摘要
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。
AIM To construct a recombinant clone of Helicobacter pylori (Hp) ureC, a fragment from Hp ureC was cloned and sequenced. So as to lay the foundation for expressing recombinant protein of Hp ureC and for further studing its functions. METHODS. A 1173bp gene fragment was amplified by the polymerase chain reaction. The template DNA was purified from Hp clinical strain. We designed the primers located in 588bp-605bp and 1737bp-1754bp, respectively. The gene fragment was digested by BamHⅠ and EcoR Ⅰ and cloned into pGEM-3Zf (-) plasmid. Its sequence was determined by autossquencing instrument from two directions. With the help of DNA TOOL 5.1, we got the complete DNA sequence. The sequence was compared with those having the corresponding region of ureC from Hp standard strains. RESULTS Ninety-five percent of the DNA sequence we got was the same with that of Hp strain M60398. BLAST analysis showed that 97% amino acids by the DNA sequence was the same with that of Hp standard strains. CONCLUSION A recombinant clone of Hp ureC was established, which laid the foundation for expressing recombinant protein of Hp ureC and further studing its functions.
出处
《世界华人消化杂志》
CAS
2001年第3期308-311,共4页
World Chinese Journal of Digestology
关键词
尿素酶
遗传学
幽门螺杆菌
酶学
分离
提纯
聚合酶链反应
胃肠道疾病
urease/genetics
Helicobacter pylori /enzymology
Helicobacter pylori/isolation & purification
polymerase chain reaction
