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目的用PCR方法扩增幽门螺杆菌(Hp)尿素酶C基因,以构建Hp UreC基因的重组克隆,为进一步表达Hp UreC基因的重组蛋白质并研究其功能奠定基础。方法以Hp临床菌株总DNA为模板,根据已发表的HpUreC基因序列设计引物(位于588bp～605bp和1737bp～1754bp),采用PCR方法扩增出一个1173bp的Hp尿素酶C基因片段,用BamHⅠ及EcoRⅠ双酶切后将其克隆入测序质粒pGEM-3Zf(-)中,以全自动测序仪双向测定目的片段序列,借助于DNA TOOL 5.1拼接成完整序列,与已知的Hp UreC基因序列做比较。结果所得核酸序列与报道的Hp国际标准菌株M60398的UreC基因同源性为95％,根据测序结果推断其编码的氨基酸序列,并行BLAST分析,发现它与标准菌株M60398的氨基酸序列同源性为97％。结论成功构建了Hp UreC基因的重组克隆,为进一步研究表达Hp UreC基因的重组蛋白质并研究其功能奠定了基础。
Objective To amplify the urease C gene of Helicobacter pylori (Hp) by PCR to construct a recombinant clone of Hp UreC gene and lay a foundation for further expression of the recombinant protein of Hp UreC gene and its function. Methods Based on the published HpUreC gene sequences, primers were designed based on the published HpUreC gene sequences (588bp ~ 605bp and 1737bp ~ 1754bp). A 1173bp Hp urease C gene fragment was amplified by PCR. The BamHⅠ and EcoRIⅠ After double digestion, it was cloned into the sequencing plasmid pGEM-3Zf (-). The sequence of the target fragment was determined bi-directionally with a full-automatic sequencer. DNA TOOL 5.1 was spliced into the complete sequence to compare with the known Hp UreC gene sequence. Results The nucleotide sequence was 95% identical to the reported UreC gene of Hp international standard strain M60398. The deduced amino acid sequence was deduced from the sequencing results and BLAST analysis showed that the amino acid sequence of the homologue to the standard strain M60398 was 97 %. Conclusion The recombinant clone of Hp UreC gene was successfully constructed, which laid the foundation of further study of the recombinant protein of Hp UreC gene and its function.