1株鹅星状病毒分子进化分析及纤突蛋白多克隆抗体的制备

Molecularevolution analysis of a goose astrovirus and preparation of polyclonal antibody against spike protein

以2018年分离鉴定的1株鹅星状病毒为研究对象(LHC株),采用分子克隆方法获得病毒衣壳蛋白基因序列,通过生物信息学方法分析分离株分子进化情况与纤突蛋白变异情况,构建纤突蛋白原核表达质粒对纤突蛋白进行原核表达,免疫新西兰白兔制备兔抗纤突蛋白多克隆抗体,以原核表达的衣壳蛋白验证其反应活性。结果显示,LHC株为鹅源星状病毒2型毒株,第425~665位氨基酸残基为衣壳蛋白纤突结构,2014—2018年不同地区分离到的鹅星状病毒2型毒株的纤突蛋白享有很高的序列及结构相似性,表明2014—2018年中国不同地区间的鹅星状病毒2型毒株尚未发生明显变异。制备的兔抗纤突蛋白血清可与纤突蛋白及衣壳蛋白特异性结合,表明重组纤突蛋白具有良好的免疫原性,制备的兔抗纤突蛋白多克隆抗体具有良好的反应活性。为鹅星状病毒纤突蛋白功能研究、血清学检测方法的建立以及亚单位疫苗的制备奠定理论基础。

A strain of goose astrovirus(named LHC strain)isolated and identified in 2018 was used to obtain the gene sequence of viral capsid protein.Besides,bioinformatics methods were applied to analyze the molecular evolution of the isolates and the mutation of the spike protein.In order to express the spike protein,the prokaryotic expression plasmid of the spike was constructed.New Zealand white rabbits were immunized for the preparation of rabbit anti-spike antibodies and the reactivity of the antibody was verified with the viral capsid protein.The results showed that the LHC was classified as a goose astrovirus type 2 strain,its amino acid residues at positions 425-665 encoding the spike of the capsid protein.In addition,the spike of GoAstV-2 strains isolated from different regions from 2014 to 2018 share high sequence and structural similarity,indicating that there was no significant mutation occurring to the GoAstV-2 strains across different parts of China.The rabbit anti-spike serum could bind specifically to the spike and the capsid protein,indicating that the recombinant spike had high immunogenicity and the rabbit anti-spike polyclonal antibody had strong reactivity,which lays a foundation for the study on the function performed by the goose astrovirus spike protein,the development of serological detection methods and the preparation of subunit vaccines.