特异性结合幽门螺杆菌黏附蛋白A(HpaA)核酸适配子的筛选及鉴定

Screening and characterization of nucleic acid aptamers specifically binding to adhesin HpaA from Helicobacter pylori

目的通过指数富集的配体系统进化技术(SELEX)筛选特异性结合幽门螺杆菌(H.pylori)黏附蛋白A(HpaA)的核酸适配子,并鉴定其结合特性。方法构建pET28a/HpaA原核表达质粒并诱导表达纯化重组HpaA蛋白;以重组HpaA蛋白为靶标利用SELEX技术筛选出能够与HpaA具有高特异性、高亲和力结合的核酸适配子。随后利用筛选所得HpaA适配子通过体外实验验证与H.pylori菌体的结合以及对临床胃黏膜切片中H.pylori的检测效能。结果本研究培养并提取ATCC26695菌株基因组,通过PCR扩增HpaA部分基因长度约699 bp,并成功克隆构建了原核表达质粒pET28a/HpaA。经异丙基β-D-半乳糖苷(IPTG)诱导表达、纯化获得纯度约98%的重组HpaA蛋白作为筛选靶标。利用SELEX技术通过10轮正向筛选和5轮的负向筛选,获得6条与HpaA高亲和力的适配子分别命名为HA1~HA6。其中适配子HA6具有较高的亲和力和特异性,且适配子HA6核心序列在体外仍表现出与H.pylori菌体较高的结合力;利用羟基荧光素(FAM)标记的HA6适配子对166例H.pylori感染的患者胃黏膜中H.pylori进行检测,检出率为94.58%,高于同批次快速脲酶检测法的检出率(87.95%)。结论本研究筛选出的特异性结合H.pylori菌体表面HpaA的适配子,可能作为一种新的方法应用于胃黏膜组织中H.pylori的检测。

Objective To screen aptamers that specifically bind to adhesin HpaA from Helicobacter pylori(H.pylori)by systematic evolution of ligands by exponential enrichment(SELEX)and identify the binding properties of aptamers.MethodsThe prokaryotic expression recombinant plasmid pET28a/HpaA was constructed and the HpaA protein was expressed and purified with IPTG.With the recombinant HpaA protein as target,we screened aptamers with high affinity and specificity binding force by SELEX.The binding force between aptamers and H.pylori in vitro and the performance of aptamers in H.pylori detection from the biopsy of gastric mucosa were examined using the aptamers we had screened.Results We extracted genome from H.pylori ATCC26695 strains and amplified 699 bp HpaA gene using PCR.The recombinant plasmid pET28a/HpaA was constructed successfully.The recombinant HpaA was expressed and purified up to 98%as target for aptamer screening.The six highest affinity aptamers were obtained and named HA1 to HA6 through 10-round positive screening and five-round negative screening by SELEX.The full-length aptamer HA6 and the core sequence of HA6 showed highest affinity and specificity in H.pylori detection in vitro.In view of this,the FAM-labelled aptamer HA6 was used to detect H.pylori in gastric mucosa from 166 patients.The aptamer HA6 showed a higher detection rate(94.58%)than URT(87.95%)in the same batch of clinical samples.Conclusion The aptamers that specifically bind to HpaA may be applied for the detection of H.pylori in gastric mucosa as a novel method.