Preparation and Identification of Polyclonal Antibody Against Bovine (Bos taurus) Pregnancy-associated Glycoprotein PAG6
LI Zhi-Ming1, YUAN Meng-Yi2, XU Hai-Tao3, CAI De-Lin1, WANG Xiang-Guo1, XIAO Long-Fei1, AN Hong1, WANG Ying-Qiu1, WANG Qian1, GUO Yong1, QI Xiao-Long1, NI He-Min1,*
1 College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China; 2 Beijing Changping Animal Disease Prevention and Control Center, Beijing 102299, China; 3 National Animal Husbandry Services, Beijing 100125, China
Abstract 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 pET30a-boPAG6 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-D-thiogalactopyranoside (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 non-pregnant 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.
LI Zhi-Ming,YUAN Meng-Yi,XU Hai-Tao, et al. Preparation and Identification of Polyclonal Antibody Against Bovine (Bos taurus) Pregnancy-associated Glycoprotein PAG6[J]. 农业生物技术学报, 2022, 30(8): 1632-1639.
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