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| Bioinformatics Analysis of Caprine parainfluenza virus type 3 NP Protein B-cell Epitopes and Preparation of Its Polyclonal Antibodies |
| LIAO Juan1,5, GAO Xiao-Long2, WANG Xiao-Long1, LIU Ya-Lin1, ZHANG Hai-Jie1, REN Shan-Hui3, YANG Yi2, BAO Shi-Jun1,*, HAN Yu4,* |
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2 College of Agriculture and Animal Husbandry, Qinghai University, Xining 810003, China;
3 Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730070, China;
4 College of Advanced Agricultural Sciences, Yulin University, Yulin 719000, China;
5 College of Life Science, Leshan Normal University, Leshan 614000, China |
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Abstract Caprine parainfluenza virus type 3 (CPIV3) belongs to the family Paramyxoviridae, genus Respirovirus, and is one of the critical pathogens of respiratory diseases in goats (Capra hircus) and sheep (Ovis aries), severely affecting the healthy development of the sheep industry in China. To provide essential biological materials for studying the pathogenic mechanisms and prevention of CPIV3, this study prepared polyclonal antibodies against the nucleoprotein (NP) of CPIV3 based on the gene sequence of CPIV3/SX/2024 strain, and its B cell epitopes were predicted and analyzed. The full-length NP gene was amplified by PCR; A prokaryotic expression recombinant plasmid was constructed; Isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce the expression of the target protein. After purification, the protein was used to immunize BALB/c mice (Mus musculus) for polyclonal antibody preparation, and the biological characteristics of the antibodies were analyzed. The result showed that there were multiple potential B-cell epitopes in the NP protein of CPIV3. The targeted NP gene fragment of CPIV3 were approximately 1 625 bp, consistent with the expected result. PCR and sequencing confirmed the successful construction of the recombinant prokaryotic expression plasmid, named pET28a-CPIV3-NP. The recombinant plasmid successfully expressed the target protein under IPTG induction with a molecular weight of approximately 60 kD. The NP protein was primarily expressed in inclusion bodies, with the highest expression achieved at 37 ℃, 0.5 mmol/L IPTG, and 8 h after induction. After denaturation, renaturation, and purification, the proteins were used to immunize mice. The resulting polyclonal antibodies specifically reacted with CPIV3, and the Western blot antibody titer reached 1∶512 000 and exhibited strong immunogenicity. This study provides important experimental materials for the research on the pathogenic mechanism, diagnosis, prevention, and control technique of CPIV3.
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Received: 18 June 2025
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Corresponding Authors:
* bsjdy@126.com; wsadzx522@163.com
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