EB病毒BZLF1基因表位疫苗的预测及构建

Prediction and construction of Epstein-Barr virus BZLF1 gene epitope vaccine

目的 采用生物信息学方法预测EB病毒BZLF1基因编码蛋白的结构、抗原表位及疫苗的初步构建,为EB病毒相关疾病的疫苗的研制提供依据。方法 通过NCBI获得EB病毒BZLF1蛋白的核苷酸序列和氨基酸序列,采用ORF Finder、Protparam、SOPMA、DTU Health Tech、TMHMM-2.0、Cell-PLoc 2.0、NetPhos 3.1 Servera、NetNGlyc、SWISS MODE、Immunomedicine Group、IEDB、BLAST、Uniprot等生物信息学软件对EB病毒BZLF1蛋白的理化性质、亲疏水性、结构域、信号肽、跨膜区、亚细胞定位、磷酸化及糖基化位点、二级结构、三级结构、抗原决定簇、B细胞与T细胞抗原表位、蛋白同源性及相互作用蛋白进行预测分析。在T4DNA连接酶作用下连接BZLF1基因与穿梭表达载体pMV261,对连接产物进行PCR及双酶切鉴定。结果 BZLF1蛋白是由245个氨基酸组成的亲水蛋白,分子式为C_(1185)H_(1850)N_(332)O_(366)S_(8),相对分子质量为26.860×10^(3),理论等电点为5.25,脂肪族氨基酸指数为71.35,平均亲水系数为-0.529;该蛋白二级结构以无规则卷曲居多,占51.84%,无信号肽和跨膜区,存在18个磷酸化位点,2个糖基化位点,含有1个碱性亮氨酸拉链结构。预测该蛋白有9个抗原决定簇,8个B细胞优势抗原表位,9个CTL细胞优势表位,8个Th细胞表位,与人类环状amp反应元件结合蛋白相似度为6.12%,同源性较低。PCR及双酶切鉴定含BZLF1基因的pMV261载体构建正确。结论 生物信息学预测BZLF1属于亲水蛋白,热稳定性较好。该蛋白含有多个磷酸化位点可参与相关信号通路,对疾病的发展起到重要作用。BZLF1含有多个B、T细胞抗原表位,且与人体蛋白同源性低,不易发生免疫交叉反应,可作为EB病毒候选疫苗表位。

Objective The bioinformatics method was used to predict the structure and epitope of the protein encoded by the Epstein-Barr virus BZLF1 gene and the preliminary construction of the vaccine, so as to provide the basis for the development of the vaccine of Epstein-Barr virus related diseases. Methods The nucleotide and amino acid sequences of EB virus BZLF1 protein were obtained by NCBI. ORF Finder, Protparam, SOPMA,DTU Health Tech, TMHM-2.0,Cell-PloC 2.0,NetPhos 3.1 Servera, NetNGlyc, SWISS MODE,Immunomedicine Bioinformatics software such as Group, IEDB,BLAST and Uniprot analyzed the physicochemical properties, hydrophilicity and hydrophobicity, domain, signal peptide, transmembrane region, subcellular localization, phosphorylation and glycoylation sites, secondary structure, tertiary structure, antigen epitopes of B cell and T cell, protein homology and interaction of EB virus BZLF1 protein Predictive analysis with protein.T4 DNA ligase was used to connect BZLF1 gene to shuttle expression vector pMV261,and PCR and double enzyme digestion were used to identify BZLF1 gene. Results BZLF1 protein is a hydrophilic protein composed of 245 amino acids, the molecular formula is C_(1185)H_(1850)N_(332)O_(366)S_(8),the relative molecular weight is 26.860×10^(3),the theoretical isoelectric point is 5.25,the aliphatic amino acid index is 71.35,the average hydrophilic coefficient is-0.529. The majority of the secondary structure of the protein was random coil, accounting for 51.84%,no signal peptide and transmembrane region, there were 18 phosphorylation sites, 2 glycoylation sites, and 1 basic leucine zipper structure. It was predicted that the protein had 9 epitopes, 8 B-cell dominant epitopes, 9 CTL dominant epitopes and 8 Th cell epitopes. The similarity to human cyclic amp reaction element binding protein was 6.12%,indicating low homology. The pMV261 vector containing BZLF1 gene was correctly constructed by PCR and double enzyme digestion. Conclusion Bioinformatics predicted that BZLF1 was a hydrophilic protein with good thermal stability. The protein contains multiple phosphorylation sites and is involved in related signaling pathways, which play an important role in the development of the disease. BZLF1 contains multiple B and T cell antigen epitopes, and has low homology with human proteins, so it is not easy to have immune cross-reaction, and can be used as a candidate Epstein-Barr virus vaccine epitopes.