猫传染性腹膜炎病毒N蛋白在昆虫细胞-杆状病毒系统中的表达与鉴定

doi:10.16431/j.cnki.1671-7236.2021.01.030

Abstract

Abstract: The aim of this study was to express Feline infectious peritonitis virus (FIPV) N protein by insect cell-baculovirus expression system,and prepare polyclonal antibody against FIPV for clinical antigen/antibody diagnosis and N protein function research.According to the N gene sequence of FIPV in GenBank,the N gene of FIPV strain (accession number:KC461235.1) was selected,and the N gene was optimized by codon optimization and gene synthesis.After enzyme digestion,the N gene was connected to pFastBac1 vector and transformed into E.coli DH5α competent cells.The positive clone was screened by ampicillin resistance.After being identified by restriction enzyme digestion and sequencing,the extracted plasmid was transformed into E.coli DH10Bac competent cells.The recombinant Bacmid-N was successfully constructed after blue and white spot screening and PCR identification.Sf9 cells were transfected with baculovirus and cultured at 28 ℃ for 4 days.The supernatant of Sf9 cells was collected and purified by Ni ion affinity chromatography.The results of SDS-PAGE and Western blotting showed that the recombinant N protein (51 ku) of FIPV was successfully expressed in insect cell-baculovirus expression system.Six weeks old BALB/c mice were immunized with the protein and Freund's adjuvant.The titer of serum antibody was 1:102 400 detected by indirect ELISA.Western blotting and indirect immunofluorescence assay were used to detect and analyze the polyclonal antibody against N protein.The results showed that the recombinant protein expressed in eukaryotic cells had good immunogenicity and the polyclonal antibody had good reactivity,which could specifically identify FIPV infected cells.This study laid a foundation for the development of FIPV antigen/antibody diagnostic kit.

Key words: Feline infectious peritonitis virus (FIPV); nucleocapsid protein; insect cell-baculovirus expression system

CLC Number:

S852.65

LI Shuangxing, LIU Yebing, LIU Fangyuan, JI Jing, DU Jige, LI Qianlin, ZHU Zhen, LI Qihong, CHEN Xiaoyun, YIN Chunsheng. Expression and Identification of Feline Infectious Peritonitis Virus N Protein in Insect Cell-Baculovirus Expression System[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 273-279.

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Expression and Identification of Feline Infectious Peritonitis Virus N Protein in Insect Cell-Baculovirus Expression System

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