幽门螺杆菌cheA基因在细菌体外趋化和体内定植过程中的作用
发布时间：2018-08-11 09:06
【摘要】：背景和目的幽门螺杆菌(Helicobacter pylori)是多种人胃炎和消化性溃疡的病原体,持续感染则与胃腺癌、胃粘膜相关淋巴组织淋巴瘤及原发性胃非何杰金淋巴瘤发病密切相关。在致病过程中,病原菌必然能感知并通过定向运动也即趋化(chemotaxis)到达合适的寄生部位进行定植(colonization),然后大量繁殖引起疾病。有文献报道,在有鞭毛的病原菌中,鞭毛提供细菌动力,但趋化运动受二元信号传导系统(two-component signaling system, TCSS)调控[4-7]。TCSS通常由跨膜组氨酸蛋白激酶(histidine protein kinase, HK)和胞浆反应调节蛋白(response regulator protein, RR)组成。在幽门螺杆菌基因组中,存在趋化相关TCSS (Che-TCSS) HK编码基因cheA及RR编码基因cheY[8],但作为Che-TCSS的CheA/CheY在该菌体外趋化和宿主体内定植中的作用,迄今未见文献报道。 我们以往研究结果证实,H离子是诱导幽门螺杆菌趋化的信号分子,同时建立了幽门螺杆菌体外趋化模型。本研究中,我们根据同源重组交换原理,采用基于自杀质粒的基因敲除技术构建了幽门螺杆菌NCTC11637株cheA基因敲除突变株(cheA-),然后分别采用幽门螺杆菌体外趋化模型及小鼠感染模型等生物学试验,探讨了cheA基因在幽门螺杆菌趋化和定植中的作用。 实验方法从幽门螺杆菌NCTC11637株基因组DNA中扩增并克隆全长cheA和cheY基因。构建该两个基因原核表达系统,Ni-NTA亲和层析法提取目的重组蛋白rCheA和rCheY. rCheA和rCheY免疫家兔制备抗血清,采用硫酸铵沉淀法及DEAE-52柱层析法制备rCheA-IgG和rCheY-IgG.构建cheA基因自杀质粒,根据同源重组交换原理利用该自杀质粒构建cheA基因敲除突变株(cheA-),采用PCR及测序对cheA突变株进行鉴定。采用rCheA-IgG和rCheY-IgG锚定靶蛋白及蛋白磷酸化检测试剂盒,测定cheA突变株与野生株CheA和CheY分子磷酸化水平。采用幽门螺杆菌趋化模型及BALB/c小鼠感染模型,比较cheA突变株与野生株体外趋化及体内定植能力的差异。 结果PCR及测序结果证实cheA-突变株基因组中cheA基因被敲除。0.001～0.1 mol/L盐酸作用10 min,野生株CheA和CheY磷酸化水平分别从59.6±11.5和55.5±10.2μmol迅速下降至10.8±2.6和5.5±1.2μmol(P0.05), cheA-突变株CheY磷酸化水平均较低且无明显变化(P0.05)。cheA突变株对盐酸、硫酸和乙酸趋化聚集环直径(10～20±2.0～3.0 mm)明显小于野生株(16～24±2.0～3.0 mm)(P0.05)。野生株感染小鼠胃黏膜标本中幽门螺杆菌分离阳性率(90%)明显高于cheA突变株(40%)(P0.05),荧光定量PCR结果也显示野生株感染小鼠胃黏膜标本中幽门螺杆菌数量(6.3±2.1×103 copies/mg)也明显高于突变株的(8.3±3.1×101 copies/mg) (P＜0.05) 结论cheA基因在幽门螺杆菌体外趋化和体内定植中有重要作用。
[Abstract]:Background and objective Helicobacter pylori (Helicobacter pylori) is a pathogen of many kinds of human gastritis and peptic ulcer. Persistent infection is closely related to gastric adenocarcinoma, gastric mucosa-associated lymphoid tissue lymphoma and primary gastric non-Hodgkin 's lymphoma. In the process of pathogenicity, the pathogen can sense and reach the suitable parasitic site through orientational movement, that is, chemotaxis (chemotaxis), and then multiply in large numbers to cause the disease. It has been reported that in pathogenic bacteria with flagella, flagella provides bacterial power. But chemotaxis is regulated by binary signal transduction system (two-component signaling system, TCSS) [4-7] .TCSS is usually composed of transmembrane histidine protein kinase (histidine protein kinase, HK) and cytoplasmic response regulatory protein (response regulator protein, RR). In the genome of Helicobacter pylori, there are chemotactic TCSS (Che-TCSS) HK encoding cheA and RR encoding cheY [8], but CheA/CheY, as a Che-TCSS, plays an important role in chemotaxis and host colonization in vitro, which has not been reported in literature. Our previous studies have confirmed that H ion is the signal molecule that induces the chemotaxis of Helicobacter pylori, and a chemotactic model of Helicobacter pylori in vitro has been established. In this study, we based on the principle of homologous recombination exchange, The cheA knockout mutant (cheA-) of Helicobacter pylori (NCTC11637) strain was constructed by using suicide plasmid-based gene knockout technique, and then the chemotactic model of Helicobacter pylori in vitro and the mouse infection model were used. The role of cheA gene in the chemotaxis and colonization of Helicobacter pylori was studied. Methods the full-length cheA and cheY genes were amplified and cloned from the genomic DNA of Helicobacter