草莓镶脉病毒外壳蛋白抗原表位预测、克隆、表达及鉴定

Prediction, cloning, expression and identification of an epitope of the coat protein of the Strawberry vein banding virus

为实现草莓镶脉病毒(Strawberry vein banding virus,SVBV)的简单和快速检测,应用生物信息学方法分析了SVBV外壳蛋白的线性抗原表位及其理化性质,以大肠杆菌的优势密码子对所预测的优势抗表原位进行设计并合成,同时进行了最佳异源表达条件优化。结果表明,第218~238位的氨基酸残基序列为SVBV外壳蛋白的优势抗原表位,其编码片段为5-AE。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)检测结果表明,重组融合蛋白的分子量约为24.8 kD,与其理论分子量20.5 kD基本一致。异源表达优化的结果表明,当IPTG浓度为0.5 mmol/L、温度为40℃、诱导时间为4 h时,重组融合蛋白的表达量最高,为23.6 mg/L。Western blot结果表明重组融合蛋白能与His多克隆抗体起特异性反应。串联质谱结果表明,5-AE肽段氨基酸残基序列正确。

For realize the simple and rapid detection of Strawberry vein banding virus（SVBV）, the linear antigen epitope and physicochemical properties of SVBV coat protein were analyzed with bioinformatics method. The dominant codon of Escherichia coli was used to design and synthesize the predicted superior antigen epitope, and the heterologous expression condition was optimized. The results showed that the sequence of amino acid residues at position 218-238 was the dominant epitope of SVBV coat protein, and its coding fragment was 5-AE. Detection results of sodium dodecyl sulfonatepolyacrylamide gel electrophoresis（SDS-PAGE） showed that the molecular weight of the recombinant fusion protein was about 24.8 kD, which was basically consistent with its theoretical molecular weight of 20.5 kD. The expression of heterologous expression showed that, when the concentration of IPTG was 0.5 mmol/L, the temperature was 40℃, and the induction time was four hours, the expression of the recombinant fusion protein was the highest（23.6 mg/L）. Western blot results showed that the recombinant fusion protein could react specifically with His polyclonal antibody. Tandem mass spectrometry showed that the amino acid sequence of 5-AE peptide fragment was correct.