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.
[1] 杜丽丽, 樊爽爽, 李赛赛, 等. 2016. 猪cGAS基因的克隆与原核表达[J]. 中国农业科学, 49(09): 1803-1809. (Du L L, Fan S S, Li S S, et al.2016. Cloning and prokaryotic expression of porcine cGAS gene[J]. Scientia Agricultura Sinica, 49(09): 1803-1809.) [2] 高方方, 吴忠义, 曾申明. 2008. 牛干扰素-tau基因的原核表达及生物功能鉴定[J]. 农业生物技术学报, 16(02): 208-213. (Gao F F, Wu Z Y, Zeng S M.2008. Prokaryotic expression of interferon tau gene in bovine[J]. Journal of Agricultural Biotechnology, 16(02): 208-213.) [3] 马凯, 胡红霞, 于婧, 等. 2015. 双酶切和同源重组方法构建pMIR-reporter载体的比较[J]. 中国病原生物学杂志, 10(06): 495-499. (Ma K, Hu H X, Yu J, et al.2015. Construction of PMIR-reporter vector by double enzyme digestion and homologous recombination[J]. Chinese Journal of Pathogen Biology, 10(06): 495-499.) [4] 祁文婧, 张帅, 赵鑫, 等. 2019. 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[J]. 河北农业大学学报, 42(06): 103-108. (Qi W J, Zhang S, Zhao X, et al.2019. Prokaryotic expression of bovine pregnancy-associated glycoprotein gene boPAG4 and preparation of monoclonal antibody[J]. Journal of Agricultural University of Hebei, 42(06): 103-108.) [5] 祁文婧, 张帅, 赵鑫, 等. 2020. 牛妊娠相关糖蛋白boPAG2双抗体夹心ELISA体系的建立[J]. 北京农学院学报, 35(02): 109-113. (Qi W J, Zhang S, Zhao X, et al.2020. Establishment of sandwich ELISA system of bovine pregnancy-associated glycoprotein boPAG2 double antibody[J]. Journal of Beijing Agricultural University, 35(02): 109-113.) [6] 王传振, 刘晓鹏, 崔怡, 等. 2016. 猪转录因子Nanog高效表达、多克隆抗体制备及其应用[J]. 农业生物技术学报, 24(1): 35-43. (Wang C Z, Liu X P, Cui Y, et al.2016. High-efficiency expression of porcine transcription factor Nanog, preparation of polyclonal antibody and its application[J]. Journal of Agricultural Biotechnology, 24(1): 35-43.) [7] 张飞龙, 姚凤桐. 2016. 奶牛养殖的成本收益分析[J]. 内蒙古科技与经济, 13(359): 48-49. (Zhang F L, Yao F T.2016. Analysis of the growth of cattle[J]. Inner Mongolian Science and Technology, 13(359): 48-49.) [8] 张昱. 2015. 奶牛妊娠相关糖蛋白1 (PAG1)单克隆抗体的制备[D]. 硕士学位论文, 山西农业大学, 导师: 赫晓燕, pp. 5-9. (Zhang Y.2015. Preparation of monoclonal antibody to pregnancy-associated glycoprotein 1 (PAG1) in dairy cows[D]. Thesis for M.S., Shanxi Agricultural University, Suppervisor: Hao X Y, pp. 5-9.) [9] Ayalon N.1978. A review of embryonic mortality in cattle[J]. Journal of Reproduction and Fertility, 54(2), 483-493. [10] Barbato O, Menchetti L, Sousa N M, et al.2017. Pregnancy-associated glycoproteins (PAGs) concentrations in water buffaloes (Bubalus bubalis) during gestation and the postpartum period[J]. Theriogenology, 97: 73-77. [11] Bériot M, Tchimbou A F, Barbato O, et al.2014. Identification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta[J]. Acta Veterinaria Scandinavica, 56(1): 4. [12] Cartmill J A, El-Zarkouny S Z, Hensley B A, et al.2001. An alternative AI breeding protocol for dairy cows exposed to elevated ambient temperatures before or after calving or both[J]. Journal of Dairy Science, 84(4): 799-806. [13] de Sousa N M, Zongo M, Pitala W, et al.2003. Pregnancy-associated glycoprotein concentrations during pregnancy and the postpartum period in Azawak Zebu cattle[J]. Theriogenology, 59(5-6): 1131-1142. [14] Feldman M F, Wacker M, Hernandez M, et al.2005. Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli[J]. Proceedings of the National Academy of Sciences of the USA, 102(8): 3016-3021. [15] Green J A, Xie S, Quan X, et al.2000. Pregnancy-associated bovine and ovine glycoproteins exhibit spatially and temporally distinct expression patterns during pregnancy[J]. Biology of Reproduction, 62(6): 1624-1631. [16] Green J A, Xie S, Roberts R M.1998. Pepsin-related molecules secreted by trophoblast[J]. Reviews of Reproduction, 3(1): 62-69. [17] Johnson M, Coulton A T, Geeves M A, et al.2010. Targeted amino-terminal acetylation of recombinant proteins in E. coli[J]. PLOS