慢病毒介导的HSV-2 gD2蛋白的表达及多克隆抗体的制备

The expression of HSV-2 gD2 protein mediated by lentivirus and preparation of gD2-specific

摘要:

目的 采用慢病毒介导293T细胞表达单纯疱疹病毒Ⅱ型包膜糖蛋白2（HSV-2 gD2），免疫新西兰兔制 备高效价的gD2多克隆抗体，为HSV-2病毒的检测及其基础研究提供便利。方法 将密码子优化的HSV-2病毒G Strain US6基因（MH790667.1）序列连接至慢病毒载体pLenti-CMV-gD2-6His中，重组慢病毒感染正常293T细胞并 筛选出稳转株，从培养上清中收获分泌型gD2蛋白后采用Ni-NTA亲和层析纯化目的蛋白，与弗氏佐剂1∶1混合乳 化后皮下注射免疫新西兰大白兔，加强免疫 1 次，ELISA 检测兔血清中 gD2 特异性抗体（纯化抗原包板）的效价， Western blot检测载体病毒中gD2抗原蛋白的表达。结果 构建的慢病毒重组质粒序列与理论设计一致，稳转株表 达的gD2-6His蛋白相对分子质量约为37 000，电泳条带位于40 000~55 000（糖基化修饰），二次免疫后兔血清中gD2 特异性抗体滴度为1∶204 800，兔血清经1∶500稀释后作为一抗孵育可检测到疫苗病毒中gD2抗原蛋白的表达，条带 特异。结论 成功表达高纯度分泌型gD2蛋白，并制备了高效价的兔多克隆抗体，便于后续HSV-2疫苗的研发。

Abstract:

Objective The expression of secreted human herpes simplex virus 2 envelope glycoprotein gD (HSV-2 gD2) mediated by lentivirus, and the preparation of gD2-specific rabbit polyclonal antibody will provide convenience for subsequent ELISA and WB detection in HSV-2 vaccine development. Methods Codon-optimized sequences of the HSV-2 G Strain US6 gene (MH790667.1) were linked into the lentiviral vector pLenti-CMV-gD2-6His. The recombinant lentivirus and packing plasmids were co-transfected into 293T cells to harvest recombinant lentivirus, then the recombinant lentivirus-infected 293T cells were screened with puromycin for establishing stable cell lines. The secreted gD2-6His proteins were harvested from the culture supernatant of the screened stable cell lines. The target protein was purified by Ni-NTA affinity chromatography technology. The purified gD2-6His protein mixed with an equal proportion of Freund’s adjuvant was subcutaneously injected into New Zealand white rabbits, strengthening the immunization once. The titer of gD2-specific antibody in rabbit serum was detected by ELISA; the specificity and affinity of the rabbit serum antibody were verified by Western blot. Results The sequence of the lentiviral recombinant plasmid was consistent with the original design. The theoretical molecular weight of gD2-6His protein was about 37 000, and the electrophoretic migration band was positioned between 40 000 and 55 000 (glycosylation modification). The gD2-specific antibody titer of rabbit serum was 1∶204 800 by ELISA assay; the 1∶500 dilution was useful for detecting the gD2 antigen protein expression of the vaccine virus. Conclusions High-purity gD2-6His protein has been successfully prepared; the efficient gD2-specified rabbit polyclonal antibody was verified by ELISA and western blot, which will provide great convenience for the subsequent development and testing of the HSV-2 vaccine.