幽门螺杆菌ureB基因的克隆及序列分析

Cloning and sequence analysis of ureB gene of Helicobacter pylori

目的 对幽门螺杆菌 (Hp)ureB核苷酸序列及推定氨基酸序列进行同源性比较分析 ,确定Hp菌株的ureB基因差异性 ,为疫苗的研究和诊断用品的评价提供依据。方法 自行设计引物 ,PCR扩增来自国内不同病人的 4株Hp菌株的ureB基因 ,与pGEM T载体克隆连接、测序 ;进一步同GenBank上发表的其他 4株国外Hp菌株ureB的全基因序列进行比较分析。结果 8株Hp菌株的ureB基因序列出现了 3种长度 ,CAPMF3和CPM6 30分别为 12 6 9bp和 16 80bp ,其他为 1710bp。 6株1710bp的ureB基因序列的分析表明 ,国外 3株Hp的ureB同源性较高 ,三者氨基酸的同源性达到10 0 %。而国内 3株Hp的ureB变异较大 ,推定氨基酸同源性为 98 7%～ 98 9%。结论 UreB作为保护性抗原时存在免疫保护覆盖率问题 ,研究疫苗和诊断用品时要注意选择使用在人群中流行占优势的Hp菌株的高度保守的UreB抗原肽 ,使其具有更高的覆盖率。

Objective To determine the diversity of ureB gene of H. pylori for development of vaccines and evaluation of diagnostic kits by sequence analysis of ureB genes of 8 H. pylori . Methods Primers were designed according to the sequence of strain 26695 and used to amplify the ureB gene of H. pylori (CAPM N62, CAMP N93, CAMP N98, CAMP F3). The PCR products were cloned into pGEM T vector respectively and sequenced. The sequences of tested strains were compared with the corresponding regions of the GenBank strains (26695, J99, MD506, CPM630) and analyzed by DNA and Winstar software. Results There are three kinds of length of ureB gene in all 8 strains. The lengths of ureB gene of CAMP F3 and CPM630 are 1269bp and 1680bp respectively. The others are 1710bp. The result of the sequence analysis of ureB gene of 6 strains of H. pylori with length of 1710bp indicated that the three foreign strains showed high homology in nucleotide acid and their homology were 100% in putative amino acid. The three domestic strains were closely related. Conclusions As a potential protective antigen, ureB has the problem of variable coverage rate in different populations. In order to gain extensive coverage rate, it is important to find the antigen peptide of conservative ureB gene of H. pylori which is predominant in population.