基因1型鹅星状病毒VP27蛋白的原核表达及其多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2024.06.032

Abstract

Abstract: 【Objective】 The aim of this study was to obtain a polyclonal antibody that could specifically recognize Goose astrovirus genotype 1 (GAstV-1) and did not cross-react with GAstV-2,so as to provide a material for the subsequent establishment of GAstV-1 immunoassay.【Method】 By comparing the amino acid sequences of GAstV-1 and GAstV-2 capsid proteins,the regions with large differences were found as target genes.According to VP27 gene sequence of GAstV-1 GDYJ-21-01,the gene codons were optimized and synthesized,the prokaryotic expression plasmid pCZN1-GAstV-1 VP27 was constructed and transformed into Escherichia coli Top10 competent cells to induce VP27 protein expression.The purified VP27 protein was used to immunize New Zealand White rabbits,and the antiserum was harvested to prepare GAstV-1 VP27 polyclonal antibody.The titer and purity of the purified antibody were analyzed by indirect ELISA and SDS-PAGE,and the specificity of the polyclonal antibody was evaluated by indirect immunofluorescence assay (IFA).【Result】 The recombinant plasmid pCZN1-GAstV-1 VP27 was successfully constructed.SDS-PAGE results showed that the molecular weight of the recombinant protein was about 35 ku,which mainly existed in the form of inclusion bodies.The results of indirect ELISA showed that the titer of purified GAstV-1 VP27 polyclonal antibody was 1∶9 841 500,and the purity was higher than 85%.IFA results showed that GAstV-1 VP27 polyclonal antibody could specifically recognize GAstV-1 and had no cross-reaction with GAstV-2.The detection results of clinical samples showed that the tissues with GAstV-1 nucleic acid positive exhibited specific fluorescence,while the tissues with GAstV-2 nucleic acid positive but GAstV-1 nucleic acid negative did not show fluorescence.【Conclusion】 In this study,a high titer of VP27 polyclonal antibody was obtained,which could specifically recognize GAstV-1 and had no cross-reaction with GAstV-2,laying a foundation for the establishment of antigen immunoassay for the differential diagnosis of GAstV-1.

Key words: Goose astrovirus genotype 1 (GAstV-1); VP27; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺9.6

XIANG Yong, LI Linlin, ZHANG Junqin, DONG Jiawen, HUANG Yunzhen, ZHAI Qi, XU Zhihong, LIAO Ming, SUN Minhua. Prokaryotic Expression of VP27 Protein of Goose Astrovirus Genotype 1 and Preparation and Identification of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(6): 2576-2584.

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Prokaryotic Expression of VP27 Protein of Goose Astrovirus Genotype 1 and Preparation and Identification of Its Polyclonal Antibody

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