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

摘要/Abstract

摘要：

目的采用原核表达系统表达鹅星状病毒（Goose astrovirus，GAstV）ORF2蛋白，并制备其兔抗多克隆抗体，为该病毒致病机制和诊断方法研究提供物质基础。方法构建重组原核表达质粒pGEX-6P-1-ORF2，并通过PCR和双酶切对重组质粒鉴定后，将其转化大肠杆菌BL21（DE3）感受态细胞，通过IPTG诱导蛋白表达并优化反应条件，获得GAstV ORF2蛋白。利用纯化的GAstV ORF2蛋白免疫新西兰大白兔，制备兔抗GAstV ORF2多克隆抗体。采用间接ELISA法测定多克隆抗体效价，通过Western blotting和间接免疫荧光（IFA）法检测多克隆抗体的免疫原性和特异性；为了明确GAstV在雏鹅组织中的嗜性和定位，利用制备的多克隆抗体，通过免疫组织化学（IHC）法检测感染雏鹅肝脏和肾脏组织中的病毒分布情况。结果 PCR和双酶切鉴定表明成功构建重组原核表达质粒pGEX-6P-1-ORF2，通过原核表达成功获得GAstV ORF2蛋白，纯化后蛋白浓度为1.38 mg/mL。免疫新西兰大白兔后获得兔抗GAstV ORF2蛋白多克隆抗体，其效价达1∶100 000。Western blotting结果显示，多克隆抗体与纯化后GAstV ORF2蛋白出现特异性反应条带；IFA结果显示，多克隆抗体与GAstV及真核表达GAstV ORF2蛋白产生特异性反应；IHC检测结果显示，GAstV在感染雏鹅的肝脏与肾脏广泛分布，GAstV特异性表达于实质细胞与炎性细胞的胞浆内。结论本研究成功构建重组原核表达质粒pGEX-6P-1-ORF2，并进行体外诱导表达制备了兔抗GAstV ORF2蛋白多克隆抗体，该多克隆抗体效价高、特异性强，可用于GAstV感染雏鹅体内病毒抗原检测。

关键词: 鹅星状病毒（GAstV）; ORF2蛋白; 原核表达; 多克隆抗体

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

中图分类号:

S852.65^＋9.6

赵忠麒, 邹蒙蒙, 张芸菁, 何丽霞, 武松山, 张欣欣, 杨桂君, 李广兴. 鹅星状病毒ORF2蛋白多克隆抗体制备及鉴定[J]. 中国畜牧兽医, 2026, 53(1): 368-377.

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.

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