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

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.