鹅星状病毒ORF2蛋白多克隆抗体制备及鉴定

doi:10.16431/j.cnki.1671-7236.2026.01.033

Abstract

Abstract:

Objective The ORF2 protein of Goose astrovirus （GAstV） was expressed using a prokaryotic expression system， and a rabbit polyclonal antibody against this protein was prepared，to provide a material basis for the study of the pathogenic mechanism and diagnostic methods of GAstV. Method The recombinant prokaryotic expression plasmid pGEX-6P-1-ORF2 was constructed. After the recombinant plasmid was identified by PCR and double enzyme digestion， it was transformed into Escherichia coli BL21（DE3） competent cells. The protein expression was induced by IPTG and the reaction conditions were optimized to obtain the GAstV ORF2 protein. Polyclonal antibody against GAstV ORF2 were generated in New Zealand White rabbits with the purified recombinant protein GAstV ORF2. The antibody titer was determined by indirect ELISA. The immunogenicity and specificity of the polyclonal antibody were assessed using Western blotting and indirect immunofluorescence assay （IFA）. In order to clarify the tropism and localization of GAstV in the tissues of goslings， using the prepared polyclonal antibody， the distribution of the virus in the liver and kidney tissues of infected goslings was detected by immunohistochemistry （IHC） method. Result PCR and double enzyme digestion assays indicated that the recombinant prokaryotic expression plasmid pGEX-6P-1-ORF2 was successfully constructed. The GAstV ORF2 protein was successfully obtained through prokaryotic expression， and the protein concentration after purification was 1.38 mg/mL. The immunization of New Zealand White rabbits successfully generated polyclonal antibodies against GAstV ORF2 protein， with a high antibody titer reaching 1∶100 000. Western blotting analysis demonstrated specific reactivity between the polyclonal antibodies and purified GAstV ORF2 protein， as evidenced by distinct immunoreactive bands. IFA results confirmed the antibody’s specific recognition of both native GAstV and eukaryotic-expressed GAstV ORF2 protein. IHC test results showed that GAst was widely distributed in the liver and kidney of infected goslings. GAstV was specifically expressed in the cytoplasm of parenchymal cells and inflammatory cells. Conclusion This study successfully constructed the recombinant prokaryotic expression plasmid pGEX-6P-1-ORF2， and carried out in vitro induction expression to prepare rabbit polyclonal antibody against GAstV ORF2 protein. This polyclonal antibody had a high titer and strong specificity， and could be used for the detection of viral antigens in GAstV-infected goslings.

Key words: Goose astrovirus （GAstV）; ORF2 protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65^＋9.6

ZHAO Zhongqi, ZOU Mengmeng, ZHANG Yunjing, HE Lixia, WU Songshan, ZHANG Xinxin, YANG Guijun, LI Guangxing. Preparation and Identification of Polyclonal Antibody for Goose Astrovirus ORF2 Protein[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(1): 368-377.

Figures/Tables 10

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Preparation and Identification of Polyclonal Antibody for Goose Astrovirus ORF2 Protein

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