食品科学2017，Vol.38Issue(16)：71-78,8.DOI:10.7506/spkx1002-6630-201716011
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
Epitope Prediction, Cloning, Expression and Immunogenicity of the Coat Protein of Strawberry Mild Yellow Edge Virus
摘要
Abstract
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 ofE.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.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Key words
strawberry mild yellow edge virus/coat protein/epitope prediction/prokaryotic expression/tandem mass spectrometry/immunogenicity分类
生物科学引用本文复制引用
杨菊梅,马建忠,王永刚,邓子兵,魏艳,潘博,李印武..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[J].食品科学,2017,38(16):71-78,8.基金项目
甘肃省科技计划项目(1504WKCA020) （1504WKCA020）
国家大学生创新创业训练项目(DC2015085) （DC2015085）
甘肃省横向科技项目(10-2203) （10-2203）
兰州理工大学实验室管理处教研项目 （）
