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目的分离鉴定临床分离株幽门螺杆菌(Helicobacter pylori,Hp),分析其CagA蛋白的磷酸化基序EPIYA,探讨东亚株Hp CagA序列的结构特点。方法选取胃相关疾病患者的胃黏膜组织,剪碎后接种于哥伦比亚血琼脂平板,微需氧培养,菌落生长后通过尿素酶试验、革兰染色及显微镜检查对Hp进行初步鉴定。提取菌落基因组,PCR扩增Hp 16SrRNA基因并进行测序鉴定;扩增cagA基因全长序列,连接入pMD18-T载体。将重组质粒转入感受态E.coli DH5α,测序鉴定重组质粒,利用DNAStar和MEGA 6软件对cagA序列进行比对及聚类分析。结果成功分离Hp共26株。构建pMD18-T/cagA克隆载体后对26株Hp的cagA基因全长测序,序列比对分析显示有6株为西方型,20株为东亚型。东亚型CagA第815~834位点存在13个氨基酸的缺失、部分缺失或变异,西方型中有3株丢失磷酸化位点EPIYA-C;聚类分析显示Hp分离株分别聚类为东亚群、西方型东亚群以及西方群3群。结论 Hp分离株以东亚株为主,但也存在西方株感染;东、西方型CagA在EPIYA基序及侧翼序列显著差异,菌株之间存在聚类关系。
OBJECTIVE: To isolate and identify Helicobacter pylori (Hp) from clinical isolates and analyze the phosphorylation motif EPIYA of CagA protein to investigate the structural characteristics of Hp CagA sequence in East Asia. Methods Gastric mucosa tissues of patients with gastric-related diseases were selected and cut into pieces and then inoculated on a blood agar plate in Colombia. After microaerophilic culture, the colonies were inoculated with urease, Gram stain and microscopy to identify Hp. The colony genome was extracted and the Hp 16S rRNA gene was amplified by PCR and sequenced. The full-length cagA gene was amplified and ligated into pMD18-T vector. The recombinant plasmid was transformed into competent E. coli DH5α, and the recombinant plasmid was identified by sequencing. DNAStar and MEGA 6 software were used to compare and cluster cagA sequences. Results A total of 26 Hp strains were successfully isolated. After construction of the cloning vector pMD18-T / cagA, the full length of cagA gene of 26 Hp strains was sequenced. Sequence alignment analysis showed that 6 strains were Western type and 20 were East type. There were 13 amino acid deletions, partial deletions or variations in the 815 ~ 834 locus of East Asian CagA, and 3 loci of EPIYA-C lost in the western type. Cluster analysis showed that Hp isolates clustered into East Asian subgroups, Western-style East Group and West Group 3. Conclusion The strains of Hp isolated from East Asia were predominant, but Western strains were also infected. The CagA of East and West showed significant differences in EPIYA motif and flanking sequences, and there was clustering relationship among the isolates.