幽门螺杆菌分离株iceA基因克隆及序列分析

Cloning and analysis of iceA gene of isolated Helicobacter pylori

目的克隆幽门螺杆菌中国郑州市慢性萎缩性胃炎患者分离株MEL-Hp27iceA基因,测序后对其进行序列分析。方法根据iceA基因上下游基因序列设计引物,PCR扩增iceA基因,并将其连接至pMD19-T载体,测序后采用相关数据库和生物信息学软件对基因序列进行分析。结果成功克隆幽门螺杆菌Hp27菌株iceA基因序列,扩增产物大小约为790 bp;重组质粒pMD19-T-iceA单酶切产生3 820 bp片段,双酶切产生3 000和820 bp的2个片段;Hp27iceA基因与大多数美国来源菌株同源性>85%,与日本、印度等地区来源菌株同源性<70%。序列分析结果表明,所克隆到的基因为iceA2亚型,与美国株Alaska strain 219、Alaska strain 213关系最近,同源性分别为87%和95%,与芬兰株Finland strain 9496等菌株存在聚类关系,同源性88%～93%,与CR9等其他菌株距离较远。结论成功克隆幽门螺杆菌Hp27菌株iceA基因序列;不同地区幽门螺杆菌分离株的iceA基因间存在着一定差异。

Objective To clone the gene（iceA） induced by contact with epithelium of Helicobacter pylori MEL-Hp27 isolated in China and to analyze its sequence.Methods Primers were designed according to the sequences located in the up-stream or down-stream of the iceA gene.The DNA was amplified by PCR and the fragment was inserted into plasmid pMD19-T for nucleotide sequencing.The bioinformatics analysis was carried out with databases of Blast or NCBI and software of DNAstar,Clustal W and MEGA4.0.Results The iceA gene of Hp27 strain was successfully cloned.The PCR product was about 790bp.The recombinant plasmid pMD19-T-iceA was digested with one restriction enzyme to produce a 3820bp DNA fragment and digested with two enzymes to produce 3000bp and 820bp DNA fragments.Sequence analysis showed that the iceA gene of MEL-Hp27 has more than 85% of homology with American H.pylori strains and less than 70% of homology with Japanese or Indian H.pylori strains.The-10 box was located in the up-stream of iceA gene and the distance from ATG is 17bp.The SD sequence is located in the up-stream of iceA gene and the distance from ATG is 7bp.There are two avariable numbers of tandem repeats between ATG and the up-stream gene cysE.There are differences in the characters of the gene between Hp27 strains and other Helicobacter pylori strains from other regions.Conclusion The iceA gene of Helicobacter pylori MEL-Hp27 was successfully cloned.There are differences among Helicobacter pylori strains from different regions.