奶牛妊娠相关糖蛋白PAG6多克隆抗体的制备及鉴定  

Preparation and Identification of Polyclonal Antibody Against Bovine(Bos taurus) Pregnancy-associated Glycoprotein PAG6

作　　者：李志明 袁梦仪 许海涛[3] 蔡德霖 王相国[1] 肖龙菲 安红 王颖秋 王骞 郭勇[1] 齐晓龙[1] 倪和民[1] LI Zhi-Ming;YUAN Meng-Yi;XU Hai-Tao;CAI De-Lin;WANG Xiang-Guo;XIAO Long-Fei;AN Hong;WANG Ying-Qiu;WANG Qian;GUO Yong;QI Xiao-Long;NI He-Min(College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China;Beijing Changping Animal Disease Prevention and Control Center,Beijing 102299,China;National Animal Husbandry Services,Beijing 100125,China)

机构地区：[1]北京农学院动物科学技术学院,北京102206 [2]北京市昌平区动物疫病预防控制中心,北京102299 [3]全国畜牧总站,北京100125

出　　处：《农业生物技术学报》2022年第8期1632-1639,共8页Journal of Agricultural Biotechnology

基　　金：北京金桥种子工程项目(ZZ19002);北京市奶牛产业创新团队项目(BAIC05-2017)。

摘　　要：基于抗原抗体反应检测奶牛(Bos taurus)血液中妊娠相关糖蛋白(pregnancy-associated glycoproteins,PAGs)是目前世界上应用最普遍的牛早孕检测技术,以boPAG6作为标志物应用于母牛早期妊娠诊断的准确性更高,因此制备高敏感性的boPAG6多克隆抗体,能为奶牛早期妊娠诊断试剂盒的研发提供支持。本研究利用PCR技术扩增荷斯坦奶牛boPAG6(GenBank No.NM_176617.2)基因,构建重组质粒pET30a-boPAG6;在大肠杆菌(Escherichia coli)BL21(DE3)中表达His-boPAG6蛋白并优化,以重组蛋白His-boPAG6作为免疫原免疫BALB/c小鼠(Mus musculus)制备多克隆抗体,用ELISA方法检测血清效价,Western blot方法检测纯化后的抗体分别与重组蛋白His-boPAG6和奶牛血清的特异性反应,利用免疫荧光技术定位boPAG6在原代滋养层细胞中的表达。结果显示,本研究成功构建出大小为6534 bp的重组质粒pET30a-boPAG6,并在异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)终浓度为0.5 mmol/L、诱导时间为8 h的条件下表达的His-boPAG6量最高,其蛋白分子质量为50 kD。以纯化后浓度为0.412 mg/mL的His-boPAG6蛋白免疫,ELISA中抗血清的效价为1∶102400,多克隆抗体能特异性识别His-boPAG6抗原并有效区分妊娠和未妊娠奶牛血清,还可检测到原代滋养层细胞中存在boPAG6蛋白。本研究成功制备了boPAG6多克隆抗体,为奶牛早期妊娠ELISA检测系统的开发提供了理论支持。The detection of pregnancy-associated glycoproteins(PAGs) in cow(Bos taurus) blood based on antigen and antibody reaction is the most widely used early pregnancy detection technology in the world.boPAG6 as a marker can be applied to the diagnosis of early pregnancy of cows with higher accuracy.Therefore, the preparation of high-sensitivity boPAG6 polyclonal antibodies can provide support for the development of early pregnancy diagnostic kits for dairy cows. In this study, the boPAG6 gene(GenBank No.NM_176617.2) of Holstein cow was amplified by PCR method, and then the recombinant plasmid pET30aboPAG6 was constructed. The yield of His-boPAG6 protein was get and optimized in Escherichia coli BL21(DE3). The recombinant protein His-boPAG6 was used as an immunogen to immunize BALB/c mice(Mus musculus) to prepare the polyclonal antibody. The titer of serum was detected by ELISA method. The specific reaction of purified antibody with recombinant protein His-boPAG6 and cow serum was detected by Western blot method. Immunofluorescence technique was used to detect the localization of boPAG6 in primary trophoblast cells. The recombinant plasmid pET30a-boPAG6 with the size of 6 534 bp was successfully constructed. The yield of His-boPAG6 was highest when induced with 0.5 mmol/L isopropyl-beta-Dthiogalactopyranoside(IPTG) for 8 h, and its protein molecular weight was 50 kD. The titers of antiserum in ELISA immunized with purified His-boPAG6 protein was 1∶102 400. The polyclonal antibody could specifically recognize His-boPAG6 antigen and effectively distinguish pregnant dairy cows’ s serum from nonpregnant dairy cows’ s serum, and detect the presence of boPAG6 protein in primary trophoblast cells. This study provides theoretical support for the exploitation of ELISA detection system in early pregnancyof dairy cows.

关 键 词：奶牛 妊娠相关糖蛋白6(PAG6) 原核表达 多克隆抗体 

分 类 号：S857.2[农业科学—临床兽医学]