G8型牛轮状病毒VP7基因克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2025.04.030

摘要/Abstract

摘要： 【目的】克隆G8型牛轮状病毒(Bovine rotavirus，BRV)新疆分离株VP7基因，分析VP7蛋白分子遗传特征，为研究其生物学功能及研发新型疫苗提供理论依据。【方法】从BRV新疆分离株第6代病毒液中提取病毒RNA，通过RT-PCR扩增VP7基因片段，克隆至pMD19-T载体，转化大肠杆菌DH5α感受态细胞，提取质粒后酶切并测序；采用生物信息学软件分析VP7蛋白理化性质、跨膜结构、亲/疏水性、亚细胞定位和信号肽、糖基化位点、磷酸化位点、二级结构、三级结构同源建模及B、T细胞抗原表位。【结果】RT-PCR扩增获得大小为1 062 bp的BRV分离株VP7基因全长核苷酸序列，提交NCBI获得GenBank登录号：OR514136.1，该序列与RVA/Cow-wt/TUR/Amasya-1/2015/G8P[5]株(GenBank登录号：KX212865.1)核苷酸序列相似性最高(89.4%)，同属A血清型G8基因型。生物信息学分析显示，BRV分离株VP7蛋白为疏水性不稳定蛋白，存在2个跨膜区，无信号肽，亚细胞定位于内质网膜，含有2个N-糖基化位点和132个磷酸化位点，二级结构以α-螺旋和无规则卷曲为主，分别占36.20%和35.28%。VP7蛋白能与SWISS-MODEL数据库中模板(SMTL ID:8bp8.1)的氨基酸序列同源建模，两者序列相似性为82.82%，符合率较高，模型GMQE、QMEAN和Ramachandran favored值分别为0.75、0.79和95.52%，表明空间构象合理，模型准确可靠。该蛋白存在多个B、T细胞抗原表位，其中表位长度设为16个氨基酸，评分＞0.8的B细胞表位数量为12条；表位长度设为9个氨基酸，选择4种等位基因，评分＞0.5的CTL表位有6条；表位长度设为12～18个氨基酸，选择4种等位基因，评分＞0.8的HTL表位有11条。【结论】本研究首次成功获得G8型BRV新疆分离株VP7基因全长核苷酸序列。VP7蛋白为不稳定蛋白，存在2个跨膜区，无信号肽，亚细胞定位于内质网膜，存在多个B、T细胞抗原表位。试验结果为G8型BRV的流行、诊断、病毒-宿主相互作用和VP7蛋白基因工程疫苗研究奠定基础。

关键词: 牛轮状病毒(BRV); VP7基因; 克隆; 生物信息学

Abstract: 【Objective】 The purpose of this study was to clone VP7 gene of genetype G8 Bovine rotavirus (BRV) isolated in Xinjiang,and analyze the molecular genetic characteristics of the VP7 protein,so as to provide a theoretical basis for studying its biological functions and new vaccines.【Method】 The viral genome RNA was extracted from the 6th passages of BRV isolate in Xinjiang,and VP7 gene fragment of BRV was amplified by RT-PCR,cloned into pMD19-T vector,and then transformed into Escherichia coli DH5α competent cells.The plasmid was extracted,digested and sequenced.VP7 protein was analyzed using bioinformatics software,including physicochemical properties,transmembrane structure,hydrophilicity/hydrophobicity analysis,subcellular localization,signal peptides,glycosylation sites,phosphorylation sites,secondary structure,tertiary structure homology modeling,and prediction of B and T cell antigen epitopes.【Result】 VP7 gene nucleotide sequence was successfully obtained by RT-PCR amplification, with a total length of 1 062 bp, and then submitted to NCBI to obtain GenBank accession No.: OR136878.1. This sequence had the highest identity (89.4%) with the nucleotide sequence of RVA/Cow-wt/TUR/Amasya-1/2015/G8P[5] strain (GenBank accession No.:KX212865.1),the two strains belonged to genotype G8 of serotype A.Bioinformatics analysis suggested that the BRV VP7 protein was a hydrophobic unstable protein with two transmembrane regions and without signal peptide.VP7 protein was located in endoplasmic reticulum membrane of the host cell.It contained 2 N-glycosylation sites and 132 phosphorylation sites.The secondary structure of VP7 protein was mainly consisted of alpha helix and random coil,which accounted for 36.20% and 35.28%,respectively.VP7 protein could be homologously modeled with the template (SMTL ID:8bp8.1) in the SWISS-MODEL database,with a sequence identity of 82.82%,which indicated a high coincidence rate between them.The GMQE,QMEAN and Ramachandran favored values of the model were 0.75,0.79 and 95.52%,respectively,indicating that the spatial conformation was reasonable and the model was accurate and reliable.The VP7 protein possessed several B and T cell antigenic epitopes.While the epitope length was 16 amino acids and the score exceeds 0.80,there were 12 B cell epitopes.While the epitope length was 9 amino acids,with 4 alleles selected and a score greater than 0.5,there were 6 cytotoxic T lymphocyte (CTL) epitopes.While the epitope length ranged from 12 to 18 amino acids,with 4 alleles selected and a score greater than 0.8,there were 11 helper T lymphocyte (HTL) epitopes.【Conclusion】 The complete nucleotide sequence of VP7 gene of genetype G8 BRV isolated in Xinjiang was successfully obtained for the first time.VP7 protein was an unstable protein with two transmembrane regions and without signal peptide. It was located in endoplasmic reticulum membrane of the host cell,and possessed several B and T cell antigenic epitopes.The experimental results laid the foundation for the epidemic,diagnosis,virus-host interaction,and the research of genetically engineering vaccine targeting VP7 protein of BRV.

Key words: Bovine rotavirus (BRV); VP7 gene; cloning; bioinformatics

中图分类号:

S852.65⁺3

苗书魁, 米晓云, 魏婕, 魏玉荣, 海力且木·买买提依明. G8型牛轮状病毒VP7基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2025, 52(4): 1784-1795.

MIAO Shukui, MI Xiaoyun, WEI Jie, WEI Yurong, HAILIQIEMU·Maimaitiyiming. Cloning and Bioinformatics Analysis of VP7 Gene of the Genotype G8 Bovine Rotavirus[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(4): 1784-1795.

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