猫TNF-α蛋白的原核表达及阻断活性单克隆抗体的制备  

Preparation of monoclonal antibodies with blocking activity against cat TNF-α

作　　者：王阅 李佳康 李秋燕 曹胜波[1,2,3] 叶静 曹龙龙 周登元 WANG Yue;LI Jiakang;LI Qiuyan;CAO Shengbo;YE Jing;CAO Longlong;ZHOU Dengyuan(College of Animal Science and Technology-College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;National Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;Wuhan Keqian Biology Co.,Ltd.,Wuhan 430040,China)

机构地区：[1]华中农业大学动物科学技术学院与动物医学院,湖北武汉430070 [2]农业微生物资源发掘与利用全国重点实验室,湖北武汉430070 [3]武汉科前生物股份有限公司,湖北武汉430040

出　　处：《中国兽医学报》2024年第8期1667-1673,共7页Chinese Journal of Veterinary Science

基　　金：武汉市“3551创新人才计划”资助项目。

摘　　要：为制备针对猫TNF-α的具有阻断活性的单克隆抗体,本研究基于可溶性猫TNF-α(sTNFα)基因成功构建、表达及纯化了重组质粒pET-28a-sTNFα,并进一步对重组蛋白诱导表达的条件进行摸索和优化,鉴定其生物活性;其次将猫TNF-α重组蛋白作为免疫原进行小鼠免疫、细胞融合、阻断活性杂交瘤细胞的筛选和腹水制备,并对获得的单克隆抗体进行鉴定。结果显示,成功构建了pET-28a-sTNFα质粒并通过大肠杆菌系统表达出具有生物活性的猫TNF-α重组蛋白,相对分子质量为34 kDa,半数细胞活性抑制质量浓度为1.22μg/L;成功筛选出3株具有阻断活性的单克隆抗体(A6-B7-9、H5-E2-94和C8-A10-100),Western blot检测显示3株单抗均能与TNF-α特异性结合,效价可达1∶512000,3株单抗在质量浓度为100 mg/L时,对TNF-α的拮抗效果最强。本研究筛选了猫TNF-α的阻断活性抗体,以期为由猫TNF-α介导的相关疾病的防治提供新型的安全的有效的候选药物。In order to prepare monoclonal antibodies with blocking activity against feline TNF-α,this study successfully constructed,expressed and purified the recombinant plasmid pET-28a-sTNFαbased on the soluble feline TNF-α(sTNFα)gene,and further investigated the induced expression.The conditions were explored and optimized to identify its biological activity;secondly,the feline TNF-αrecombinant protein was used as an immunogen for mouse immunization,after cell fusion,screening of blocking active hybridoma cells and ascites preparation,the obtained monoclonal antibodies were tested.The results showed that the pET-28a-sTNFαplasmid was successfully constructed and the bioactive feline TNF-αrecombinant protein was expressed in E.coli system.The molecular weight was 34 kDa and the 50%inhibitory concentration was 1.22μg/L.Three monoclonal antibodies(A6-B7-9,H5-E2-94 and C8-A10-100)with blocking activity were successfully screened out.The results of Western blot showed that all the three mAbs could specifically bind to TNF-αwith a titer of 1∶512000.When the concentration of the three mAbs was 100 mg/L,the inhibitory effect on TNF-αwas the strongest.In this study,we screened antibodies that can block the activity of cat TNF-α,in order to provide novel,safe and effective candidate drugs for the treatment of TNF-αmediated diseases in cats.

关 键 词：肿瘤坏死因子-Α 单克隆抗体 阻断活性 重组蛋白表达 猫 

分 类 号：S852.4[农业科学—基础兽医学]