摘要
目的构建幽门螺杆菌(Helicobacter pylori,Hp)hp0169基因的敲除突变株,研究其在Hp感染GES-1细胞中的作用。方法敲除质粒pSJHK为模板,通过同源重组双交换的方法构建hp0169基因敲除突变株(Δ0169),分析其与野生26695株的生长曲线及琼脂表面菌落扩散情况;通过FITC标记法检测两菌株对GES-1细胞的吸附率;以感染复数(MOI=200)构建Hp感染GES-1细胞体系,比较Δ0169突变株与野生株感染后致细胞凋亡及细胞活性变化的差异。结果通过质粒敲除、电击转化成功获得hp0169基因敲除突变株Δ0169。该突变株与野生菌株的生长曲线、琼脂表面菌落扩散情况基本一致,对GES-1细胞的吸附率差异无统计学意义(t值为1.102,P>0.05)。Δ0169突变株与野生株感染GES-1细胞活力分别为6h(0.74±9.56)%、(0.90±6.31)%,12h(0.53±7.89)%、(0.73±8.31)%,差异均有统计学意义(t值分别为-3.474,-2.880,P<0.05);野生组和突变组GES-1细胞凋亡率分别为6h(19.2±11.03)%、(20.4±8.67)%和12h(29.3±14.47)%、(28.6±10.32)%,差异均无统计学意义(t值分别为-0.995,0.218,P>0.05)。结论 Hp hp0169基因敲除后不影响其生长、运动以及对GES-1细胞的吸附,不影响细菌感染GES-1细胞致细胞凋亡的程度,但影响细菌感染致细胞活力下降的程度。这对研究Hp感染及致病机制有重要意义。
Objectives To construct an hp0169 gene knockout mutant strain and to analyze the function of the hp0169 gene in Helicobacter pylori infection of GES-1 cells. Methods The plasmid pSJHK was used as a template to generate an hp0169 knockout plasmid via double-crossover recombination. The knockout mutant △0169 was obtained via electropo- ration of the knockout plasmid. The growth curve and cell motility of wild-type H. pylori （WT） and △0169 were compared. Adhesion of two strains to GES-1 cells was detected using FITC labeling. In addition, H. pylori was co-cultured with GES-1 cells at an MOI of 200. The viability and apoptosis of infected GES-1 cells were determined and compared between WT H. pylori and A0169. Cell viability was determined by counting living cells using Cell Counting Kit 8 （CCK- 8）. The apoptosis of infected GES-1 cells was determined using the Annexin V-FITC Apoptosis Detection Kit, and the relative number of apoptotic cells was detected using flow cytometry. Results An hp0169 knockout mutant, △0169, was constructed via electroporation of a plasmid and knock out of a gene. The △0169 mutant had a growth curve and level of cell motility similar to the growth curve and level of cell motility of WT H. pylori, and cell adhesion did not differ sig-nificantly for WT H. pylori and △0169. When GES-1 cells were infected with WT H. pylori, their viability at 6 h was 0. 74±9. 56% ; when those cells were infected with △0169, their viability at 6 h was 0. 90±6. 31%. When GES-1 cells were infected with WT H. pylori, their viability at 12 h was 0.53±7.89% when those cells were infected with △0169, their viability at 12 h was 0. 73±8. 31%. The viability of GES-1 cells differed significantly （t= -3. 474, -2. 880, P〈0.05） when they were infected with WT H. pylori or the mutant. When GES-1 cells were infected with WT H. pylori, the rate of their apoptosis at 6 h was 19.2±11.03%; when those cells were infected with △0169, the rate of their apopto- sis at 6 h was 20.4±8.67%. When GES-1 cells were infected with WT H. pylori, the rate of their apoptosis at 12 h was 29.3±14.47%; when those ceils were infected with △0169, the rate of their apoptosis at 12 h was 28.6±10.32. The rate of apoptosis did not differ significantly （t=-0. 995, 0. 218, P〉0.05） between WT H. pylori and the mutant. Conclusion Deletion of hp0169 did not affect bacterial growth, motility, adhesion to GES-1 cells, or apoptosis of infected GES-1 cells. However, GES-1 cells infected with the △0169 mutant had a higher cell viability compared to those infected with WT H. pylori, suggested that hp0169 plays a role in H. pylori infection. This finding is crucial to studying the pathogenesis of H. pylori.
作者
赵慧琳
吴玉龙
徐正
丁雲飞
张艳丽
杜镇镇
张珍
李波清
季晓飞
ZHAO Hui-lin;WU Yu-long;XU Zheng;DING Yun-fei;Zhang Yan-li;DU Zhen-zhen;ZHANG Zhen;LI Bo-qing;JI Xiao-fei(Department of Pathogen Biology,Binzhou Medical University,Yantai,Shandong,China 26400)
出处
《中国病原生物学杂志》
CSCD
北大核心
2018年第10期1062-1066,共5页
Journal of Pathogen Biology
基金
国家自然科学基金面上项目(No.81672044)
国家自然科学基金青年基金项目(No.81702054
81501718)
山东省自然科学基金培养基金项目(No.ZR2017BC011)
