Tandem Expression of N Proteins from Virulent and Attenuated Strains of Peste des petits ruminants virus and Preparation of Polyclonal Antibodies
ZHU Kai-Yu1, GAO Xiao-Long1,*, CHEN Yun1, DU Xin-Chao1, YANG Yi1, ZHANG Yang1, HUANG Hong-Jian1, TONG Li-Na1, REN Shan-Hui2,*
1 College of Animal Science and Veterinary Medicine, Qinghai University, Xining 810003, China; 2 Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730070, China
Abstract：Peste des petits ruminants (PPR) counts as an acute virulent animal disease that spreads efficiently through direct contact, where goats (Capra hircus) and sheep (Ovis aries) are the primary susceptible small ruminant species. This study aimed to prepare polyclonal antibodies against the nucleocapsid (N) protein of the Peste des petits ruminants virus (PPRV) to provide biological materials for research on PPRV pathogenesis and disease control. Based on the N gene sequences of virulent and attenuated PPRV strains, the dominant antigenic epitopes were screened and analyzed using the online bioinformatics tools SEMA and IEDB. The dominant antigenic regions of the N gene from both strains were amplified separately by overlap PCR and then linked in tandem. The tandem gene encoding the dominant antigenic epitopes of the N protein was cloned into the pET-28a vector via BamHⅠand EcoRⅠrestriction sites to construct the prokaryotic expression plasmid, pET28a-PPRV-2N. After sequencing verification, the recombinant plasmid was transformed into Escherichia coli Rosetta competent cells for induced expression and optimization of the conditions. BALB/c mice (Mus musculus) were immunized using emulsified mixtures of purified target protein and adjuvant. The harvested polyclonal antibodies were subjected to reactivity identification assays. The size of the tandem 2N gene fragment amplified by PCR was approximately 1 607 bp, which was consistent with the expected result. The recombinant expression strain successfully expressed the target protein upon induction with IPTG, and the protein was mainly present in the form of inclusion bodies with an apparent molecular weight of approximately 60 kD. Optimal expression was achieved under the conditions of 30 ℃, 1 mmol/L IPTG, and 14 h induction. After denaturation, renaturation, and purification, the target protein was used to immunize BALB/c mice. Following the 3 immunizations, blood was collected, and the sera were separated to obtain polyclonal antibodies. The titer of the polyclonal antibodies against the PPRV N protein reached 1:10 000, as determined by Western blot. ELISA showed that the purified 2N protein exhibited good reactivity with positive serum. This study successfully obtained the expression product, 2N recombinant protein. The prepared mouse polyclonal antibody exhibited good immunoreactivity against both the N protein and the virus, providing theoretical and technical support for further exploration of the pathogenic mechanism of PPRV.
朱开宇, 高小龙, 陈韵, 杜新潮, 杨毅, 张洋, 黄泓键, 仝丽娜, 任善会. 小反刍兽疫病毒强、弱毒株N蛋白串联表达及多克隆抗体制备[J]. 农业生物技术学报, 2026, 34(9): 2040-2052.
ZHU Kai-Yu, GAO Xiao-Long, CHEN Yun, DU Xin-Chao, YANG Yi, ZHANG Yang, HUANG Hong-Jian, TONG Li-Na, REN Shan-Hui. Tandem Expression of N Proteins from Virulent and Attenuated Strains of Peste des petits ruminants virus and Preparation of Polyclonal Antibodies. 农业生物技术学报, 2026, 34(9): 2040-2052.
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