幽门螺杆菌球形体cagA基因片段的克隆与测序
Cloning and sequencing of cagA gene fragment of Helicobacter pylori with coccoid form.
作者信息
Wang Ke-Xia, Wang Xue-Feng
机构信息
School of Medicine, Anhui University of Science and Technology, Huainan 232001, Anhui Province, China.
出版信息
World J Gastroenterol. 2004 Dec 1;10(23):3511-3. doi: 10.3748/wjg.v10.i23.3511.
AIM
To clone and sequence the cagA gene fragment of Helicobacter pylori (H pylori) with coccoid form.
METHODS
H pylori strain NCTC11637 were transformed to coccoid form by exposure to antibiotics in subinhibitory concentrations. The coccoid H pylori was collected. cagA gene of the coccoid H pylori strain was amplified by PCR. After purified, the target fragment was cloned into plasmid pMD-18T. The recombinant plasmid pMD-18T-cagA was transformed into E.coli JM109. Positive clones were screened and identified by PCR and digestion with restriction endonucleases. The sequence of inserted fragment was then analysed.
RESULTS
cagA gene of 3,444 bp was obtained from the coccoid H pylori genome DNA. The recombinant plasmid pMD-18T-cagA was constructed, then it was digested by BamH I+Sac I, and the product of digestion was identical with the predicted one. Sequence analysis showed that the homology of coccoid and the reported original sequence H pylori was 99.7%.
CONCLUSION
The recombinant plasmid containing cagA gene from coccoid H pylori has been constructed successfully. The coccoid H pylori contain completed cagA gene, which may be related to pathogenicity of them.
目的
克隆并测序球形幽门螺杆菌（H pylori）的cagA基因片段。
方法
将幽门螺杆菌菌株NCTC11637暴露于亚抑菌浓度的抗生素中使其转化为球形。收集球形幽门螺杆菌。通过PCR扩增球形幽门螺杆菌菌株的cagA基因。纯化后，将目标片段克隆到质粒pMD - 18T中。将重组质粒pMD - 18T - cagA转化到大肠杆菌JM109中。通过PCR和限制性内切酶消化筛选并鉴定阳性克隆。然后分析插入片段的序列。
结果
从球形幽门螺杆菌基因组DNA中获得了3444 bp的cagA基因。构建了重组质粒pMD - 18T - cagA，用BamH I + Sac I消化后，消化产物与预测的一致。序列分析表明，球形幽门螺杆菌与已报道的原始序列幽门螺杆菌的同源性为99.7%。
结论
成功构建了含有球形幽门螺杆菌cagA基因的重组质粒。球形幽门螺杆菌含有完整的cagA基因，这可能与它们的致病性有关。
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