幽门螺杆菌多表位疫苗的设计及其表达分析  

Design and Expression Analysis of Multi-Epitope Vaccine Against Helicobacter Pylori

作　　者：贾志达 于雪莹 尚慧敏 李旭阳 斯日古楞 王晓玲 JIA Zhida;YU Xueying;SHANG Huimin(College of Basic Medical Sciences,Chifeng University,Chifeng City,Inner Mongolia Autonomous Region 024000)

机构地区：[1]赤峰学院基础医学院,内蒙古自治区赤峰市024000

出　　处：《医学理论与实践》2025年第5期740-743,共4页The Journal of Medical Theory and Practice

基　　金：中央引导地方科技发展资金项目(2022ZY0161);赤峰学院教育教学研究项目(JYJXZ202207)。

摘　　要：目的:针对幽门螺杆菌与定植和致病相关的6种蛋白设计作用位点较为全面的多表位疫苗,将其构建到复制缺陷型腺病毒穿梭载体中并分析其表达情况,为后续包装腺病毒并通过动物实验验证疫苗的有效性奠定实验基础。方法:使用IEDB数据库查找和预测幽门螺杆菌UreB、OipA、HomB、HopQ、VacA和CagA的B细胞表位,采用在线工具VaxiJen v2.0、AllerTOP v2.0和AllergenFP v1.0对6种表位与接头KK随机串联得到的720组合进行抗原性和过敏原性分析,选出优势表位组合,将优势表位组合与具有黏膜佐剂活性的NSP4C融合并加Flag标签便于后续检测,综合考虑融合蛋白的抗原性、可溶性和理化性质,评选出最优疫苗蛋白,将其构建到复制缺陷型腺病毒穿梭载体中,通过转染HEK-293T细胞和Western blot实验分析其表达情况。结果:通过对表位组合的抗原性分析找到3个评分>1.0的表位组合,即优势表位组合,三者均无过敏原性。将优势表位组合、NSP4C和Flag随机组合后,选出最优疫苗蛋白NSP4C-Dec663。将NSP4C-Dec663编码序列连入pShuttle-CMV载体中,重组质粒转染HEK-293T细胞进行目的蛋白表达,Western blot结果表明NSP4C-Dec663成功表达。结论:本研究针对幽门螺杆菌设计了多表位疫苗NSP4C-Dec663,其能在HEK-293T细胞中表达,这为后续包装复制缺陷型腺病毒和进行动物实验奠定了基础。Objective:Designing a comprehensive multi-epitope vaccine targeting six proteins associated with colonization and pathogenesis of helicobacter pylori,constructing it into a replication-deficient adenoviral shuttle vector,analyzing its expression,and laying the experimental foundation for subsequent adenovirus packaging and validation of vaccine effectiveness through animal experiments.Methods:Using the IEDB database to search for and predict B cell epitopes of helicobacter pylori UreB,OipA,HomB,HopQ,VacA,and CagA,the 720 combinations obtained by randomly linking the 6 epitopes with KK linkers were analyzed for antigenicity and allergenicity using online tools VaxiJen v2.0,AllerTOP v2.0,and AllergenFP v1.0.Dominant epitope combinations were selected,and these were fused with NSP4C,which has mucosal adjuvant activity,and tagged with a Flag tag for subsequent detection.Considering the antigenicity,solubility,and physicochemical properties of the fused protein,the optimal vaccine protein was selected.This optimal protein was then constructed into a replication-deficient adenoviral shuttle vector,and its expression was analyzed through transfection of HEK-293T cells and Western blot experiments.Results:Through antigenicity analysis of epitope combinations,three combinations with scores greater than 1.0 were identified as dominant epitope combinations,all of which showed no allergenicity.After randomly combining the dominant epitope combinations,NSP4C,and the Flag tag,the optimal vaccine protein NSP4C-Dec663 was selected.The coding sequence for NSP4C-Dec663 was inserted into the pShuttle-CMV vector,and the recombinant plasmid was transfected into HEK-293T cells for the expression of the target protein.Western blot results confirmed successful expression of NSP4C-Dec663.Conclusion:This study designed a multi-epitope vaccine NSP4C-Dec663 targeting helicobacter pylori,which can be expressed in HEK-293T cells,laying the foundation for subsequent packaging replication of defective adenovirus and animal experiments.

关 键 词：幽门螺杆菌 生物信息学 多表位疫苗 复制缺陷型腺病毒载体 

分 类 号：R-33[医药卫生]