pylori NCTC11637 strain. The recombinant protein rCheA, rCheY. rCheA and rCheY were immunized with rCheA and rCheY to prepare antiserum. RCheA-IgG and rCheY-IgG were prepared by ammonium sulfate precipitation and DEAE-52 column chromatography. The suicide plasmid of cheA gene was constructed and the cheA gene knockout mutant (cheA-) was constructed by using the suicide plasmid according to the homologous recombination exchange principle. PCR and sequencing were used to identify the cheA mutant. The phosphorylation levels of CheA and CheY molecules in cheA mutants and wild strains were determined by using rCheA-IgG and rCheY-IgG anchored target protein and protein phosphorylation detection kit. Helicobacter pylori chemotaxis model and BALB/c mouse infection model were used to compare the difference between cheA mutant and wild strain in vitro chemotaxis and in vivo colonization ability. Results the results of PCR and sequencing confirmed that the phosphorylation level of CheA and CheY in the wild strain decreased rapidly from 59.6 卤11.5 渭 mol and 55.5 卤10.2 渭 mol to 10.8 卤2.6 and 5.5 卤1.2 渭 mol (P0.05), respectively, and the CheY phosphorylation level of cheA- mutant was decreased rapidly from 59.6 卤11.5 渭 mol to 10.8 卤2.6 渭 mol and 5.5 卤1.2 渭 mol (P0.05). Low and no significant change (P0.05) .cheA mutant against hydrochloric acid, The diameter of the chemotactic aggregation ring of sulfuric acid and acetic acid (10 ~ 20 卤2.0 ~ 3.0 mm) was significantly smaller than that of wild plant (16 ~ 24 卤2.0 ~ 3.0 mm) (, P0.05). The positive rate of Helicobacter pylori isolated from gastric mucosa of wild strain infected mice (90%) was significantly higher than that of cheA mutant (40%) (P0.05). The results of fluorescence quantitative PCR also showed that the number of Helicobacter pylori in gastric mucosa of wild strain infected mice (6.3 卤2.1 脳 10 ~ 3 copies/mg) was significantly higher than that of cheA mutant (P 0.05). Conclusion cheA gene plays an important role in vitro chemotaxis and in vivo colonization of Helicobacter pylori.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2176534
[Abstract]:Background and objective Helicobacter pylori (Helicobacter pylori) is a pathogen of many kinds of human gastritis and peptic ulcer. Persistent infection is closely related to gastric adenocarcinoma, gastric mucosa-associated lymphoid tissue lymphoma and primary gastric non-Hodgkin 's lymphoma. In the process of pathogenicity, the pathogen can sense and reach the suitable parasitic site through orientational movement, that is, chemotaxis (chemotaxis), and then multiply in large numbers to cause the disease. It has been reported that in pathogenic bacteria with flagella, flagella provides bacterial power. But chemotaxis is regulated by binary signal transduction system (two-component signaling system, TCSS) [4-7] .TCSS is usually composed of transmembrane histidine protein kinase (histidine protein kinase, HK) and cytoplasmic response regulatory protein (response regulator protein, RR). In the genome of Helicobacter pylori, there are chemotactic TCSS (Che-TCSS) HK encoding cheA and RR encoding cheY [8], but CheA/CheY, as a Che-TCSS, plays an important role in chemotaxis and host colonization in vitro, which has not been reported in literature. Our previous studies have confirmed that H ion is the