摘要
目的 探索核酸适配子特异性结合幽门螺杆菌(H.pylori)血型抗原结合黏附素(BabA)对H.pylori宿主细胞黏附的阻断作用。方法 培养H.pylori菌株并提取基因组,并设计引物PCR扩增BabA基因,扩增获得的BabA基因克隆、构建至原核表达质粒,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并纯化后作为靶标,利用指数富集的配体系统进化技术(SELEX)筛选能够特异性结合BabA的单链脱氧核苷酸(ssDNA)适配子;酶联寡聚核苷酸吸附试验(ELONA)法检测并评估候选适配子的特征;随后在体外细胞水平和小鼠感染模型中,分别采用流式细胞术和菌落计数验证ssDNA适配子阻断H.pylori黏附的效果,同时利用ELISA检测小鼠胃黏膜细胞匀浆中白细胞介素6(IL-6)、 IL-8、肿瘤坏死因子α(TNF-α)、 IL-10和IL-4的水平。结果提取H.pylori ATCC 43504基因组并构建了pET32a-BabA质粒,诱导后并纯化重组蛋白相对分子质量(Mr)约为39 000;经肽指纹图谱(PMF)分析其与BabA蛋白一致;以此为靶标运用SELEX筛选获得5个候选适配子,经分析鉴定适配子A10、 A30和A42识别相同位点,A3和A16与上述三个适配子各自识别不同的位点。适配子体外能够显著阻断H.pylori的黏附,且动物模型实验证明适配子经灌胃处理后,对胃黏膜的H.pylori定植有阻断效果,且能够减轻诱导的炎症反应,适配子治疗组胃黏膜匀浆中IL-4、 IL-6、 IL-8和TNF-ɑ水平低于模型组。结论 适配子特异性结合BabA封闭H.pylori与胃黏膜上皮细胞的黏附能够阻断H.pylori在胃黏膜的定植。
Objective To explore the aptamer specific binding blood group antigen-binding adhesin(BabA)of Helicobacter pylori(H.pylori)for blocking of H.pylori adhering host cell.MethodssH.pylori strainwasculturedand its genome was extracted as templates to amplify the BabA gene by PCR with designed primers.The BabA gene obtained was cloned and constructed into prokaryotic expression plasmid,which was induced by isopropyl beta-D-galactoside(IPTG)and purified as target.The single stranded DNA(ssDNA)aptamers that specifically bind to BabA were screened by SELEX.Enzyme-linked oligonucleotide assay(ELONA)was used to detect and evaluate the characteristics of candidate aptamers.The blocking effect of ssDNA aptamers on H.pylori adhesion was subsequently verified by flow cytometry and colony counting at the cell level in vitro and in mouse model of infection,respectively.Meanwhile,the levels of cytokines,interleukin 6(IL-6),IL-8,tumor necrosis factorα(TNF-α),IL-10 and IL-4 in the homogenate of mouse gastric mucosa cells were detected by ELISA.Results The genome of H.pylori ATCC 43504 strains was extracted and the recombinant plasmid pET32a-BabA was constructed.After induction and purification,the relative molecular mass(M,)of the recombinant BabA protein was about 39000.The amino acid sequence of recombinent protein was consistent with BabA protein by peptide mass fingerprint(PMF).Five candidate aptamers were selected to bind to the above recombinent BabA protein by SELEX.The aptamers A10,A30 and A42 identified the same site,while A3,A16 and the above three aptamers identified different sites respectively.The aptamer significantly blocked the adhesion of H.pylori in vitro.Animal model experiments showed that the aptamers can block the colonization of H.pylori in gastric mucosa by intragastric injection and reduce the inflammatory response.The levels of IL-4,IL-6,IL-8 and TNF-αin gastric mucosal homogenates in the model group with aptamer treatment were lower than that of model group without treatment.Conclusion Aptamers can reduce the colonization of H.pylori in gastric mucosa via binding BabA to block the adhesion between H.pylori and gastric mucosal epithelial cells.
作者
袁媛
李卫鹏
周晓静
孙伟莉
唐晓磊
YUAN Yuan;LI Weipeng;ZHOU Xiaojing;SUN Weli;TANG Xiaolei(Department of Nuclear Medicine,First Affiliated Hospital of Bengbu Medical College,Bengbu 233030;Translational Medicine Centre Second Affiliated Hospital of Wannan Medical College,Wuhu 241000;Clinical Pathogen Detection Engineering Center of Wuhu,Wuhu 241000,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2023年第9期793-800,共8页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81601806)
安徽高校合作攻关和卫生协同创新项目(GXXT-2020-023)
安徽省卫健委重点项目(A HWJ2021a015)
蚌埠医学院校级重点项目(byzd123)。
