草莓轻型黄边病毒外壳蛋白抗原表位预测、克隆、表达及其免疫原性分析

应用IEDB、DNAStar、DNAMAN和Snap Gene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分析。选择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(Escherichia coli)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E.coli表达载体pET32a(+)的Eco RⅠ和XhoⅠ位点,获得重组质粒pET32a(+)-AE。含AE片段的开放阅读框长561 bp,编码了一个187氨基酸残基的重组融合蛋白,理论分子质量为20.29kD。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中的最佳表达条件为1.5 mmol/L异丙基-β-D-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG)、35℃诱导表达2 h,产率为13.22 mg/L;在E.coli BL21(DE3)中的最佳表达条件是为0.5 mmol/L IPTG、30℃诱导表达2 h,产率为9.55 mg/L。重组融合蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离、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Ni~(2+)亲和色谱纯化、EK酶切、分子筛除去融合蛋白标签后,免疫产蛋鸡。从免疫鸡所产鸡蛋中提取鸡卵黄免疫球蛋白(immunoglobulin of yolk,Ig Y),Dot-Blot检测抗体结合活性。结果表明,所提取的IgY可特异性识别AE肽段和SMYEV外壳蛋白。这一结果表明所预测的AE肽段具有较好的免疫原性。

Epitope Prediction,Cloning, Expression and Immunogenicity of the Coat Protein of Strawberry Mild Yellow Edge Virus

The amino acid sequence of epitope peptides of the coat protein of strawberry mild yellow edge virus was predicted by bioinformatic tools including IEDB, DNAStar, DNAMAN and SnapGene. The peptide from the 27th to 38th amino acid residues with predicted high 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 EcoRⅠ and XhoⅠ sites. The open reading frame (ORF) containing the AE fragment was 561 bp, which encoded a fusion protein of 187 amino acid residues. The theoretical molecular mass of the recombinant fusion protein was 20.29 kD. 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 optimal expression conditions of the fusion protein in E. coli Rosetta were 1.5 mmol/L IPTG at 35 ℃ for 2 h, resulting in a yield of 13.22 mg/L, while those for expression in E. coli BL21(DE3) were 0.5 mmol/L IPTG at 30 ℃ for 2 h, giving a yield of 9.55 mg/L. The recombinant fusion protein was isolated by SDS-PAGE and identified by tandem mass spectrometry. The results showed that the recombinant fusion protein contained the designed peptide AE. The recombinant fusion protein was purified by Ni2+-affinity chromatography, digested by EK enzyme, and separated from the fusion protein tag by molecular sieve. Chicken immunoglobulin (IgY) was extracted from chicken eggs collected on the 21st day after the first injection. The binding activity of the IgY to AE and SMYEV was analyzed by the Dot-Blot method. The results showed that the IgY could specifically recognize both AE peptide and SMYEV indicating that the predicted AE peptide has desired immunogenicity.