新型鸭呼肠孤病毒DH13株结构蛋白σB的原核表达及免疫原性分析

Prokaryotic Expression and Immunogenicity Analysis of the Structural ProteinσB of a Novel Duck Reovirus DH13 Strain

本研究旨在利用原核表达系统表达新型鸭呼肠孤病毒(NDRV)DH13株重组σB蛋白,并检测其免疫原性,为NDRV基因工程疫苗研制等提供物质材料。采用RT-PCR方法扩增获得NDRVσB基因,构建原核表达重组质粒pET-30a-σB和pET-32a-σB,将2个重组质粒转化大肠杆菌BL21(DE3)感受态细胞,利用IPTG诱导获得相应的重组蛋白,通过SDS-PAGE分析重组蛋白的表达形式。纯化无His的σB蛋白,并以此蛋白作为免疫原免疫新西兰白兔,制备兔源多克隆抗体。利用Ni2+柱亲和层析法纯化含His的σB蛋白,以此蛋白作为检测原,通过间接ELISA方法测定兔源多克隆抗体效价;Western blotting鉴定多克隆抗体与重组蛋白的特异性识别能力。结果显示,PCR扩增获得大小约为1100 bp的σB基因片段。SDS-PAGE结果显示,高效表达出了2种重组σB蛋白,大小分别约为41和46 ku,均以包涵体形式存在。间接ELISA结果显示,制备的多克隆抗体效价达1∶204800,能特异性地识别原核表达的重组蛋白,表明重组无His的σB蛋白具有良好的免疫原性。Western blotting特异性鉴定结果显示,含His的σB蛋白和无His的σB蛋白均与制备的兔源多克隆抗体发生特异性反应,表明原核表达得到的重组σB蛋白具备良好的反应原性。本试验利用原核表达系统成功地表达出了重组σB蛋白,并证实了重组NDRVσB蛋白具有良好的免疫原性及反应原性,该结果将有助于后续对NDRVσB蛋白生物学功能的鉴定、NDRV诊断用抗原的制备及NDRV新型疫苗的研制。

The purpose of this study was to express recombinantσB protein of DH13 strain of Novel duck reovirus(NDRV)using prokaryotic expression system,and detect its immunogenicity,so as to provide material basis for the development of NDRV gene engineering vaccine.σB gene of NDRV DH13 strain was amplified by RT-PCR and prokaryotic expression recombinant plasmids pET-30a-σB and pET-32a-σB were constructed and transformed into E.coli BL21(DE3)and with IPTG induction corresponding recombinant proteins were obtained and analyzed by SDS-PAGE.The recombinant protein without His tag was purified by digestion and as immunogen to immunize New Zealand White rabbits to prepare polyclonal antibody.The recombinant protein with His tag was purified by Ni^(2+) affinity chromatography and as detection antigen to measure rabbit polyclonal antibody titer by indirect ELISA.It was determined whether the polyclonal antibody specifically recognize the recombinant protein by Western blotting.The results showed thatσB gene with a size of about 1100 bp was amplified.The results of SDS-PAGE showed that two recombinantσB proteins were efficiently expressed,the sizes of which were about 41 and 46 ku respectively,and both existed in the form of inclusion bodies.The indirect ELISA results showed the titer of the prepared polyclonal antibody was up to 1∶204800,it could specifically recognize the recombinant protein,which further reflected that the recombinant HisσB protein had good immunogenicity.Western blotting results showed that the recombinantσB protein could reacted with the rabbit polyclonal antibody,indicating that the recombinantσB protein expressed by prokaryotic expression had good reactivity.This experiment successfully expressed the recombinantσB protein using the prokaryotic expression system,and confirmed that the NDRVσB protein had good immunogenicity and reactogenicity.This result would help the subsequent identification of the biological function of the NDRVσB protein,preparation of diagnostic antigens and development of new NDRV vaccines.