球形幽门螺杆菌vacA基因的克隆及序列测定

Cloning and Sequencing of vacA Gene from a Helicobacter pylori Strain with Coccoid Form

目的:克隆球形幽门螺杆菌(HpC)vacA基因全长片段,并进行序列分析。方法:采用亚抑菌浓度的抗生素作用,使幽门螺杆菌(Hp)标准株NCTC11637发生球形变异,收集球形Hp。从HpC基因组DNA中特异扩增出vacA基因片段,扩增的目的片段经纯化回收后克隆到pMD-18T中,转化人大肠杆菌JMl09。阳性重组质粒用PCR及双酶切鉴定,并进行序列测定。结果:从HpC基因组DNA中扩增出3 888 bp的vacA基因,构建重组质粒pMD-18T-vacA,酶切产物的大小与预期相符。测序结果显示,HpC与已公布的Hp vacA基因序列的同源性达99.8％。结论:成功地对HpC、vacA基因进行体外扩增及构建原核重组质粒pMD-18T-vacA,并经酶切及序列分析所验证,证明HpC含有完整的vacA基因,其可能与Hp的致病性有关。

To clone and sequence the vacA gene from a Helicobacter pylori (Hp) strain with coccoid form. Methods: Hp strains NCTC 11637 were transformed to coccoid form by exposure to antibiotics in subinhibitory concentrations. The coccoid Hp was collected. The vacA gene of coccoid Hp strain was amplified by PCR. After being purified, the target fragment was cloned into plasmid pMD-18T. The recombinant plasmid pMD-18T-vacA 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 analyzed. Results: The vacA gene of 3 888 bp was obtained from the coccoid Hp genome DNA. The recombinant plasmid pMD-18T-vacA was constructed, then digested by restriction endonucleases of BamH I + Sac I, and the product of digestion was identical with the expected one. Sequence analysis showed that the homology between coccoid and the reported original sequence Hp was 99. 8% . Conclusion: The recombinant plasmid containing vacA gene from coccoid H. pylori has been constructed successfully. The coccoid Hp contain completed vacA gene, which may be related to pathogenicity of them.