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

Abstract

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

CLC Number:

S852.65⁺5

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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Eukaryotic Expression and Polyclonal Antibody Preparation of N Protein of Canine Parainfluenza Virus

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