浙江地区鹅星状病毒分离鉴定及其衣壳蛋白多克隆抗体的制备

doi:10.3969/j.issn.1004-1524.2022.10.09

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (10): 2149-2159.DOI: 10.3969/j.issn.1004-1524.2022.10.09

浙江地区鹅星状病毒分离鉴定及其衣壳蛋白多克隆抗体的制备

朱寅初¹(), 王宏宇¹^,², 云涛¹, 华炯钢¹, 叶伟成¹, 倪征¹, 陈柳¹, 张存¹^,^*()

1.浙江省农业科学院畜牧兽医研究所,浙江杭州 310021
2.南京农业大学动物医学院,江苏南京 210095

收稿日期:2021-07-26 出版日期:2022-10-25 发布日期:2022-10-26
作者简介:*张存,E-mail: cz65@foxmail.com
朱寅初(1990—),男,浙江湖州人,博士,助理研究员,主要从事畜禽疫病防控研究。E-mail: zhuyinchu@zaas.ac.cn
通讯作者: 张存
基金资助:
浙江省基础公益研究计划(LGN22C180005);浙江省重点研发计划(2019C02052)

Isolation and identification of goose astrovirus in Zhejiang Province, China, and preparation of polyclone antibodies against capside protein

ZHU Yinchu¹(), WANG Hongyu¹^,², YUN Tao¹, HUA Jionggang¹, YE Weicheng¹, NI Zheng¹, CHEN Liu¹, ZHANG Cun¹^,^*()

1. Institute of Animal Husbandry and Veterinary Sciences, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2021-07-26 Online:2022-10-25 Published:2022-10-26
Contact: ZHANG Cun

摘要/Abstract

摘要：

鹅星状病毒(GAstV)是当前鹅养殖业的重要病原,易引起雏鹅内脏痛风症状和死亡,造成巨大经济损失。于浙江省内采集30份病料,进行核酸检测、病原分离和测序,并克隆其ORF2序列至pET-28a原核表达载体,转化BL21菌株后经诱导获得衣壳蛋白,免疫新西兰大白兔制备多克隆抗体。结果显示,临床死亡雏鹅剖检均发现典型内脏痛风症状,核酸检测鉴定为鹅星状病毒阳性,且出现不同基因型鹅星状病毒混合感染情况。共分离得到ZJC14和ZJLD20两个毒株,其中,ZJLD20在鹅胚和LMH细胞中均稳定增殖,但ZJC14并不能适应LMH细胞。病毒基因组测序显示,ZJC14与ZJLD20亲缘关系较远,ZJC14属于GAstV-Ⅰ基因型,而ZJLD20为GAstV-Ⅱ基因型。重组表达载体pET28a-ORF2诱导后获得纯化目的蛋白,经免疫成功制备兔源多克隆抗体,该抗体可与目的蛋白结合。此外,间接免疫荧光和Western-blot试验结果显示,ZJLD20衣壳蛋白制备的多克隆抗体可与病毒结合反应。研究成果有利于后续对该病原致病能力的研究,同时,试验制备的ORF2衣壳蛋白与多克隆抗体为鹅源星状病毒感染的诊断试剂开发奠定了基础。

关键词: 鹅星状病毒, 分离鉴定, 衣壳蛋白, 原核表达, 多克隆抗体

Abstract:

Goose astrovirus (GAstV) is an important pathogen in gosling, which causes viscera gout and death and leads to huge economic loss in the gosling-breeding industry in China. In the present study, a total of 30 gosling samples were collected from Zhejiang Province, China, and were identified by RT-PCR and pathogen isolation and sequencing. The prokaryotic expression plasmid pET-28a-ORF2 was constructed and transformed into BL21 strain for expression of the recombinant protein with induction. The results showed that all samples had typical viscera gout symptoms and were positive for goose astrovirus. Some samples were infected by two different genotypes of goose astrovirus. Strains ZJC14 and ZJLD20 were isolated from goose embryos. ZJLD20 could stably proliferate in both goose embryos and LMH cells, but ZJC14 could not adapt to LMH cell. ZJC14 and ZJLD20 were relatively distantly related. ZJC14 belonged to the genotype of GAstV-Ⅰ, yet ZJLD20 belonged to the genotype of GAstV-Ⅱ. The recombinant protein was purified and used to immunize rabbit for preparation of polyclonal antibodies. The prepared polyclonal antibodies could effectively bind to the protein. In addition, indirect immunofluorescence and Western-blot test results showed that the polyclonal antibody from the ZJLD20 capsid protein could effectively recognize the virus replicated in LMH. These findings would benefit the further study of the pathogenicity of GAstV. Meanwhile, the prepared capsid protein and polyclonal antibody would lay foundation for the development of diagnostic reagents for goose astrovirus infection.

