猫泛白细胞减少症病毒北京株NS1基因克隆与生物信息学分析

Cloning and Bioinformatic Analysis of NS1 Gene of Feline Parvovirus in Beijing

为分析猫泛白细胞减少症病毒(FPV)北京株(FPV-BJ 05株)NS1基因的分子特征及其编码蛋白的生物学功能,本研究对FPV-BJ 05株NS1基因进行PCR扩增、克隆及序列测定。应用生物信息学软件分析FPV-BJ 05株与GenBank上登录的11株FPV参考株NS1基因的同源性,并预测NS1蛋白理化性质、信号肽、跨膜结构、B细胞抗原表位、磷酸化位点、亚细胞定位及蛋白结构与功能、高级结构等。结果显示,NS1基因全长2 007bp,编码668个氨基酸,且与其他FPV分离株NS1基因核苷酸序列同源性为98.8%～99.3%,氨基酸序列同源性为97.9%～98.8%。系统进化树分析结果显示,FPV-BJ 05株NS1蛋白与GenBank上登录的3株FPV参考株处于同一大分支,但由于氨基酸的突变导致其属于独立的一小分支。NS1蛋白既无信号肽也无跨膜结构域,属于非分泌型的疏水性蛋白。B细胞抗原表位预测结果显示,NS1蛋白柔韧性较好,抗原性及表面可及性较高,预测该蛋白含有13个优势抗原位点。修饰结构预测表明,NS1蛋白含有62个潜在磷酸化位点,27个O-糖基化位点和2个N-糖基化位点。亚细胞定位结果显示,NS1蛋白在细胞质和细胞核的概率较高,分别为69.6%和17.4%。NS1蛋白二级结构中α-螺旋、延伸链、无规则卷曲和β-转角分别占39.37%、15.42%、39.37%和5.84%。应用在线软件SWISS-Modle对NS1蛋白进行建模,预测其三级结构,结果发现NS1蛋白主要以α-螺旋为主。本试验结果将为北京地区FPV免疫诊断及核酸疫苗研究提供理论依据。

In order to study the molecular characteristics and biological function of NS1 gene of feline parvovirus(FPV),the NS1 gene of FPV-BJ 05 strain was amplified,cloned and sequenced.Nucleotide and amino acid homology analysis,signal peptide,transmembrane domain,B-cell preponderant epitope,phosphorylation site subcellular localization and protein structure and fuction,advanced structure of NS1 gene were predicted using bioinformatic softwares.The results showedthat the length of NS1 gene was 2 007 bp,encoding 668 amino acids.The NS1 gene of FPV-BJ 05 shared 98.8%to 99.3% nucleotide homology with that of the other FPV strains,and the amino acid homology was 97.9%to 98.8%.Phylogenetic tree analysis showed that the NS1 protein of FPV-BJ 05 strain was classified as the same major branch with 3 FPV reference strains registered in GenBank,but it was a small independent branch due to amino acid mutation.The NS1 protein had neither a signal peptide nor a transmembrane domain,which was a non-secretory hydrophobic protein.The prediction results of B cell antigen parameters showed that NS1 protein had good flexibility,high antigenicity and surface accessibility,and it was predicted that the protein contained 13 dominant antigenic sites.62 phosphorylation sites,27 O-glycosylation sites and 2 N-glycosylation sites were found in NS1 protein.The result of subcellular localization showed that the probability of NS1 protein in the cytoplasm and nucleus was higher,which were 69.6% and17.4%respectively.The prediction of secondary structure revealed thatα-helix(39.37%),extended chain(15.42%),random coil(39.37%)andβ-turn(5.84%),and the tertiary structure of NS1 protein was curved spiral structure.This study provided theoretical basis for immunologic diagnosis and further research of nucleic acid vaccine of FPV in Beijing.