用噬菌体展示技术筛选幽门螺杆菌细胞毒素相关蛋白基因结合蛋白的研究

Screening of DNA-binding protein of Helicobacter pylori CagA gene by phage display technique from human stomach cDNA library

目的筛选幽门螺杆菌细胞毒素相关蛋白(CagA)基因结合蛋白,探索CagA致病机理。方法应用噬菌体展示技术,以幽门螺杆菌CagA基因片段作为固相筛选分子,对噬菌体人胃细胞cDNA文库进行4轮"吸附-洗脱-扩增"富集,噬斑裂解液PCR扩增后,进行DNA序列分析和同源性生物信息学搜索,确定编码结合蛋白的种类。结果噬菌体经富集后,筛选出36个阳性克隆,构建了克隆载体。序列测定后经过同源性搜索,确定幽门螺杆菌CagA基因共编码核纤层蛋白B1和胰岛素样生长因子结合蛋白2两种已知蛋白。结论用噬菌体人胃cDNA文库筛选得到幽门螺杆菌CagA基因DNA结合蛋白,为研究CagA致病机理,幽门螺杆菌疫苗的研制和治疗等提供依据。

Objective To screen DNA-binding protein of Helicobacter pylori cytotoxin associated gene A（CagA） gene by phage display technique from human stomach cDNA library and investigate the pathogenesis mechanism of CagA gene. Methods By using CagA gene as the selective molecule, the T7 select human stomach cDNA library was biopanned and positive clones were selected. After screening, positive plaques were performed to amplify for inserted DNA fragment and the DNA fragment was cloned into pGEM-T easy vector. Results Sequence analysis was performed in thirty-six positive plaque, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether twelve coding sequences were gotten. Conclusion CagA gene binding protein have been screened. These proteins need to be further studied and will be useful for further study of the pathogenesis mechanism of CagA.