牛病毒性腹泻病毒E^rns多表位蛋白的设计及验证

Design and Verification of Erns Multi-epitope Protein of Bovine Viral Diarrhea Virus

利用生物信息学方法分析牛病毒性腹泻病毒(BVDV)E^rns蛋白,筛选出优势B淋巴细胞(简称B细胞)和T淋巴细胞(简称T细胞)表位组装成多表位蛋白,并在大肠埃希菌中进行表达和验证。首先从NCBI GenBank数据库获得E^rns蛋白的氨基酸序列,将所得到的序列进行理化性质、跨膜结构域、亚细胞定位、磷酸化、酰基化位点分析,预测二级结构及二硫键的位置,使用4种B细胞和2种T细胞软件预测其抗原表位,将预测的表位进行筛选并组装成多表位疫苗,进行密码子优化确保其在大肠埃希菌中高效表达,最后进行蛋白的纯化和Western blot验证。结果显示,E^rns蛋白由227个氨基酸残基组成,分子质量约为26 ku,理论等电点8.62,化学式为C1131H1768N326O335S14,不稳定系数为29.98,亲水性平均值(GRAVY)和脂肪指数分别-0.529和71.37,在大肠埃希菌体内半衰期大于10 h,不具有跨膜结构域,主要定位在细胞质中,有21个翻译后修饰(PTM)位点;在E^rns蛋白的二级结构中,α螺旋占约36.12%,β折叠占约18.06%,β转角占约7.93%,无规卷曲占约37.89%,而且具有4个二硫键。将筛选出的5个B细胞和6个T细胞优势表位用柔性linker组装成多表位疫苗,Western blot结果显示多表位蛋白对BVDV阳性血清具有良好的反应原性,为牛病毒性腹泻病毒E^rns多表位蛋白后续的研究奠定了基础。

This study aimed to perform bioinformatics analysis of bovine viral diarrhea virus(BVDV)E^rns protein and screening of dominant epitopes of T cells and B cells,and expressed and verified in E.coli.The amino acid sequence of the E^rns protein was first obtained from the NCBI GenBank database,and the resulting sequence was subjected to anslyses of physicochemical properties,transmembrane domain,subcellular localization,phosphorylation,and acylation site,predicting secondary structures,using four B cell prediction software and two T cell software to predict epitopes,screening predicted epitopes,and then assembling into multi-epitope vaccines,and finally the protein was purified and verified by Western blot.The results showed that the E^rns protein consists of 227 amino acid residues with a molecular mass about 26 ku,a theoretical isoelectric point of 8.62,a chemical formula of C1131H1768N326O335S14,an instability coefficient of 29.98,a hydrophilic mean(GRAVY)of-0.529,and a fat index of 71.37.In E.coli,the half-life is greater than 10 h,does not have a transmembrane domain,and is mainly located in the cytoplasm.There are 21 post-translational modification(PTM)sites.In the secondary structure of E^rns protein,α-helix accounts for about 36.12%,βfolding accounts for about 18.06%,βturns about 7.93%,random curl accounts for about 37.89%,and has four disulfide bonds.Finally,five B-cell epitopes and six T-cell epitopes were screened,and a multi-epitope vaccine were linked by a flexible linker.Western blot results showed that the multi-epitope protein had good reactivity to BVDV positive serum.This study laid the foundation for the subsequent study of the bovine viral diarrhea virus E^rns polyepitopes.