幽门螺杆菌γ-谷氨酰转肽酶基因的克隆及序列分析

Cloning and sequence analysis of γ-glutamyltranspeptidase gene of Helicobacter pylori

目的:克隆不同疾病来源的幽门螺杆菌(Helicobacter pylori,Hp)γ-谷氨酰转肽酶(Gamma-glutamyltranspep-tidase,GGT)基因,并进行序列分析。方法:从人胃黏膜组织中分离培养获得Hp,提取其基因组DNA,对GGT基因进行PCR扩增,克隆进pMD18-T载体,进行测序和生物信息学分析,并与基因库中公布的标准株26695和J99的GGT氨基酸序列进行比对分析。结果:成功克隆了分别来源于慢性胃炎,胃溃疡,十二指肠溃疡和胃癌的4种Hp菌株GGT片段,并进行了序列分析。4种病源的HpGGT序列大小均为1 704 bp,编码567个氨基酸,理论分子质量是61kDa,等电点是9.3,在N端具有信号肽,含有ATP蛋白激酶结合结构域和γ-谷氨酰转肽酶保守结构域。序列比对分析显示,不同来源的Hp菌株GGT具有很高的保守性。结论:GGT在Hp中高度保守,具有分泌作用,可能对宿主细胞产生影响。该基因的成功克隆为进一步研究其功能打下了基础。

Objective： To clone and analyze the γ-glutamyltranspeptidase （GGT） gene of Helicobacter pylori（H, pylori）. Methods： The strains of H. pylori were isolated from human gastric mucosa with the diseases of chronic gastritis, gastric ulcer, duodenal ulcer and gastric cancer;and cultured on solid agar medium. The GGT amplified by PCR from H. pylori DNA was cloned into T vector. The gene was sequenced and analyzed by the bioinformatics method. The sequences were compared with the typic H. pylori strains 26695 and J99. Results： The GGT gene was successfully cloned into pMD18-T vector. The GGT contains 1 704 bp and encodes 567 amino acids with a predicted of 61 kDa, PI was 9.3. The gene had a signal peptide in the N-terminal and there were both protein kinases ATP-binding region signature and the γ-glntamyltranspeptid- ase signature. The sequence analysis showed that the amino acid sequences were highly conserved in different H. pylori strains. Conclusion ： GGT of H. pylori is a conserved amino acid and has an important role in host cells. Cloning this gene will be helpful in its functional study.