Key words: goose astrovirus, isolation and identification, capside protein, prokaryotic expression, polyclonal antibody

中图分类号:

S858.33

朱寅初, 王宏宇, 云涛, 华炯钢, 叶伟成, 倪征, 陈柳, 张存. 浙江地区鹅星状病毒分离鉴定及其衣壳蛋白多克隆抗体的制备[J]. 浙江农业学报, 2022, 34(10): 2149-2159.

ZHU Yinchu, WANG Hongyu, YUN Tao, HUA Jionggang, YE Weicheng, NI Zheng, CHEN Liu, ZHANG Cun. Isolation and identification of goose astrovirus in Zhejiang Province, China, and preparation of polyclone antibodies against capside protein[J]. Acta Agriculturae Zhejiangensis, 2022, 34(10): 2149-2159.

图/表 8

表1 用于试验的引物基本信息

Table 1 Basic information of primers used in study

目的基因 Target gene	片断大小 Fragment length/bp	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
TMUV-E	401	GCCACGGAATTAGCGGTTGT	TAATCCTCCATCTCAGCGGTGTAG
GPV-VP1	779	AGACTTATCAACAACCATTG	TCACTTATTCCTGCTGTAG
GRV-sigma C	380	TGAGACGCCTGACTACGATT	ATGCTTGGAGTGAGACGACT
GAstVⅠ-RbRp	441	GATACTTATGCTTCCACAAC	ATCTTGTTCAAAAGATGCTC
GAstVⅡ-RbRp	277	ACGACAGATGCGTTACTT	GGTGACATTATCCCTGAG
ZJLD20-Capsid	2 112	AGCAAATGGGTCGCGGATCCATGGCAGACAGGGCGGTGGC	CGGAGCTCGAATTCGGATCCCTCATGTCCGCCCTTCTCAA
ZJC14-Spike	834	AGCAAATGGGTCGCGGATCCGTAGTTCATTACCTGCCACT	CGGAGCTCGAATTCGGATCCTTGTCCACCCTTTTCAAAGC

图1 发病雏鹅的剖检结果与双重PCR检测结果 A,心、肝表面尿酸盐沉积包裹;B,肾红肿;C,胆囊内尿酸盐积聚;D,部分临床样品双重RT-PCR检测结果。M,DNA marker;N,阴性对照。

Fig.1 Results of necropsy and double PCR for diseased goslings A, Urate deposition on the surfaces of the heart and liver; B, Severe nephritis with hemorrhage and swelling; C, Urate crystals accumulated in the gallbladder; D. Results of double RT-PCR for some clinical samples. M, DNA marker; N, Negative control.

图2 鹅胚和LMH细胞病毒分离结果

Fig.2 Isolation of GAstV from goose embryo and LMH cells

图3 基于RT-PCR的鹅胚和LMH中鹅星状病毒检测结果 M,DNA marker;1,ZJLD20鹅胚检测结果;2,ZJC14鹅胚检测结果;3, ZJLD20 LMH检测结果;4,ZJC14 LMH检测结果。N,阴性对照。

Fig.3 RT-PCR results of GAstV replicated in goose embryo and LMH cells M, DNA marker; 1, PCR result of ZJLD20 in goose embryo; 2, PCR result of ZJC14 in goose embryo; 3. PCR result of ZJLD20 in LMH cell; 4, PCR result of ZJC14 in LMH cell. N, Negative control.

图4 鹅星状病毒核酸序列的遗传进化分析

Fig.4 Phylogenic analysis of goose astrovirus based on nucleotide sequences

图5 鹅星状病毒ORF2氨基酸序列的遗传进化分析

Fig.5 Phylogenetic analysis based on amino acid sequences of ORF2

图6 鹅星状病毒ORF2的原核表达载体构建与蛋白表达 A,PCR检测重组载体;B,SDS-PAGE检测蛋白;C,Western-blot检测多克隆抗体。M,Marker;N,阴性对照。

Fig.6 Construction of ORF2 recombinant plasmid and protein expression A, PCR detection for recombinant vectors; B, SDS-PAGE detection for protein expression; C, Western-blot detection for polyclonal antibody. M, DNA marker; N, Negative control.

图7 基于Western blot和IFA的ORF2多克隆抗体与鹅星状病毒GAstV ZJLD20的免疫反应结果 Mock,空白对照。

Fig.7 Western blot and IFA identification result of ORF2 polyclonal antibody with GAstV ZJLD20 Mock, Blank control.

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浙江地区鹅星状病毒分离鉴定及其衣壳蛋白多克隆抗体的制备

Isolation and identification of goose astrovirus in Zhejiang Province, China, and preparation of polyclone antibodies against capside protein

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