幽门螺杆菌cagA基因全长克隆及序列分析

目的探索扩增cagA基因全长的PCR方法,克隆中国2株胃癌幽门螺杆菌(Helicobacter pylori,Hp)分离株和1株十二指肠溃疡分离株及国际标准株SS1的cagA基因,初步探讨cagA序列和磷酸化位点变异及其临床意义.方法针对已知cagA基因两侧及cag致病岛右侧反转重复序列设计PCR引物,扩增cagA基因并克隆测序,分析已知cagA序列同源性及其酪氨酸磷酸化位点.结果3株中国Hp分离株cagA氨基酸序列的同源性约为94%(93.9%、94%、94.5%),并都有pY117/118/122和EPIYA-A、B、D 4个酪氨酸磷酸化位点.SS1株cagA与西方菌株同源性大于85%,而与中国菌株的同源性小于80%,具有pY117/118/122和EPIYA-A、B、C 4个酪氨酸磷酸化位点.结论建立的PCR方法可扩增cagA基因全长.CagA序列变异和磷酸化位点具有地区聚类特征,但与Hp感染临床结局无特殊关联.

Cloning and sequence analysis of cagA gene in full-length of Helicobacter pylori

Objective To establish PCR method for amplifying cagA gene in full-length, and clone the cagA genes of two Chinese H. pylori strains isolated from patients with gastric cancer and one from patients with duodenal ulcer as well as that of Sydney strain 1 so as to investigate the heterogeneity of the amino acid sequences and phosphorylation sites of CagA and its clinical significance. Methods PCR primers designed according to the nucleotide acid sequence flanking the open reading frame of cagA and the lower reverse repeat sequence of cag pathological island were used to amplify cagA genes of H. pylori strains. PCR products were then cloned and sequenced, and the homology of all published CagA amino acid sequences and their putative phosphorylation sites were analyzed. Results The amino acid sequences of the three Chinese H. pylori strains revealed about 94% homologous to each other (93.9%, 94%, and 94.5%), and four tyrosine phosphorylation motifs (pY117/118/122, EPIYA-A, B, and D) present in CagA protein of all three Chinese isolates. The SS1 CagA showed more than 85% homologous to Western H. pylori strains while less than 80% to Chinese H. pylori strains, and four tyrosine phosphorylation motifs (pY117/118/122, EPIYA-A, B, and C) present in it. Conclusion A PCR method to amplify cagA genes in full-length has been established. Although distinct geographical cluster characteristics exist, the heterogeneity of amino acid sequence and phosphorylation sites of CagA are not specifically associated with the manifestation of H. pylori infection.