旨在利用原核表达系统表达基因Ⅱ型鹅星状病毒(goose astrovirus,GoAstV)ORF2蛋白,制备兔源多克隆抗体,并对制备的多抗进行效价检测及鉴定.根据GoAstV-GDZJ37株ORF2基因序列设计特异性表达引物,利用RT-PCR方法进行目的基因扩增,将其连接至原核表达载体pET-32a(+);经酶切鉴定和测序正确后,转化至表达感受态细胞,并进行诱导表达及SDS-PAGE分析.以无His标签的ORF2蛋白作为免疫原免疫新西兰大白兔,制备兔源多克隆抗体.将含His标签的ORF2蛋白经Ni-NTA亲和层析法进行纯化,并以纯化后的蛋白作为检测原,通过I-ELISA方法检测兔源多克隆抗体的效价,并通过 Western blot及IFA检测方法鉴定其特异性.结果显示,克隆获得GoAstV-GDZJ37株ORF2基因,并利用原核表达系统分别表达2种重组蛋白,大小分别为77、95 kDa,且均为不可溶性表达.I-ELISA结果显示,多克隆抗体的效价为1:102 400,表明无His标签的ORF2蛋白具有良好的免疫原性;Western blot及IFA结果显示,多克隆抗体可与含His标签的ORF2蛋白及GoAstV毒株均能发生特异性反应,表明制备的多抗具有良好的反应性.本试验成功建立GoAstV ORF2蛋白原核表达系统,并制备高效价兔源多克隆抗体,为后续GoAstV血清学诊断及GoAstV致病机制的深入研究提供了依据.
Prokaryotic expression of ORF2 protein of genotype Ⅱ goose astrovirus and prep-aration and identification of its polyclonal antibody
The aim of this study was to express ORF2 protein of GoAstV genotype Ⅱ using pro-karyotic expression system to prepare rabbit polyclonal antibody.According to the sequence of GoAstV-GDZJ37 strain,the ORF2 gene was amplified using specific primers by RT-PCR method and cloned to the prokaryotic expression vector pET-32a(+).After verification by enzyme diges-ting and sequencing,the plasmid was transformed into the expression competent cells,and the ex-pression was induced,and then was analyzed by SDS-PAGE.Polyclonal antibody was prepared by immunizing New Zealand white rabbits with ORF2 protein without His label as immunogen.The ORF2 protein containing His label was purified by Ni-NTA affinity chromatography.The purified protein was used as antigen for determination of the titer of rabbit polyclonal antibody by I-ELISA.The specificity for generated antibody was determined by Western blot and IFA.The re-sults showed that the ORF2 gene of GoAstV GDZJ37 strain was cloned,and the two recombinant proteins were expressed by the prokaryotic expression system.The expressed proteins with the sizes of 77 kDa and 95 kDa,respectively,they were insoluble.I-ELISA showed that the titer of polyclonal antibody was 1∶102 400,indicating that ORF2 protein without His label had good im-munogenicity.Western blot and IFA results showed that the polyclonal antibody reacted specifical-ly with ORF2 protein containing His label and GoAstV strain,indicating that the prepared poly-clonal antibody had good reactivity.In this study,the prokaryotic expression system of GoAstV ORF2 protein was successfully established,and the highly valent rabbit polyclonal antibody was prepared,which provided technical support and material basis for the subsequent serological diag-nosis of GoAstV and study on the pathogenesis of GoAstV.
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