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 anti-body
杜文广 1彭佳佳 2曾悦 2李佳康 1史开拓 2周登元 1曹胜波 3张燕 1李秋燕1
作者信息
- 1. 华中农业大学动物医学院,湖北武汉 430070;武汉科前生物股份有限公司,湖北武汉 430040
- 2. 武汉科前生物股份有限公司,湖北武汉 430040
- 3. 华中农业大学动物医学院,湖北武汉 430070
- 折叠
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∶2 048 000.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.
Abstract
Feline leukemia virus(FeLV)is a retrovirus that spreads horizontally in domestic and fe-ral 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 pro-duce 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 baculovir-uses,and the titer of 2H5 monoclonal antibody can reach 1:2 048 000.In this study,a monoclonal antibody against the gp70 protein 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Key words
feline leukemia virus/gp70 protein/prokaryotic expression/monoclonal antibodies/bacu-lovirus expression system引用本文复制引用
基金项目
武汉市"3551"创新人才计划资助项目()
出版年
2024
NETL
NSTL
万方数据