Hua Ying, Chen Liping, Li Sheng, Zhou Fayou, Tang Xiaolei
Basic Medical Laboratory, Second Affiliated Hospital, Nursing School, Wuhu 241000, China.
Department of Gastroenterology, Second Affiliated Hospital, Wannan Medical College, Wuhu 241000, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2019 Jun;35(6):533-539.
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. Methods The 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.
目的 通过指数富集配体系统进化技术（SELEX）筛选与幽门螺杆菌黏附素HpaA特异性结合的适体，并鉴定适体的结合特性。方法 构建原核表达重组质粒pET28a/HpaA，用异丙基-β-D-硫代半乳糖苷（IPTG）表达并纯化HpaA蛋白。以重组HpaA蛋白为靶标，通过SELEX筛选具有高亲和力和特异性结合力的适体。用筛选得到的适体检测其与幽门螺杆菌在体外的结合力以及在胃黏膜活检标本中检测幽门螺杆菌的性能。结果 从幽门螺杆菌ATCC26695菌株中提取基因组，通过聚合酶链反应（PCR）扩增出699 bp的HpaA基因。成功构建重组质粒pET28a/HpaA。表达并纯化重组HpaA，纯度达98%，作为适体筛选的靶标。通过10轮阳性筛选和5轮阴性筛选，获得6个亲和力最高的适体，命名为HA1至HA6。全长适体HA6及其核心序列在体外幽门螺杆菌检测中显示出最高的亲和力和特异性。据此，用荧光素标记的适体HA6检测166例患者胃黏膜中的幽门螺杆菌。在同一批临床样本中，适体HA6的检测率（94.58%）高于尿素呼吸试验（URT，87.95%）。结论 特异性结合HpaA的适体可作为一种新方法应用于胃黏膜中幽门螺杆菌的检测。
