非洲猪瘟病毒H3基因序列分析蛋白结构预测及其亚细胞定位

Sequence analysis,protein structure prediction and subcellular localization of African swine fever virusH3 gene

在前期研究中本实验室构建了敲除H3(Hypothetical protein 3)基因的非洲猪瘟病毒(ASFV)毒株,并进一步通过动物实验表明敲除该基因对于ASFV的毒力没有影响,推测该蛋白可能是ASFV的非结构蛋白。为进一步了解ASFVH3基因及其编码蛋白的理化性质和结构特征,从NCBI数据库中搜集到71株包含H3基因的ASFV毒株进行序列比对并绘制了H3基因的分子进化树。利用在线软件分析ASFV H3蛋白的理化性质和组成结构,通过在HEK-293T细胞中转染真核表达质粒pRK-H3-GFP确定其亚细胞定位。结果表明,H3基因在Ⅰ型和Ⅱ型ASFV毒株中具有较高的同源性。H3蛋白包含35个氨基酸,等电点为5.49,相对分子质量为8648.38,氨基酸组成中丙氨酸、苏氨酸含量分别占37.1%、34.3%;其二级结构中,α-螺旋、β-折叠、无规卷曲分别占37.14%、25.71%、25.71%。制备了该蛋白的多克隆抗体,用荧光显微镜验证ASFV H3蛋白的表达。通过核定位序列预测该蛋白定位于细胞质和细胞核中;构建H3蛋白的真核表达质粒,通过间接免疫荧光试验证实该蛋白定位于细胞质和细胞核。这为进一步揭示H3基因功能奠定了一定基础,也为后续深入探讨H3基因功能提供了理论依据。

In the previous study,our laboratory constructed the African swine fever virus(ASFV)strain which knocked down H3(Hypothetical protein 3)gene,and further confirmed by animal experiments that knocked down the gene has no effect on the virulence of ASFV,and speculated that the protein may be a non-structural protein of ASFV.In order to further understand the physical and chemical properties and structural characteristics of ASFV H3 gene and its encoded protein,71 ASFV strains containing H3 gene sequences were collected from the NCBI database for sequence alignment and then the phylogenetic tree of H3 gene was generated.The physicochemical properties and composition as well as structure of the H3 protein encoded by ASFV were analyzed by online software.The subcellular localization of H3 protein was determined by transfection of eukaryotic expression plasmid pRK-H3-GFP in HEK-293T cells.The results showed that H3 gene showed high homology in typeⅠand typeⅡASFV strains.The H3 protein contained 35 amino acids,of these,Ala and Thr in amino acid composition accounted for 37.1%and 34.3%respectively.H3 protein contains 35 amino acids with an isoelectric point of 5.49 and a relative molecular weight of 8648.38.The content of alanine and threonine accounted for 37.1%and 34.3%,respectively.In its secondary structure,α-spiral,extended strand and random coil accounted for 37.14%,25.71%,25.71%respectively.Polyclonal antibody was prepared and the expression of ASFV H3 was verified by fluorescence microscopy.The nuclear localization sequence predicted that the protein was located in cytoplasm and nucleus.The eukaryotic expression plasmid of H3 protein was constructed.Indirect immunofluorescence assay also confirmed that the protein was localized in cytoplasm and nucleus.This lays a foundation for further revealing the function of H3 gene.It provides a theoretical basis for further study on the function of H3 gene.