ONE, 5(12): e15801. [18] Kiracofe G H, Wright J M, Schalles R R, et al.1993. Pregnancy-specific protein B in serum of postpartum beef cows[J]. Journal of Animal Science, 71(8): 2199-2205. [19] Klisch K, Boos A, Friedrich M, et al.2006. The glycosylation of pregnancy-associated glycoproteins and prolactin-related protein-I in bovine binucleate trophoblast giant cells changes before parturition[J]. Reproduction (Cambridge, England), 132(5): 791-798. [20] Maldonado-Estrada J, Menu E, Roques P, et al.2004. Evaluation of cytokeratin 7 as an accurate intracellular marker with which to assess the purity of human placental villous trophoblast cells by flow cytometry[J]. Journal of Immunological Methods, 286(1-2): 21-34. [21] Mialon M M, Camous S, Renand G, et al.1993. Peripheral concentrations of a 60-kDa pregnancy serum protein during gestation and after calving and in relationship to embryonic mortality in cattle[J]. Reproduction, Nutrition, Development, 33(3): 269-282. [22] Nagappan M,Michael M,Robert S.2009. Methods for early detection of pregnancy in cows. United States, 7604950[P]. [23] Ni Y, Chen R.2009. Extracellular recombinant protein production from Escherichia coli[J]. Biotechnology Letters, 31(11): 1661-1670. [24] Patel O V, Takahashi T, Imai K, et al.2004. Generation and purification of recombinant bovine pregnancy associated glycoprotein[J]. Veterinary Journal (London, England : 1997), 168(3): 328-335. [25] Pohler K G, Peres R, Green J A, et al.2016. Use of bovine pregnancy-associated glycoproteins to predict late embryonic mortality in postpartum Nelore beef cows[J]. Theriogenology, 85(9): 1652-1659. [26] Ramírez O T, Zamora R, Quintero R, et al.1994. Exponentially fed-batch cultures as an alternative to chemostats: The case of penicillin acylase production by recombinant E. coli[J]. Enzyme and Microbial Technology, 16(10): 895-903. [27] Santos J E, Thatcher W W, Chebel R C, et al.2004. The effect of embryonic death rates in cattle on the efficacy of estrus synchronization programs[J]. Animal Reproduction Science, 82-83, 513-535. [28] Sasser R G, Crock J, Ruder-Montgomery C A.1989. Characteristics of pregnancy-specific protein B in cattle[J]. Journal of reproduction and fertility. Supplement, 37, 109-113. [29] Sasser R G, Ruder C A.1987. Detection of early pregnancy in domestic ruminants[J]. Journal of Reproduction and Fertility. Supplement, 34, 261-271. [30] Silke V, Diskin M G, Kenny D A, et al.2002. Extent, pattern and factors associated with late embryonic loss in dairy cows[J]. Animal Reproduction Science, 71(1-2): 1-12. [31] Szafranska B, Panasiewicz G, Majewska M.2006. Biodiversity of multiple Pregnancy-Associated Glycoprotein (PAG) family: Gene cloning and chorionic protein purification in domestic and wild eutherians (Placentalia)--a review[J]. Reproduction, Nutrition, Development, 46(5): 481-502. [32] Telugu B, Palmier M O, Van Doren S R, et al.2010. An examination of the proteolytic activity for bovine pregnancy-associated glycoproteins 2 and 12[J]. Biological Chemistry, 391(2-3): 259-270. [33] Wooding F B.1992. Current topic: the synepitheliochorial placenta of ruminants: binucleate cell fusions and hormone production[J]. Placenta, 13(2): 101-113. [34] Xie S, Green J, Bixby J B, et al.1997. The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes[J]. Proceedings of the National Academy of Sciences of the USA, 94(24): 12809-12816. [35] Zoli A P, Beckers J F, Wouters-Ballman P, et al.1991. Purification and characterization of a bovine pregnancy-associated glycoprotein[J]. Biology of Reproduction, 45(1): 1-10. [36] Zoli A P, Guilbault L A, Delahaut P, et al.1992. Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: Its application for pregnancy diagnosis[J]. Biology of Reproduction, 46(1): 83-92.
