Abstract:

【Objective】 The purpose of this paper is to grasp the genetic variation patterns of the prevalent strains of bovine coronavirus (BCoV) in Xinjiang, and analyze the characteristics of structural and functional of spike (S) protein.Providing data support for the genetic evolution, structure and function research, vaccine research and development, and targeted drug design of S gene of BCoV.【Methods】 The viral genomic RNA was extracted from the feces of BCoV positive calves in a cattle farm in Bachu County, Xinjiang, and the S gene was amplified by RT-PCR.The full length of the S gene was obtained through sequencing and splicing, and after that, the bioinformatics analysis and genetic evolution analysis were carried out.【Results】 The BCoV S gene nucleotide sequence (Published on GenBank.Accession number: OR136878.1) was successfully obtained, with a total length of 4 092 nucleotides (nt).The S gene was encoded by 1 363 amino acids (aa).The molecular weight of S protein was 150.67 Ku, and its isoelectric point was 5.29.It had a transmembrane helical region with weak hydrophilicity and slightly stronger hydrophobicity.It has a transmembrane spiral region, with weak hydrophilicity and slightly strong hydrophobicity.S protein was mainly located in the endoplasmic reticulum membrane and golgi apparatus of the host cell, and the probability of signal peptide in N-terminal was 0.9842.The S protein contained 14 potential N-glycosylation sites and 132 phosphorylation sites, and had 3 structural domains, among which the receptor binding domain (RBD) had a total of 215 aa.The proportion of random curls in RBD was the highest (62.79%), followed by extended chains (20.47%) and α-helices (12.56%), the lowest proportion comprised β-turns (4.19%).15 dominant B cell epitopes and 11 T cell epitopes were picked out by software ABCpred and SYFPEITHI.The S protein could be homologously modeled with the template (MTL ID: 7sbw.1.C) in the SWISS-MODEL database, with a sequence identity of 92.00%.The GMQE score of the model was 0.76, and the QMEAND score was 0.82, which indicated a high coincidence rate between them.The Ramachandran favored value of the ramachandran plot is 96.14%, indicating that the spatial conformation was reasonable and the model was accurate and reliable.Evolutionary analysis showed that the BCoV S gene amplified in this experiment was located in the same branch as BCV-AKS-01 strain in southern Xinjiang of China in 2016, and the nucleotide sequence identity of those S gene was 99.07%.【Conclusion】 The S protein of BCoV has multiple antigenic epitopes and immunogenicity.RBD plays an important role in the entry of viruses into host cells, and vaccine targets can be designed based on the RBD sequence to prevent the virus from binding to host receptors.The results of homologous modeling of the S protein of BCoV are accurate and reliable.In this study, the physicochemical properties, signal peptides and subcellular localization, phosphorylation sites, glycosylation sites, domains, antigenic epitopes, tertiary structures, and RBD secondary structures of the S protein of BCoV are analyzed using bioinformatics methods.

Key words: bovine coronavirus; spike protein; bioinformatics analysis

摘要：

【目的】研究新疆牛冠状病毒(Bovine coronavirus,BCoV)流行株的遗传变异规律,分析纤突(Spike,S)蛋白结构与功能特征。【方法】对新疆巴楚县某牛场BCoV阳性的犊牛粪便,提取病毒RNA,采用RT-PCR方法扩增S基因,通过序列测定和拼接,获得BCoV S基因全长序列,对其开展生物信息学分析和遗传进化分析。【结果】获得BCoV S基因核苷酸序列(GenBank索引号:OR136878.1)。S基因全长4 092核苷酸(nucleotide,nt),编码1 363氨基酸(amino acid,aa)。BCoV S蛋白分子量为150.67 Ku,等电点为5.29,有1个跨膜螺旋区,亲水性较弱,疏水性略强。S蛋白主要分布于宿主细胞的内质网膜和高尔基体,具有信号肽的概率为0.984 2,是分泌型蛋白,含有14个潜在的N-糖基化位点和132个磷酸化位点。S蛋白具有3个结构域,其中受体结合结构域(Receptor binding domain,RBD)共含有215 aa,无规则卷曲(Coiled coil, Cc)占比最高(62.79%),其次为延伸链(Extended strand, Es)(20.47%)和α-螺旋(α-helix, Hh)(12.56%),β-转角(β-turn, Tt)占比最少(4.19%)。筛选出S蛋白15个优势B细胞表位和11个T细胞表位。S蛋白能与SWISS-MODEL数据库中模板(SMTL ID:7sbw.1.C)同源建模,二者序列相似性为92.00%,模型GMQE值为0.76,QMEAN值为0.82,符合率较高,拉氏图(Ramachandran plots)的Ramachandran favored值为96.14%,表明空间构象合理,模型准确可靠。遗传进化试验扩增的BCoVS基因序列,与2016年我国新疆的BCV-Aks-01株处于同一小分支,两者S基因核苷酸序列同源性为99.1%。【结论】BCoV S蛋白存在多个抗原表位。RBD在病毒进入宿主细胞时具有重要作用,可针对RBD序列设计疫苗靶点,阻止病毒与宿主受体结合。采用生物信息学方法首次分析BCoV S蛋白理化性质、信号肽和亚细胞定位、磷酸化位点、糖基化位点、结构域、抗原表位、三级结构和RBD二级结构等特征,为BCoV S基因遗传进化、结构与功能研究、疫苗研发、靶向药物设计提供参考。

关键词: 牛冠状病毒, 纤突蛋白, 生物信息学分析