新型鸭细小病毒NS1基因的原核表达及生物信息学分析

发布时间：2018-04-09 03:27

本文选题：新型鸭细小病毒　切入点：克隆　出处：《中国动物传染病学报》2017年02期

【摘要】：为研究新型鸭细小病毒(Novel duck parvovirus,NDPV)NS1蛋白的生物学功能,根据NDPV DS15株NS1基因(Gen Bank登录号:KU947420)设计1对特异性引物,对NS1全基因进行PCR扩增,将PCR扩增产物克隆入pGEX-4T-1载体,构建重组质粒pGEX-4T-NS1,并进行测序鉴定。经鉴定正确后重组质粒转化到BL21感受态细胞,SDS-PAGE和Western blot进行表达鉴定。将测序获得NS1基因编码蛋白进行生物信息学分析。结果显示,本研究构建的重组质粒可在原核细胞中成功表达97kDa的NS1融合蛋白。进一步生物信息学分析结果显示,NS1蛋白为酸性、可溶性蛋白,由627个氨基酸组成,分子质量约为72kDa。该蛋白主要位于胞浆,不含信号肽序列,无跨膜区,含有丝/苏氨酸激酶底物模序和1个潜在磷酸化位点(s546),抗原表位主要集中在多肽的C端。NS1基因的同源性及系统发生树分析表明,NDPV与鹅细小病毒(Goose parvovirus,GPV)处于同一进化分支,遗传进化关系最近。本研究结果为揭示NDPV NS1在病毒的复制、增殖以及致病机制中的作用奠定了基础。
[Abstract]:In order to study the biological function of novel duck parvovirusNDPV-NS1 protein of duck parvovirus, a pair of specific primers were designed according to the accession number of NS1 gene Genin Bank of NDPV DS15 strain: KU947420). The whole NS1 gene was amplified by PCR, and the PCR amplification product was cloned into pGEX-4T-1 vector.The recombinant plasmid pGEX-4 T-NS1 was constructed and sequenced.After identification, the recombinant plasmid was transformed into BL21 competent cells and expressed by SDS-PAGE and Western blot.The NS1 gene encoding protein was sequenced for bioinformatics analysis.The results showed that the recombinant plasmid could successfully express the NS1 fusion protein of 97kDa in prokaryotic cells.Further bioinformatics analysis showed that the NS1 protein was acidic and soluble, consisting of 627 amino acids with a molecular weight of about 72 kDa.The protein is mainly located in the cytoplasm, does not contain signal peptide sequence, and has no transmembrane region.The sequence of silk / threonine kinase substrates and a potential phosphorylation site (S546) were found. The homology of NS1 gene and phylogenetic tree analysis showed that NDPV and Goose parvovirusGPVs were in the same evolutionary branch as goose parvovirus (Goose parvovirus).Genetic and evolutionary relationships are recent.The results laid a foundation for revealing the role of NDPV NS1 in the replication, proliferation and pathogenesis of the virus.
【作者单位】：中国农业科学院上海兽医研究所;青岛农业大学动物科技学院;
【基金】：国家自然科学青年基金(31502068) 上海市科委科技创新医药与农业领域项目(13391901602)
【分类号】：S852.65

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