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

作　　者：朱寅初 王宏宇 云涛[1] 华炯钢[1] 叶伟成[1] 倪征[1] 陈柳[1] 张存[1] ZHU Yinchu;WANG Hongyu;YUN Tao;HUA Jionggang;YE Weicheng;NI Zheng;CHEN Liu;ZHANG Cun(Institute of Animal Husbandry and Veterinary Sciences,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[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

出　　处：《浙江农业学报》2022年第10期2149-2159,共11页Acta Agriculturae Zhejiangensis

基　　金：浙江省基础公益研究计划(LGN22C180005);浙江省重点研发计划(2019C02052)。

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。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.

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 

分 类 号：S858.33[农业科学—临床兽医学]