Ⅰ型牛疱疹病毒VP22蛋白原核表达及多克隆抗体制备

Prokaryotic Expression and Preparation of Polyclonal Antibodies against BHV-1 VP22

为制备Ⅰ型牛疱疹病毒(BHV-1)VP22蛋白抗体,选用国内分离株BHV-SHJS,在其UL49基因核酸序列第517至747位间设计引物扩增目的片段,构建pET-28a-UL49(172-249 aa)原核表达载体,转化载体至BL21(DE3)感受态细胞;经IPTG诱导表达并纯化VP22(172-249 aa)融合蛋白后;用该融合蛋白作抗原,以皮下注射的方式免疫Balb/c小鼠制备多克隆抗体;经间接ELISA、Western blot及间接免疫荧光(IFA)方法检测其灵敏度和特异性。结果表明,表达的VP22蛋白以包涵体形式存在;ELISA测定结果显示,制备的VP22蛋白抗体效价达到1∶16000;Western blot和IFA结果显示,制备的多克隆抗体能与BHV-SHJS VP22特异性结合,具有良好的反应原性。试验结果为后期BHV-1感染宿主细胞在机制方面的研究和VP22功能的探究奠定了基础。

To prepare Bovine herpesvirus type I(BHV 1)VP22 antibody,domestic BHV-SHJS strain UL49 gene in positions 517 to 747 were selected to design primers,amplify target fragment and to construct prokaryotic expression vector of pET-28a-UL49(172-249 aa).Then,that was transformed it into E.coli BL21(DE3)competent cells.BL21 was induced by isopropyl-D-thiogalactoside(IPTG);Then,VP22(172-249 aa)proteins were expressed and purified.The fusion proteins were used as antigen to immunize BALB/c mice by the subcutaneous injection to prepare antibodies.The sensitivity and specificity were tested by the indirect ELISA,Western blot and the indirect immunofluorescence(IFA)methods.The results revealed that the recombinant protein was expressed in the form of inclusion body;The titer of mouse anti-VP22 polyclonal antibody was 1∶16000 detected by indirect ELISA.Results of both Western blot and IFA test showed that the polyclonal antibody could recognize BHV-SHJS specifically,with effective specific reactivity.It laid a foundation for the establishment of the mechanistic study and exploring function of VP22 of late BHV-SHJS infection of host cells.