猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.04.023

Abstract

Abstract: 【Objective】 The aim of this study was to prokaryotically express VP2 protein of Luoyang Feline panleukopenia virus (FPV) isolates and prepare polyclonal antibodies from mice.【Method】 A pair of specific primers were designed according to the published FPV VP2 gene sequence (GenBank accession No.:EU018143.1),DNA was extracted from the feces of 7 cats suspected of FPV infection in Luoyang area,and VP2 gene was identified by PCR and sequenced.The genetic evolution of VP2 gene was analyzed by Mega 11.0 software.The online software was used to predict the structure characteristics of VP2 protein and find the dominant epitopes.The VP2 dominant antigen (sVP2) was cloned into pET-32a(+) vector,the recombinant plasmid pET-32a-sVP2 was constructed and transformed into E.coli Rosetta (DE3) competent cell for induction and optimal expression.The recombinant protein was identified by SDS-PAGE and Western blotting.BABL/c mice were immunized with pET-32a-sVP2 purified by nickel column affinity chromatography to prepare polyclonal antibody,and the titer of the antibody was detected by ELISA method.【Result】 A FPV strain was isolated and named LY-1.Genetic evolution analysis showed that this strain belonged to FPV-G2 subgroup,which was the closest relative to Beijing strain (GenBank accession No.:MT270581).Bioinformatics analysis showed that VP2 was a stable intramembrane protein with no signal peptide and transmembrane region,and there were abundant B cell epitopes (1-800 bp,sVP2) at the N-terminus.The recombinant plasmid pET-32a-sVP2 was successfully constructed and expressed.SDS-PAGE showed that the molecular mass of the recombinant protein was about 50 ku,mainly in the form of inclusion bodies,the optimal protein expression was induced by 1 mmol/L IPTG at 30 ℃ for 6 h;Western blotting results showed that the recombinant protein could react specifically with mouse anti-His monoclonal antibody.ELISA results showed that the antibody titer of the recombinant protein was 1∶128 000.The antibody could recognize the specific reaction between sVP2 and prokaryotic expression of VP2 protein.【Conclusion】 FPV sVP2 fragment was cloned and polyclonal antibody was prepared,which provided a reference for further study of the role of VP2 in FPV replication and pathogenesis and establishing a diagnostic method for FPV.

Key words: Feline panleukopenia virus (FPV); Luoyang isolate; VP2 gene; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺9.2

WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.

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Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation

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