关键词: 牛冠状病毒; 纤突蛋白; 生物信息学分析

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