南非2型口蹄疫病毒VP1蛋白单抗的制备与特性研究

Preparation and Characterization of A VP1⁃Specific Monoclonal Antibody of South African Type 2 Foot⁃and⁃Mouth Disease Virus

为制备南非2型口蹄疫病毒型特异性单克隆抗体,本研究通过原核系统表达南非2型口蹄疫病毒VP1蛋白,亲和层析的方法纯化目的蛋白。将纯化的VP1蛋白使用弗氏佐剂乳化后,免疫BALB/c小鼠,待加强免疫后取血清效价最稳定的小鼠脾细胞与小鼠骨髓瘤SP2/0细胞进行融合,应用间接ELISA筛选针对南非2型VP1蛋白的单克隆抗体。采用特异性试验、叠加试验和抗体亚型鉴定等方法对筛选的单克隆抗体特性进行分析。结果发现,本研究成功表达南非2型VP1蛋白,筛选出2株能稳定分泌特异性针对南非2型VP1蛋白的杂交瘤细胞株(3B2和6F2),其亚型分别为IgG2a和IgG1,叠加试验结果表明2株单抗的叠加系数>40%,说明两株单抗针对不同的抗原位点;单抗的效价可达1∶256000,特异性试验证明2株单抗均不与O型、A型口蹄疫病毒及其他常见猪病病毒产生交叉反应。由此可见,本研究制备的单克隆抗体具有良好的特异性和反应性,为南非2型口蹄疫病毒定型诊断和未来疫苗的研制奠定了基础。

In order to prepare South African type 2 foot⁃and⁃mouth disease virus(SAT2⁃FMDV)specific monoclonal antibodies,in this study,the VP1 protein of SAT2⁃FMDV was expressed by a prokaryotic system.The target protein was purified by affinity chromatography.The purified VP1 protein was emulsified with Freund's adjuvant and immunized to Balb/c mice.Mouse splenocytes with the most stable serum potency in serum were fused with mouse myeloma SP2/0 cells after reinforcement of the immunization.Indirect ELISA was applied to screen for monoclonal antibodies against the VP1 protein.of SAT2⁃FMDV.The characteristics of the screened monoclonal antibodies were analyzed by specificity tests,overlay tests,and identification of antibody subtype.The VP1 protein of SAT2⁃FMDVwas expressed in this study.Two hybridoma cell lines(3B2 and 6F2)were screened that could stably secrete antibodies specifically targeting the VP1 protein of SAT2⁃FMDV,and their subtypes were IgG2a and IgG1,respectively.A superposition assay showed that the stacking coefficients of the two monoclonal antibodies was>40%,which indicated that the two monoclonal antibodies were directed against different antigenic sites.The potency of the monoclonal antibodies was up to 1∶256000.A specificity assay used to characterize the monoclonal antibodies revealed no cross⁃reaction with O⁃type or A⁃type foot⁃and⁃mouth disease viruses or other common viruses that cause diseases in pigs.The monoclonal antibodies prepared in this study had good specificity and reactivity.Our data lay a foundation for the SAT2⁃FMDV diagnosis and vaccine development.