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目的:对重组表达的StxB和预测的模拟表位肽进行免疫保护效应评价。方法:用分子生物学方法构建基因工程菌,IPTG诱导表达重组StxB,经复性后进行离子交换层析纯化。同时采用生物信息学手段对StxB的表位相关参数进行分析,预测StxB可能的抗原表位。以纯化的StxB和合成的模拟表位肽免疫小鼠后攻毒,评价StxB及其模拟表位肽的免疫保护效应。结果:表达并制备了90%以上纯度重组StxB;发现p14和p17多肽易形成转角和无规卷曲的柔性区段,可及性、极性和亲水性较强,是可能的抗原表位。免疫保护试验显示,经重组StxB和合成多肽免疫的小鼠发病延迟,症状减轻,存活率显著提高。结论:重组StxB和预测的模拟表位肽具有良好的免疫保护效果,为亚单位疫苗及其作用机制的研究奠定了基础。
OBJECTIVE: To evaluate the immunoprotective effect of recombinantly expressed StxB and predicted mimotope peptides. Methods: The genetically engineered bacteria were constructed by molecular biology method. Recombinant StxB was induced by IPTG and purified by ion exchange chromatography after refolding. At the same time, bioinformatics methods were used to analyze the epitope-related parameters of StxB to predict the possible epitopes of StxB. The mice were challenged with purified StxB and synthetic mimotope peptides to evaluate the immunoprotective effects of StxB and its mimotope peptides. Results: The recombinant StxB with more than 90% purity was expressed and prepared. The p14 and p17 polypeptides were found to be flexible and easy to be turned into curly and flexible sections. Their accessibility, polarity and hydrophilicity were the possible epitopes. Immunoprotection experiments showed that mice immunized with recombinant StxB and synthetic peptides delayed onset, reduced symptoms and significantly improved survival. Conclusion: Recombinant StxB and predicted mimotope peptides have good immunoprotective effect, which lays the foundation for the study of subunit vaccine and its mechanism of action.