草莓轻型黄边病毒外壳蛋白抗原表位的预测、克隆表达及免疫原性研究

摘要/Abstract

摘要： 摘要：应用Antibody Epitope Prediction (IEDB)、DNAStar、DNAMAN和SnapGene等生物信息学工具对草莓轻型黄边病毒外壳蛋白的氨基酸残基序列进行了分析。选择了一段抗原性较强的肽段(位于外壳蛋白27-38氨基酸残基处)，依据大肠杆菌的密码子偏好性化学合成了该肽段的DNA编码序列(AE)。AE片段克隆至大肠杆菌表达载体pET32a(+)的EcoRI和XhoI位点，获得重组质粒pET32a(+)-AE。含AE片段的开放阅读框长561bp，编码了一个187氨基酸残基的重组融合蛋白，理论分子量为20.29kDa。重组质粒pET32a(+)-AE分别在E.coli BL21(DE3) 和E.coli Rosetta中进行了诱导表达和条件优化。重组融合蛋白在E.coli Rosetta中的最佳表达条件是：1.5mmol/L IPTG，35℃诱导表达2h，产率为13.22mg/L；在E.coli BL21(DE3)中的最佳表达条件是：0.5mmol/L IPTG，30℃诱导表达2h，产率为9.55mg/L。重组融合蛋白经SDS-PAGE分离、质谱鉴定表明其含有所预测的草莓轻型黄边病毒外壳蛋白抗原表位肽段AE。AE重组融合蛋白经Ni2+亲和色谱纯化、EK酶切除去融合标签后，免疫产蛋鸡、提取鸡卵黄免疫球蛋白(IgY)通过Dot-Blot检测其活性，结果表明鸡卵黄免疫球蛋白(IgY)可特异性识别SMYEV外壳蛋白，进而表明本文所预测的AE肽段具有免疫原性。为SMYEV 的检测提供了技术保障。

关键词: 关键词：草莓轻型黄边病毒, 外壳蛋白, 抗原表位预测, 原核表达, 质谱鉴定, 免疫原性

Abstract: Abstract: Epitope peptides of the coat protein of strawberry mild yellow edge virus were predicted by bioinformatic tools, IEDB, DNAStar, DNAMAN and SnapGene. A peptide from 27 to 38 amino acid residues with a highly predicted antigenic activity, referred to as AE, was reversely translated into its encoding DNA sequence according to the codon bias of E. coli. The encoding DNA fragment AE was then synthesized and cloned into the expression vector pET32a(+) between the sites of EcoRI and XhoI. The open reading frame containing the AE fragment is 561bp, in which encodes a fusion protein of 187 amino acid residues. The theoretical molecular weight of the recombinant fusion protein is 20.29kDa. Expression of the target gene in the recombinant plasmid pET32a (+)-AE was induced and optimized in E.coli BL21 (DE3) and E.coli Rosetta, respectively. The best expression conditions of the fusion protein in E.coli Rosetta were reached at 1.5mmol/L IPTG and 35℃ for 2h. The yield is 13.22mg/L. The best expression conditions of the protein in E.coli BL21(DE3) were at 0.5mmol/L IPTG and 30℃ for 2h. Its yield is 9.55mg/L. The recombinant fusion protein was isolated by SDS-PAGE and identified by the tandem mass spectrometry. The results showed that the recombinant fusion protein contained the designed peptide AE.AE recombinant fusion protein was purificated by Ni2+ affinity chromatography,was digested by enterakinase. immunning laying hens, extracting of egg yolk immunoglobulin (IgY) that was detected by Dot-Blot. The results showed that egg yolk immunoglobulin (IgY) could specifically recognize SMYEV coat protein, which showed that AE peptide predicted in this paper had immunogenicity . Provide technical support for the detection of SMYEV.

Key words: Key words：Strawberry mild yellow edge virus, Coat protein, Epitope prediction, Prokaryotic expression, Tandem mass spectrometry, immunogenicity

中图分类号:

Q785/6

马建忠杨菊梅王永刚邓子兵. 草莓轻型黄边病毒外壳蛋白抗原表位的预测、克隆表达及免疫原性研究[J]. 食品科学, 0, (): 0-0.

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