幽门螺杆菌硫氧还蛋白两种不同亚型基因序列的测定及分析

摘要

目的 测定不同胃疾病分离的幽门螺杆菌(Hp)临床菌株中硫氧还蛋白(Trx)1、2基因序列,并确定其氨基酸序列,探讨其与相关疾病的关系.方法 通过胃镜获取慢性胃炎、消化性溃疡、胃癌3种疾病共25例患者的胃黏膜组织,从中分离培养Hp菌株,提取基因组DNA.根据GenBank基因组中Hp基因序列设计Trx1、Trx2引物,应用聚合酶链反应(PCR)进行扩增,对扩增产物进行序列测定及生物信息学分析,并与国际标准菌株比对.结果 应用PCR的方法成功扩增出Trx1全基因序列及Trx2基因前295核苷酸.应用BioEdit软件进行核苷酸和氨基酸同源性分析,不同疾病分离的Hp Trx1均含有321个核苷酸,总突变位点率13.4%(43/321);编码106个氨基酸,含有一个Cys-Gly-Pro-Cys氧化还原活性位点,25例不同胃疾病Hp TRX1的氨基酸同源性高达97.2%(103/106).Trx2的前295个核苷酸,总突变位点率18.6%(55/295);编码的氨基酸无Cys-Gly-Pro-Cys,含有一个CXXC区域,氨基酸同源性为84.7%(83/98).结论 Hp临床分离菌株的Trx两个亚型核苷酸序列均有不同程度的位点突变,但氨基酸的序列同源性较高,均为无效突变.不同疾病分离的Hp Trx1均含有一个Cys-Gly-Pro-Cys氧化还原活性位点,TRX2仅含有一个CXXC区域.研究结果为进一步探讨Hp Trx的生物学功能及其致病机制提供了可靠依据.%Objective To analyze the gene sequences of Trx1 and Trx2 of Helicobacter pylori (HP)from different gastric diseases, define the amino acid sequences and investigate the relationship with these diseases. Methods The HP strains were isolated from gastric mucosa of 25 patients with chronic gastritis,peptic ulcer and gastric cancer respectively and cultured on solid blood agar medium. The primers of Trx1 and Trx2 were designed according to the sequences of GenBank. The Trx1 and Trx2 were respectively amplified by PCR (polymerase chain reaction) and were sequenced by the bioinformatics method. The sequences were compared with those of the international standard HP strains. Results The whole sequence of Trx1 and the first 295 bp of Trx2 were successfully amplified to allow for sequence comparison by BioEdit.The Trx1 from different HP strains contained 321 bp encoding 106 amino acids. The mutation ratio was 13.4% (43/321). All amino acids contained the same active motif Cys-Gly-Pro-Cys. The homology of Trx1 amino acids from 25 strains was 97.2% (103/106). The mutation ratio for first 295 bp of Trx2 was 18.6% (55/295). All amino acids encoded by Trx2 contained one CXXC zone while the Cys-Gly-Pro-Cys motif was not found. The homology of Trx2 amino acids was 84. 7% (83/98). Conclusion Two different subtypes of Trx from clinically isolated Hp strains have mutation sites. But the homology of encoded amino acids is relatively high because of invalid mutations. Trx1 of HP strains from different diseases all contains a redox active motif Cys-Gly-Pro-Cys while Trx2 contains only a CXXC zone. The above results will provide valuable rationales for future studies of the biological function and pathogenic mechanisms of HP Trx1 and Trx2.