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| Preparation of Bovine (Bos taurus) ARPC3 Polyclonal Antibody and Expression of ARPC3 in Mammary Tissue |
| BAI Wen-Kai1,2, LIU Pei-Wen1,2, CHEN Yan1,2, WANG An1,2, FANG Bo1,2, YANG Ting-Ji1,2, ZHANG Quan-Wei2,3, WANG Qi1,2, ZHAO Xing-Xu1,2, DONG Wei-Tao1,2*, ZHANG Yong1,2* |
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2 Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China;
3 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China |
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Abstract Actin associated protein 2/3 complex subunit 3 (ARPC3) is an important component of actin skeleton tissue, which plays an important role in bacterial invasion, inflammation development and apoptosis. To investigate the role of ARPC3 protein in bovine mastitis, in this study, rabbit polyclonal antibody against bovine (Bos taurus) ARPC3 was prepared and its activity was identified. The biological characteristics of bovine ARPC3 (NM 001034271.2) were analyzed by online software. pQE-80L-ARPC3 recombinant plasmid was constructed. The prokaryotic expression system was used to express ARPC3. The concentration of isopropyl-beta-D-thiogalactopyranoside (IPTG) and induction time were optimized. The expressed protein was purified by affinity chromatography, identified by SDS-PAGE and mass spectrometry, and then immunized New Zealand white rabbits (Oryctolagus cuniculus) to prepare polyclonal antibody. The titer of polyclonal antibody was detected by ELISA, and the specificity of polyclonal antibody was identified by Western blot. The results showed that the recombinant plasmid pQE-80L-ARPC3 was successfully constructed, and the recombinant protein (about 20.5 kD) was expressed in the form of inclusion bodies after IPTG induction . The optimal expression conditions were as follows: IPTG concentration was 1 mmol/L for 8 h. The titer of ARPC3 polyclonal antibody was 1∶128 000. Western blot identification of natural protein showed that ARPC3 polyclonal antiserum could specifically recognize ARPC3 in target protein and mammary tissue. Immunohistochemical (IHC) results showed that the polyclonal antibody could be effectively applied for the detection of bovine mastitis, and the expression of ARPC3 protein was positively correlated with the expression of related apoptotic proteins. In this study, the polyclonal antibody of ARPC3 with good specificity was successfully prepared, which can be used to the establishment and application of a new technology for rapid detection of bovine mastitis.
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Received: 12 March 2024
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Corresponding Authors:
* d.wt2008@163.com;zhychy@163.com
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