兔轮状病毒VP8*蛋白原核表达、基因序列及蛋白特性分析  

Analysis on the sequence,protein characteristic and prokaryotic expression of the truncated VP8* gene of rabbit rotavirus

作　　者：赵巧雅 田野 鞠艳[2] 姜亦飞[2] 黄涛[3] 陈秋生[1] ZHAO Qiaoya;TIAN Ye;JU Yan;JIANG Yifei;HUANG Tao;CHEN Qiusheng(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Institute of Poultry Sciences,Shandong Academy of Agricultural Sciences,Jinan 250023,China;College of Veterinary Medicine,Southwest University,Chongqing 402460,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]山东省农业科学院家禽研究所,山东济南250023 [3]西南大学动物医学院,重庆402460

出　　处：《畜牧与兽医》2023年第7期100-107,共8页Animal Husbandry & Veterinary Medicine

基　　金：山东省现代农业产业技术体系资助项目(SDAIT-21-06);山东省重点研发计划(2022CXGC010606)。

摘　　要：为深入研究兔轮状病毒VP8*蛋白的结构和功能,本研究利用生物信息学软件DNAStar对兔轮状病毒Z3171株的VP8*基因序列进行核苷酸和氨基酸同源性分析,运用生物信息学方法对该蛋白的理化性质、亲/疏水性、磷酸化及糖基化位点、结构域、中和表位及三维结构等进行了分析。设计合成引物,采用PCR方法扩增VP8*蛋白编码基因,并将目的片段克隆到pET28a(+)原核表达载体,经体外原核表达,采用亲和层析纯化获得VP8*目的蛋白。结果显示:Z3171株的VP8*基因与国内外参考序列对比,核苷酸序列同源性为32.8%~90.3%,氨基酸序列同源性为39.2%~91.9%。生物信息学分析显示该蛋白理化性质稳定,无跨膜结构域,不含有信号肽,主要定位于细胞骨架中。其中1个中和表位区域内有2个氨基酸插入,该区域三维构象亦发生变化。PCR扩增的VP8*基因条带大小为558 bp,经测序与目的基因完全一致;经SDS-PAGE和Western blot鉴定,重组VP8*蛋白以可溶形式表达,血凝性试验结果表明该蛋白无血凝性。本研究为VP8*免疫原性分析及研制兔轮状病毒疫苗提供了参考。In order to further study the structure and function of the rabbit rotavirus VP8*protein and to develop a rabbit rotavirus vac-cine,the nucleotide and amino acid homology of the VP8*gene sequence of a rabbit rotavirus strain Z3171 was analyzed using the bioinfor-matics software DNAStar.Online bioinformatics analysis software was used to analyze the physical and chemical properties,hydrophilicity/hy-drophobicity,phosphorylation site and glycosylation site,signal peptide,domain and other bioinformatics of the protein.Primers were de-signed and synthesized.The VP8*protein coding gene was amplified by PCR.The target fragments were cloned onto the pET28a(+)pro-karyotic expression vector,and were double digested by restriction endonuclease.After prokaryotic expression in vitro,the VP8*protein was purified by affinity chromatography.The sequencing results showed that the homology of the nucleotide sequence was 32.8%to 90.3%,and that of the amino acid sequence was 39.2%to 91.9%.The biological information analysis showed that the protein was stable in physical and chemical properties.It was a hydrophilic protein with no transmembrane domain or signal peptide.It was mainly located in the cytoskeleton.The results showed that the size of the VP8*gene band amplified by PCR was 558 bp long.The gene was expressed in E.coli.In this study,the expression vector was constructed to achieve the stable expression of the VP8*protein in the prokaryotic system,which provided a refer-ence for immunogen analysis of the protein and for development of rabbit rotavirus vaccines.

关 键 词：兔轮状病毒 VP8* 蛋白特性 原核表达 

分 类 号：S855.3[农业科学—临床兽医学]