VP6 proteins,two parts with high antigenicity in VP4 open reading frame were amplified:58-1080 bp gene fragment(1023 bp),1657-2328 bp gene fragment(672 bp)and complete VP6 open reading frame(1191 bp).The recombinant expression plasmids pET32 a-VP4-672,pET32 a-VP4-1023 and pET32 a-VP6 were successfully constructed.After sequencing,the recombinant plasmids were transformed into BL21 competent cells.After induction by IPTG,the recombinant proteins were expressed as inclusion bodies.The fusion protein sizes were 44,58 and 64 kDa,respectively,which were consistent with the expected size.New Zealand white rabbits were immunized with the purified protein to prepare rabbit polyclonal antibody.The titer of the polyclonal antibody determined by indirect ELISA was more than1:10^(6).The results of Western blot and indirect immunofluorescence assay showed that the polyclonal antibody could produce good antigen antibody reaction with the viral protein.The results of this study can provide a reference for the establishment of pig rotavirus antigen and antibody detection methods and the evaluation of vaccine efficacy in Guangxi region,and also lay a foundation for the study of the function of structural proteins of the virus.