猫白血病病毒gp70蛋白单克隆抗体的制备及应用  

Preparation and application of feline leukemia virus gp70 protein monoclonal antibody

作　　者：杜文广 彭佳佳 曾悦 李佳康 史开拓 周登元 曹胜波[1] 张燕 李秋燕 DU Wenguang;PENG Jiajia;ZENG Yue;LI Jiakang;SHI Kaituo;ZHOU Dengyuan;CAO Shengbo;ZHANG Yan;LI Qiuyan(College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;Wuhan Keqian Biology Co.,Ltd,Wuhan 430040,China)

机构地区：[1]华中农业大学动物医学院,湖北武汉430070 [2]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1期74-79,共6页Chinese Journal of Veterinary Science

基　　金：武汉市“3551”创新人才计划资助项目。

摘　　要：猫白血病病毒(FeLV)是一种在家猫和野生猫科动物中水平传播的逆转录病毒,可引起淋巴肿瘤、免疫抑制、贫血等疾病,且国内缺乏针对FeLV的诊断试剂及预防或治疗用生物制剂。膜表面糖蛋白gp70是FeLV中和抗体的主要作用靶点,本研究通过构建gp70重组表达载体,表达纯化gp70重组蛋白,并以纯化的gp70蛋白免疫6周龄的BALB/c小鼠,进行单克隆抗体的制备。结果表明,成功构建了pET-32a-gp70重组表达质粒,并应用IPTG诱导表达、包涵体纯化获得了原核表达的gp70重组蛋白;成功制备出3株能稳定分泌针对gp70蛋白的单克隆抗体杂交瘤细胞株,分别命名为2H5、3B1、5A8;Western blot检测表明仅有2H5单克隆抗体能与gp70蛋白特异性结合;IFA检测表明2H5单克隆抗体能与真核杆状病毒表达的gp70蛋白发生特异性反应,2H5单抗效价可达1∶2048000。本研究成功制备了针对FeLV gp70蛋白的单克隆抗体,为进一步建立准确快速的FeLV诊断方法及研究预防或治疗用生物制剂奠定基础。Feline leukemia virus(FeLV)is a retrovirus that spreads horizontally in domestic and feral cats and can cause lymphoma,immunosuppression,anemia and other diseases,and there is a lack of diagnostic tools and biological agents to prevent or treat FeLV in China.The membrane surface glycoprotein gp70 is the main target of the neutralizing FeLV antibody.In this study,the recombinant gp70 expression vector was constructed,the purified recombinant gp70 protein was expressed,and the purified gp70 protein was used to immunize 6-week-old BALB/c mice to produce monoclonal antibodies.The results showed that the recombinant expression plasmid pET-32a-gp70 was successfully constructed,and the expression of recombinant protein was induced by IPTG,and inclusion body purification was used to obtain the prokaryotic recombinant protein.Three hybridoma cell lines capable of stably secreting monoclonal antibodies against gp70 protein,named 2H5,3B1,and 5A8,were successfully produced.Western blot detection showed that the 2H5 monoclonal antibodies could specifically bind to gp70 protein;IFA detection showed that the 2H5 monoclonal antibodies could specifically react with gp70 protein expressed by eukaryotic baculoviruses,and the titer of 2H5 monoclonal antibody can reach 1:2048000.In this study,a monoclonal antibody against the gp70protein of FeLV was successfully produced,which provides the basis for further development of accurate and rapid FeLV diagnostic methods and exploration of biological agents for prevention or treatment.

关 键 词：猫白血病病毒(FeLV) gp70蛋白 原核表达 单克隆抗体 杆状病毒表达系统 

分 类 号：S852.65[农业科学—基础兽医学]