中国幽门螺杆菌vacA基因的等位变异

Allelic variation in the vacA gene of Helicobacter pylori isolated from China

目的 明确中国幽门螺杆菌 (Helicobacterpylori,Hp)全长vacA基因的等位变异及其与西方主要菌株基因序列的差异。方法 从 5例胃病患者胃黏膜活检组织中分离培养 2 2个单个Hp克隆 ,以Westernblot确定vacA表型特点 ,以体外细胞毒性试验确定其活性。然后选择 5株有代表性的克隆进行基因组DNA抽提、PCR扩增及vacA基因全序列测定、分析。结果 5株中 4株vacA表达阳性。只有 1株 (5 0 6 0d)可在体外诱导HeLa细胞产生显著空泡变性。 5株vacA基因有相同的信号序列 (sla)及相似的编码相对分子质量为 37× 10 3 和跨膜输出段 (outermembraneexporter ,OME)的基因序列。 4株vacA基因的中间区 (编码相对分子质量为 5 8× 10 3 蛋白 )表现为m2型等位基因 ,1株为m1型。同型中国HpvacA基因相似率为 97.6 %～ 99.2 % ,高于西方同型相似率 (92 .1% )。 结论 中国人HpvacA基因与西方菌株相比存在显著的等位变异 ,形成相对独立的一个亚簇。m2型菌株比m1型菌株常见。细胞毒活性与vacA基因中间区类型有关 。

Objective To characterize the vacA alleles that tare present in Chinese H.pylori group and compare the allelic variation in the vacA gene with the Western strains. Methods 22 single colonies of H.pylori isolated from 5 biopsies with different gastric disorders were characterized for expression of VacA protein and for evaluating the cytotoxic activity in HeLa cell. Five representative strains were selected and the vacA gene was amplified by PCR and subjected to automatic DNA sequencing using a primer walking strategy. Results Four strains expressed VacA protein. Only one of the five strains induced the vacuoles in HeLa cell. All the five vacA gene coded for identical signal peptides of the s1a allele and an eight amino acids represent at the reported proteolytic cleavage site. One of the five strains expressed a VacA protein with a middle region most similar to the m1 allele and the others to the m2 allele. The four m2 allele strains had few vacuolating activity in HeLa cell. The phylogenetic analysis indicate that the homologues of same type Chinese vacA genes were 97.6%～99.2% compared with those 91.3%～92.0% of the Western strains. Conclusions There are allelic variations of vacA gene between Chinese H.pylori and the Western's. It forms phylogenetically a independent cluster. The type m2 strains seem to be more prevalent in China. The cytotoxicity in HeLa cell appears to associate with the middle region but not signal sequence.