论文部分内容阅读
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 ,而与信号序列无关
Objective To determine the allelic variation of the full-length vacA gene of Helicobacter pylori (Hp) and its difference with the major Western strains. Methods Two single Hp clones were isolated and cultured from gastric mucosa biopsies from 5 gastric patients. The vacA phenotype was determined by Western blot, and its cytotoxicity was tested by in vitro cytotoxicity assay. Then, five representative clones were selected for genomic DNA extraction, PCR amplification and vacA gene complete sequence analysis. Results 4 of 5 strains were positive for vacA. Only one strain (600d) induced HeLa cells to produce significant vacuolar degeneration in vitro. The five vacA genes share the same signal sequence (sla) and similar gene sequences encoding a relative molecular mass of 37 x 10 3 and the outer membrane exporter (OME). The four vacA genes in the middle region (encoding a relative molecular mass of 5 8 × 10 3 protein) showed the m2-type allele, one strain was m1 type. Homology of HpvacA gene similarity rate was 97.6% ~ 99.2%, higher than the Western homotype similarity rate (92.1%). Conclusion The Chinese HpvacA gene has significant allelic variation compared with Western strains, forming a relatively independent sub-cluster. m2 type strains than m1 type strains common. The cytotoxic activity is related to the type of vacA gene in the middle, but not to the signal sequence