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目的构建幽门螺杆菌ATCC26695hp0788基因敲除突变菌株(ATCC26695△0788km),观察hp0788基因对GES-1细胞功能的影响。方法以基因敲除质粒载体pSJHK构建基因敲除质粒,在hp0788基因的上下游分别扩增800~1100bp的基因片段作为同源臂,经限制性内切酶酶切后与质粒载体连接构建基因敲除质粒pSJHK-0788,通过电击转化构建hp0788基因敲除突变菌株,以FITC标记法检测其对GES-1细胞黏附能力的影响;以感染复数(MOI)为200:1构建幽门螺杆菌与GES-1细胞共培养体系,比较ATCC26695和ATCC26695△0788km对GES-1细胞凋亡及活性的影响。结果构建了hp0788基因双交换敲除质粒,电击转化获得hp0788基因敲除突变菌株。FITC标记ATCC26695和幽门螺杆菌ATCC26695△0788km与GES-1细胞混匀后采用流式细胞术检测FITC的荧光强度,ATCC26695组的黏附率记为100%,ATCC26695△0788km组黏附率为90.40%,差异均有统计学意义(t=2.80,P<0.05);ATCC26695与ATCC26695△0788km分别与GES-1细胞共培养8h和16h,流式细胞术检测细胞凋亡率分别为(15.73±7.84)%、(25.26±5.81)%和(12.46±12.30)%、(21.13±10.09)%,细胞增殖-毒性检测试剂盒检测细胞活力分别为53%、40%和66%、50%,差异均有统计学意义(t值分别为3.30,2.80,-2.93,-2.76,P<0.05)。结论hp0788基因敲除幽门螺杆菌ATCC26695对GES-1细胞的黏附率下降,并使GES-1凋亡率下降,而细胞活力增高。表明hp0788基因是影响幽门螺杆菌ATCC26695感染GES-1细胞后引起细胞凋亡和活性变化的重要基因。
Objective To construct the gene knock-out mutant (ATCC26695 △ 0788km) of Helicobacter pylori ATCC26695hp0788 and observe the effect of hp0788 gene on the function of GES-1 cells. Methods Gene knockout plasmids were constructed by gene knockout plasmid pSJHK. The 800 ~ 1100bp gene fragment was amplified upstream and downstream of hp0788 gene as a homology arm. After restriction endonuclease digestion, the plasmid was ligated with plasmid vector In addition to the plasmid pSJHK-0788, the hp0788 gene knockout mutant strain was constructed by electroporation, and the effect on the adhesion of GES-1 cells was detected by FITC labeling method. The Helicobacter pylori and GES- 1 cell co-culture system to compare ATCC26695 and ATCC26695 △ 0788km on the apoptosis and activity of GES-1 cells. Results The hp0788 gene double knockout plasmid was constructed and transformed into hp0788 knockout mutant. FITC-labeled ATCC26695 and Helicobacter pylori ATCC26695 △ 0788km and GES-1 cells were mixed by flow cytometry FITC fluorescence intensity, the adhesion rate of ATCC26695 group was recorded as 100%, ATCC26695 △ 0788km group adhesion rate of 90.40%, the difference (T = 2.80, P <0.05). The apoptosis rates of ATCC26695 and ATCC26695 △ 0788km co-cultured with GES-1 cells for 8 and 16 h respectively were (15.73 ± 7.84)% and (25.26 ± 5.81)% and (12.46 ± 12.30)%, (21.13 ± 10.09)% respectively. The cell viability was 53%, 40%, 66% and 50% Significance (t = 3.30, 2.80, -2.93, -2.76, P <0.05). Conclusion The hp0788 gene knockdown of Helicobacter pylori ATCC26695 on GES-1 cells adhesion rate decreased, and the apoptosis rate of GES-1 decreased, while cell viability increased. Hp0788 gene is an important gene affecting the apoptosis and the activity of Helicobacter pylori ATCC26695 infected GES-1 cells.