signal molecule that induces the chemotaxis of Helicobacter pylori, and a chemotactic model of Helicobacter pylori in vitro has been established. In this study, we based on the principle of homologous recombination exchange, The cheA knockout mutant (cheA-) of Helicobacter pylori (NCTC11637) strain was constructed by using suicide plasmid-based gene knockout technique, and then the chemotactic model of Helicobacter pylori in vitro and the mouse infection model were used. The role of cheA gene in the chemotaxis and colonization of Helicobacter pylori was studied. Methods the full-length cheA and cheY genes were amplified and cloned from the genomic DNA of Helicobacter pylori NCTC11637 strain. The recombinant protein rCheA, rCheY. rCheA and rCheY were immunized with rCheA and rCheY to prepare antiserum. RCheA-IgG and rCheY-IgG were prepared by ammonium sulfate precipitation and DEAE-52 column chromatography. The suicide plasmid of cheA gene was constructed and the cheA gene knockout mutant (cheA-) was constructed by using the suicide plasmid according to the homologous recombination exchange principle. PCR and sequencing were used to identify the cheA mutant. The phosphorylation levels of CheA and CheY molecules in cheA mutants and wild strains were determined by using rCheA-IgG and rCheY-IgG anchored target protein and protein phosphorylation detection kit. Helicobacter pylori chemotaxis model and BALB/c mouse infection model were used to compare the difference between cheA mutant and wild strain in vitro chemotaxis and in vivo colonization ability. Results the results of PCR and sequencing confirmed that the phosphorylation level of CheA and CheY in the wild strain decreased rapidly from 59.6 卤11.5 渭 mol and 55.5 卤10.2 渭 mol to 10.8 卤2.6 and 5.5 卤1.2 渭 mol (P0.05), respectively, and the CheY phosphorylation level of cheA- mutant was decreased rapidly from 59.6 卤11.5 渭 mol to 10.8 卤2.6 渭 mol and 5.5 卤1.2 渭 mol (P0.05). Low and no significant change (P0.05) .cheA mutant against hydrochloric acid, The diameter of the chemotactic aggregation ring of sulfuric acid and acetic acid (10 ~ 20 卤2.0 ~ 3.0 mm) was significantly smaller than that of wild plant (16 ~ 24 卤2.0 ~ 3.0 mm) (, P0.05). The positive rate of Helicobacter pylori isolated from gastric mucosa of wild strain infected mice (90%) was significantly higher than that of cheA mutant (40%) (P0.05). The results of fluorescence quantitative PCR also showed that the number of Helicobacter pylori in gastric mucosa of wild strain infected mice (6.3 卤2.1 脳 10 ~ 3 copies/mg) was significantly higher than that of cheA mutant (P 0.05). Conclusion cheA gene plays an important role in vitro chemotaxis and in vivo colonization of Helicobacter pylori.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
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1 徐樝,严杰,叶嗣颖,毛亚飞,李立伟,李淑萍;重组尿素酶B亚单位和黏附素双价疫苗对幽门螺杆菌SS1株感染BALB/c小鼠的免疫保护作用[J];中华微生物学和免疫学杂志;2004年05期
2 Cristina Lenzi;Alberto Palazzuoli;Nicola Giordano;Giuliano Alegente;Catia Gonnelli;Maria Stella Campagna;Annalisa Santucci;Michele Sozzi;Panagiotis Papakostas;Fabio Rollo;Ranuccio Nuti;Natale Figura;;H pylori infection and systemic antibodies to CagA and heat shock protein 60 in patients with coronary heart disease[J];World Journal of Gastroenterology;2006年48期
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