猫细小病毒VP2基因克隆与生物信息学分析

Cloning and Bioinformatics Analysis of VP2 Gene of Feline Parvovirus

为了解猫细小病毒(feline parvovirus,FPV) VP2蛋白的分子特点及变异规律,克隆FPV山东分离株SD-01的VP2基因,对其进行生物信息学分析。首先,以FPV 阳性病料基因组DNA为模板,PCR扩增VP2基因并克隆获得重组质粒pMD18-T-FPV SD-01-VP2。测序结果显示:VP2基因全长1755 bp,编码584个氨基酸;氨基酸同源性比对发现,与其他FPV分离株的VP2基因氨基酸同源性较高,可达99.0%~100%。生物信息学分析结果显示:VP2蛋白为非分泌型的亲水性蛋白,无信号肽和跨膜区域;二级结构中α螺旋、延伸链、β转角和无规则卷曲分别占8.7%、24.5%、4.3%和62.5%;三级结构预测发现,VP2蛋白以无规则卷曲为主,与二级结构预测结果一致。亚细胞定位预测显示,VP2蛋白不含有线粒体导肽(mTP)或分泌通道信号肽(SP),定位在细胞质和细胞核中的概率较高,分别为47.8%和26.1%。B细胞抗原表位预测结果显示,VP2蛋白的抗原性及表面可及性较高,共含有11个潜在的优势B细胞抗原表位。蛋白翻译后修饰预测结果显示,VP2蛋白可能含有49个潜在磷酸化修饰位点,6个N-糖基化修饰位点和21个O-糖基化修饰位点,且这些修饰位点在FPVVP2蛋白内高度保守。本研究有助于了解我国FPV流行情况,掌握其重要结构蛋白VP2的理化性质和特性,从而为下一步VP2蛋白功能研究和疫苗研发奠定了基础。

In order to study the molecular characteristics and variation rule of VP2 gene of feline parvovirus(FPV),the VP2 gene of Shandong strain,SD-01,was cloned to conduct bioinformatics analysis.The VP2 gene was amplified by PCR and cloned by taking genomic DNA of FPV positive lesions as a template to obtain the recombinant plasmid,pMD18-T-FPV SD-01-VP2.It was sequenced that the VP2 gene was 1 755 bp in length,encoding 584 amino acids;it was compared that it shared the highest amino acid homology with VP2 gene of other FPV strains,which could be up to 99.0% to 100%.It was found that,by bioinformatics analysis,VP2 protein was a non-secretory hydrophilic protein without a signal peptide and a transmembrane region.In the secondary structure,a-helix,extended chain,β-turn and irregular coil accounted for 8.7%,24.5%,4.3% and 62.5%,respectively;it was found that,by prediction of tertiary structure,VP2 protein was mainly irregular coil,which was consistent with the results obtained by prediction of secondary structure.It was shown that,by prediction of subcellular localization,neither a mitochondrial guide peptide(mTP) nor a secretion channel signal peptide(SP) was contained in the VP2 protein that was most likely located in cytoplasm and nucleus,accounting for 47.8% and 26.1%,respectively.It was shown that,by prediction of B-cell epitopes,VP2 protein had high antigenicity and surface accessibility,containing 11 potential dominant epitopes.And it was shown that,by prediction of post-translational modification,49 potential phosphorylation sites,6 N-glycosylation sites and 21 O-glycosylation sites were likely contained in VP2 protein,all of which were highly conserved in FPV VP2 protein.In a conclusion,the study was helpful to learn the prevalence status of FPV in China,to identify the physical and chemical properties and characteristics of its important structural protein VP2,and subsequently to lay a foundation for future study on functions of VP2 protein and development of vaccines.