犬副流感病毒N蛋白的真核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.05.032

摘要/Abstract

摘要： 【目的】通过真核表达系统表达犬副流感病毒(Canine parainfluenza virus，CPIV)N蛋白，并制备N蛋白多克隆抗体，为后续CPIV N蛋白的鉴定及病毒在生命周期过程中的功能研究提供基础。【方法】构建CPIV N蛋白真核表达质粒pCAGGS-N，经PCR和Western blotting验证质粒功能后，免疫小鼠，制备CPIV N蛋白多克隆抗体；ELISA法测定抗体效价达到理想效价后，终止免疫；通过Western blotting、间接免疫荧光试验对抗体进行鉴定和分析，并测定多克隆抗体的中和活性。【结果】酶切连接、测序验证结果显示，成功构建了CPIV N蛋白真核表达重组质粒pCAGGS-N，且该质粒可在真核细胞中正常转录和翻译；通过DNA免疫成功制备了小鼠抗CPIV N蛋白多克隆抗体，效价为1∶128 000。Western blotting结果显示，该抗体可与纯化的CPIV病毒粒子及外源性表达的CPIV N蛋白结合，分子质量约为59 ku。间接免疫荧光试验结果显示，制备的多克隆抗体可特异性识别并结合具有空间构象的CPIV N蛋白，但该多克隆抗体不具有病毒中和活性。【结论】本试验成功构建了真核表达质粒pCAGGS-N，并通过DNA免疫方法成功制备了小鼠抗CPIV N蛋白的多克隆抗体，该抗体具有良好的反应性和特异性，但未显示出中和病毒的活性，为进一步研究CPIV N基因的功能及其他细胞免疫学试验提供了研究基础。

关键词: 犬副流感病毒(CPIV); 核衣壳蛋白; 真核表达; DNA免疫; 多克隆抗体

Abstract: 【Objective】 This study aimed to express protein N of Canine parainfluenza virus (CPIV) through the eukaryotic expression system,and prepare polyclonal antibodies against N protein,so as to provide the basis for the subsequent identification of CPIV N protein and the study of the function of the virus during its life cycle.【Method】 pCAGGS-N was constructed,after the function of the plasmid was verified by PCR and Western blotting,mice were immunized to prepare polyclonal antibody against CPIV N protein.The immunity was terminated when the antibody titer reached the ideal titer detected by ELISA.The antibodies were identified and analyzed by Western blotting and indirect immunofluorescence assay,and the neutralizing activity of the polyclonal antibodies was measured.【Result】 pCAGGS-N was successfully constructed and validated through restriction enzyme digestion and sequencing,and the recombinant plasmid could be transcribed and translated normally in eukaryotic cells.Mouse polyclonal antibody against CPIV N protein was successfully prepared by DNA immunization with a titer of 1∶128 000.Moreover,Western blotting analysis showed that the antibody could bind to purified virions and exogenous expression of CPIV N protein,with a molecular mass of about 59 ku.Indirect immunofluorescence assay result showed that the polyclonal antibody could specifically recognize and bind N protein with spatial conformation,but the polyclonal antibody did not have virus-neutralizing activity.【Conclusion】 In this experiment,the eukaryotic expression plasmid pCAGGS-N was successfully constructed,and the mouse polyclonal antibody against CPIV N protein was successfully prepared by DNA immunization.The antibody showed good reactivity and specificity,but did not show the activity of neutralizing the virus.This results provided a basis for further study of the function of CPIV N gene and other cellular immunological experiments.

Key words: Canine parainfluenza virus (CPIV); nucleocapsid protein; eukaryotic expression; DNA immunity; polyclonal antibody

中图分类号:

S852.65⁺5

刘敏, 程淮, 郑珍珍, 张梦思, 张贺伟, 任静强. 犬副流感病毒N蛋白的真核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2025, 52(5): 2287-2294.

LIU Min, CHENG Huai, ZHENG Zhenzhen, ZHANG Mengsi, ZHANG Hewei, REN Jingqiang. Eukaryotic Expression and Polyclonal Antibody Preparation of N Protein of Canine Parainfluenza Virus[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2287-2294.

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