摘要
目的:构建cagA基因缺失的中国幽门螺杆菌突变株.方法:运用基因同源重组方法将卡那霉素抗性基因(kmR)连接到PCR扩增cagA基因两端区域产生的2个基因片段之间,并共同插人到pBluescriptSKⅡ(-)载体的多克隆位点中,构建出带卡那霉素抗性标志的缺失突变载体pBs-cagA-mutant.将突变载体转化到含完整cagA基因的突变受体MEL-Hpylori27菌株中.卡那霉素筛选出cagA基因缺失的中国幽门螺杆菌突变株,并经PCR和DNA测序鉴定.结果:构建的突变载体经限制性内切酶酶切分析显示,产生的条带与设计结果完全一致.PCR方法扩增突变株cagA,kmR基因显示,cagA基因已被完整敲除掉,DNA测序证实筛选得到了cagA基因缺失的幽门螺杆菌突变株.连续培养25代后证实,幽门螺杆菌突变株具有良好稳定性.结论:首次成功构建出1株中国人来源的、cagA基因缺失的幽门螺杆菌突变株.
AIM： To knock out the entire cagA gene of Chinese Helicobacter pylori strain via homologous recombination and construct a cagA-deleted mutant strain of Chinese H pylori.
METHODS： Two DNA fragments locating in the upper and downstream of cagA gene were amplified and a kanamycin resistance gene of pEGFP-N2 between them were engineered into pBluescript SK Ⅱ（-） plasmid （pBs-cagA-mutant）. Electrotransformation of H pylori cells with pBs- cagA-mutant resulted in isolation of kanamycinresistant H pylori transformants, which was identified by polymerase chain reaction （PCR） and sequencing analysis.
RESULTS： Restriction endonuclease analyses showed that pBs-cagA-mutant vector had been successfully recombined. The cagA-deleted status of Chinese H pylori mutants was confirmed by PCR with primers specific for the genes of cagA and kanamycin resistance. After 25 generations of culture, the constructed Chinese H pylori mutants were confirmed stable.
CONCLUSION： A cagA-negative mutants of Chinese H pylori strain is firstly constructed, which can help to further study the role of cagA in the pathogenesis of H pylori infection.
出处
《世界华人消化杂志》
CAS
北大核心
2006年第33期3190-3194,共5页
World Chinese Journal of Digestology
