牛妊娠相关糖蛋白基因boPAG4的原核表达及单克隆抗体的制备  

Prokaryotic expression of bovine pregnancy-associated glycoprotein gene boPAG4 and preparation of monoclonal antibodies

作　　者：祁文婧 张帅 赵鑫 张红星 谢远红 金君华 QI Wenjing;ZHANG Shuai;ZHAO Xin;ZHANG Hongxing;XIE Yuanhong;JIN Junhua(College of Food Science and Engineering,Beijing University of Agriculture/Beijing Key Laboratory of Detection and Control of Spoilage Organisms and Pesticide Residues in Agricultural Products/Beijing Engineering Laboratory of Key Technology Development of Microecologics,Beijing 102206,China)

机构地区：[1]北京农学院食品科学与工程学院/农产品有害微生物及农残安全检测与控制北京市重点实验室/微生态制剂关键技术开发北京市工程实验室

出　　处：《河北农业大学学报》2019年第6期103-108,共6页Journal of Hebei Agricultural University

基　　金：北京市教委科研计划项目(KM201810020016)

摘　　要：为了获得具有高特异性和高免疫原性的boPAG4单克隆抗体,本试验以PCR技术扩增荷斯坦奶牛boPAG4基因,构建重组质粒pCold TF-boPAG4后,在E.coli BL21(DE3)中表达pCold TF-boPAG4蛋白并优化。以重组蛋白pCold TF-boPAG4作为免疫原免疫BALB/c小鼠,经细胞融合最终筛选出3株能稳定分泌pCold TF-boPAG4单克隆抗体的杂交瘤细胞,pCold TF-boPAG4单克隆抗体滴度均为1×105以上。纯化蛋白pCold TF-boPAG4经HRV3C蛋白酶酶切后制备出boPAG4抗原,Westernblot(WB)结果显示,3种单克隆抗体都能特异性识别boPAG4抗原,pCold TF-boPAG4单克隆抗体的成功制备为开发现场奶牛妊娠ELISA检测系统提供了理论支持。To obtain boPAG4 monoclonal antibody with high specificity and immunogenicity,the gene of boPAG4 in Holstein cow was amplified by PCR in this experiment,and the recombinant plasmid pCold TF-boPAG4 was constructed.After that,the protein of pCold TF-boPAG4 was expressed and optimized in E.coli BL21(DE3).BALB/c mice were immunized with recombinant protein pCold TF-boPAG4 as immunogen,and three hybridoma cells were screened out by cell fusion.The titer of the monoclonal antibody of pCold TF-bopag 4 was more than 1×105.The purified protein,pCold TF-boPAG4,was digested by HRV3C protease to produce boPAG4 antigen.Western Blot(WB)results showed that all three monoclonal antibodies could specifically recognize boPAG4 antigen.The successful preparation of pCold TF-boPAG4 monoclonal antibody provided theoretical support for the development of field cow pregnancy ELISA system.

关 键 词：牛 妊娠相关糖蛋白 原核表达 单克隆抗体 

分 类 号：Q784[生物学—分子生物学] S852.43[农业科学—基础兽医学]