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目的构建用于幽门螺杆菌(helicobacter pylori,Hp)同源重组双交换基因敲除的质粒载体,实现Hp内基因的快速敲除。方法以质粒pLYL03为骨架,重新排列质粒上的基因元件以减少冗余序列,用卡那霉素抗性基因(aphA)替换原质粒上的红霉素抗性基因作为抗性筛选标记,在抗性筛选标记两端构建多克隆位点用于同源重组同源臂的插入,由此构建用于Hp同源重组双交换基因敲除的质粒载体pSJHK。以质粒pSJHK为载体构建hp0169基因的同源重组双交换敲除质粒pSJHK-0169,通过电击转化方法将敲除质粒pSJHK-0169转入Hp中,用卡那霉素筛选转化子。结果以质粒pLYL03为基础构建用于Hp同源重组双交换基因敲除的质粒载体pSJHK,大小4 371bp。以基因hp0169作为目标基因构建同源重组双交换敲除质粒pSJHK-0169,经电击转化Hp中,获得卡那霉素抗性阳性转化子,测序确证基因敲除成功。结论构建的基因敲除质粒载体pSJHK实现了对Hp中目标基因的敲除,为Hp新基因功能研究和致病因子的发掘提供了有效的基因操作工具。
Objective To construct a plasmid vector for knocking out homologous recombination double-exchange gene of Helicobacter pylori (Hp) and achieve rapid knockdown of Hp gene. Methods Plasmid pLYL03 was used as the backbone to rearrange the genetic elements in the plasmids to reduce the redundant sequences. The erythromycin resistance gene on the original plasmid was replaced by the kanamycin resistance gene (aphA) A multiple cloning site was constructed at both ends of the selection marker for insertion of homologous recombination homology arms, thereby constructing a plasmid vector pSJHK for Hp homologous recombination double crossover gene knockout. The pSJHK-0169 homologous recombination knockout plasmid hp0169 was constructed by using plasmid pSJHK as a vector. The knockout plasmid pSJHK-0169 was transformed into Hp by electroporation and the transformants were screened by kanamycin. Results Plasmid pSJHK was constructed based on plasmid pLYL03. The plasmid pSJHK was used to knockdown Hp homologous recombination double crossover gene and was 4 371 bp in size. The homologous recombination double crossover knockout plasmid pSJHK-0169 was constructed with the gene hp0169 as a target gene and transformed into Hp by electroporation to obtain a kanamycin-resistant transformant. Sequencing confirmed that the gene knockout was successful. CONCLUSION: The constructed knockout plasmid pSJHK can knock out the target gene in Hp and provide an effective gene manipulator for the study of new Hp gene function and virulence factor discovery.