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| Bioinformatics Analysis and Polyclonal Antibody Preparation of Small Peptide EPC5 Encoded by LncBMP4 in Chicken (Gallus gallus) |
| XIA Qian, GONG Wei, JIN Jing, ZHANG Chen, GAO Xiao-Min, CHEN Chen, ZHOU Shu-Jian, HU Cai, ZHANG Yu, LI Bi-Chun, ZUO Qi-Sheng* |
| College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China |
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Abstract Chicken (Gallus gallus) primordial germ cells (PGCs) have a wide application prospect in the fields of transgenic animal preparation and germplasm resources protection. In order to analyze the molecular mechanism of chicken PGCs formation, a key long noncoding RNA-bone morphogenetic protein 4 (LncBMP4) that regulates chicken PGCs formation was identified earlier, and it can encode a small peptide, named EPC5. In this study, the chemical structure and subcellular localization of EPC5 (expression in primordial germ cells chromosome 5) peptides were analyzed by online prediction software. The prokaryotic fusion expression vector of PET-EPC5-His-B2M was constructed by chemical synthesis and transformed into the BL2 competent state of Escherichia coli. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), EPC5 antigen was expressed and purified. The purified antigen was used for animal immunization and antiserum (G1480, G1481) was collected. The titer of antiserum was detected by ELISA. The antibody was purified and identified by Western blot. The results indicated that EPC5 mainly localized in cells and had RNA polymerase subunits. The PET-EPC5-His-B2M vector was successfully constructed, and the size of the recombinant protein was about 25 kD after induction. ELISA showed that the titer of G1480 antiserum was 1:5.12×105 (ODantiserum/ODpre-immune blood≥2.1), and that of G1481 antiserum was 1:1.024×106 (ODantiserum/ODpre-immune blood≥2.1). After purification of G1480 antiserum, the concentration of EPC5 polyclonal antibody was 10 mg/mL, and the titer was 9.8 ng/mL (OD value>0.2) by ELISA. The results of Western blot showed that the prepared polyclonal antibody could specifically bind to EPC5 protein expressed in vitro and in vivo. In conclusion, the polyclonal antibody against EPC5 was successfully prepared in this study, which laid a foundation for further study on the function and mechanism of EPC5 in the formation of PGCs.
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Received: 10 February 2022
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Corresponding Authors:
* 006664@yzu.edu.cn
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