猪丁型冠状病毒ns7、ns7a蛋白原核表达及多克隆抗体的制备

Prokaryotic expression and polyclonal antibodies preparation of ns7 and ns7a proteins of porcine deltacoronavirus

为获得免疫原性好的猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)辅助蛋白ns7和ns7a,并制备特异性的多克隆抗体,以用于ns7和ns7a蛋白功能的初步研究,本试验构建原核表达载体pGEX-PDCoV-ns7和pGEX-PDCoV-ns7a,测序正确后转入E.coli BL21(DE3)中诱导表达。利用GST-Resin进行蛋白纯化,将获得的重组蛋白以皮下注射方式免疫BALB/c小鼠,成功制备4份PDCoV ns7和2份ns7a小鼠多克隆抗体,并用ELISA、Western blot和IFA对抗体效价、反应性及特异性进行鉴定。结果显示,本试验成功应用大肠杆菌表达了可溶性ns7和ns7a蛋白,制备的PDCoV ns7和ns7a多克隆抗体效价分别为1∶409600和1∶12800,并在Western blot和IFA试验中具有良好的反应性及特异性。结果表明,制备的PDCoV辅助蛋白ns7和ns7a多克隆抗体为深入研究ns7及ns7a蛋白的生物学功能提供了重要工具。

The purpose of this study is to express porcine deltacoronavirus(PDCoV)accessory proteins ns7 and ns7a with good immunogenicity,and to prepare specific polyclonal antibodies of ns7 and ns7a for further functional study.Prokaryotic expression vectors pGEX-PDCoV-ns7 and pGEX-PDCoV-ns7a were constructed,sequenced,and transferred into E.coli BL21(DE3),soluble ns7 and ns7a were expressed and purified with GST-Resin,then used to immunize BALB/c mice by subcutaneous injection.Four kinds of ns7 and two kinds of ns7a polyclonal antibodies were prepared successfully.The titers,reactivity and specificity of the antibodies were identified by ELISA,Western blot,and IFA.Results showed that E.coli was successfully used to express soluble ns7 and ns7a proteins in this experiment,and the prepared PDCoV ns7 and ns7a polyclonal antibodies with titers of 1∶409600 and 1∶12800 showed good reactivity and specificity in Western blot and IFA reactions.The successful preparation of polyclonal antibodies for PDCoV accessory protein ns7 and ns7a provides an important tool for further analysis of the biological functions of ns7 and ns7a proteins.