鹅源星状病毒ORF2基因原核表达及遗传进化分析  

Prokaryotic expression and phylogenetic analysis of ORF2 gene of goose astrovirus

作　　者：蒲路莎 苏世博 陈肖韩 赵丽丽[2] 陈洪岩[2] PU Lusha;SU Shibo;CHEN Xiaohan;ZHAO Lili;CHEN Hongyan(College of Animal Science, Heilongjiang Bayi Agricultural University, Daqing 163000, China;State Key Laboratory of Veterinary Biotechnology, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, Harbin Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Harbin 150069, China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163000 [2]中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室,黑龙江省实验动物与比较医学重点实验室,黑龙江哈尔滨150069

出　　处：《浙江农业学报》2020年第5期789-797,共9页Acta Agriculturae Zhejiangensis

基　　金：黑龙江省青年基金(QC2018033);国家十三五重点研发计划(2016YFD0500800);黑龙江省自然科学基金重点项目(ZD201606);中央级公益性科研院所基本科研业务费专项(1610302017013)。

摘　　要：应用RT-PCR方法扩增鹅源星状病毒(goose astrovirus,GoAstV)HLJ01株的ORF2基因,将扩增产物克隆至pET32a原核表达载体,并在大肠埃希菌BL21进行表达。通过SDS-PAGE分析表明,重组菌诱导后得到大小约为106 ku的ORF2蛋白,与理论值相符。将诱导表达的蛋白经镍亲和层析柱纯化后免疫小鼠制备多克隆抗体,并测定其抗体效价。Western-blotting结果显示,ORF2蛋白可被鼠抗GoAstV阳性血清识别。序列分析表明,该毒株与其他鹅源星状病毒参考毒株的核苷酸相似性介于37.8%~99.3%之间,HLJ-1801分离株与已发表的8株鹅源星状病毒毒株位于同一分支,表明其属于一种新型星状病毒成员。利用原核表达系统成功高效地表达了GoAstV ORF2蛋白,融合蛋白以包涵体形式存在。本试验成功获得了纯化的ORF2重组蛋白和小鼠抗GoAstV多克隆抗体,为进一步研究ORF2蛋白对星状病毒感染的诊断试剂的研发奠定了基础。The goose astrovirus(GoAstV)ORF2 gene was amplified by RT-PCR,and the amplified product was cloned into the prokaryotic expression vector pET32a expressed in E.coli BL21.SDS-PAGE analysis showed that the ORF2 protein with the size of 106 ku was obtained after the induction of recombinant bacteria,which was consistent with the theoretical value.The polyclonal antibody was prepared by immunizing mice with the induced protein that was purified by Ni column affinity chromatography and the antibody titer was determined.Western blotting results showed that ORF2 protein could be recognized by mouse anti GoAstv positive serum.Sequence analysis showed that the nucleotide similarity between the virus and other astrovirus of goose origin were between 37.8%and 99.3%.The HLJ-1801 isolate was located in the same branch with the eight published strains of astrovirus of goose origin,indicating that it belonged to a new type of astrovirus.In this study,the GoAstV ORF2 protein was successfully and efficiently expressed using a prokaryotic expression system,and the fusion protein existed as an inclusion body.The purified ORF2 recombinant protein and mouse anti goastv polyclonal antibody were successfully obtained,which laid a foundation for further research on the diagnostic reagent of ORF2 protein for astrovirus infection.

关 键 词：鹅星状病毒 ORF2 原核表达 遗传进化分析 

分 类 号：S855.3[农业科学—临床兽医学]