幽門(mén)螺桿菌cheA基因在細(xì)菌體外趨化和體內(nèi)定植過(guò)程中的作用
發(fā)布時(shí)間：2018-08-11 09:06
【摘要】：背景和目的幽門(mén)螺桿菌(Helicobacter pylori)是多種人胃炎和消化性潰瘍的病原體,持續(xù)感染則與胃腺癌、胃粘膜相關(guān)淋巴組織淋巴瘤及原發(fā)性胃非何杰金淋巴瘤發(fā)病密切相關(guān)。在致病過(guò)程中,病原菌必然能感知并通過(guò)定向運(yùn)動(dòng)也即趨化(chemotaxis)到達(dá)合適的寄生部位進(jìn)行定植(colonization),然后大量繁殖引起疾病。有文獻(xiàn)報(bào)道,在有鞭毛的病原菌中,鞭毛提供細(xì)菌動(dòng)力,但趨化運(yùn)動(dòng)受二元信號(hào)傳導(dǎo)系統(tǒng)(two-component signaling system, TCSS)調(diào)控[4-7]。TCSS通常由跨膜組氨酸蛋白激酶(histidine protein kinase, HK)和胞漿反應(yīng)調(diào)節(jié)蛋白(response regulator protein, RR)組成。在幽門(mén)螺桿菌基因組中,存在趨化相關(guān)TCSS (Che-TCSS) HK編碼基因cheA及RR編碼基因cheY[8],但作為Che-TCSS的CheA/CheY在該菌體外趨化和宿主體內(nèi)定植中的作用,迄今未見(jiàn)文獻(xiàn)報(bào)道。 我們以往研究結(jié)果證實(shí),H離子是誘導(dǎo)幽門(mén)螺桿菌趨化的信號(hào)分子,同時(shí)建立了幽門(mén)螺桿菌體外趨化模型。本研究中,我們根據(jù)同源重組交換原理,采用基于自殺質(zhì)粒的基因敲除技術(shù)構(gòu)建了幽門(mén)螺桿菌NCTC11637株cheA基因敲除突變株(cheA-),然后分別采用幽門(mén)螺桿菌體外趨化模型及小鼠感染模型等生物學(xué)試驗(yàn),探討了cheA基因在幽門(mén)螺桿菌趨化和定植中的作用。 實(shí)驗(yàn)方法從幽門(mén)螺桿菌NCTC11637株基因組DNA中擴(kuò)增并克隆全長(zhǎng)cheA和cheY基因。構(gòu)建該兩個(gè)基因原核表達(dá)系統(tǒng),Ni-NTA親和層析法提取目的重組蛋白rCheA和rCheY. rCheA和rCheY免疫家兔制備抗血清,采用硫酸銨沉淀法及DEAE-52柱層析法制備rCheA-IgG和rCheY-IgG.構(gòu)建cheA基因自殺質(zhì)粒,根據(jù)同源重組交換原理利用該自殺質(zhì)粒構(gòu)建cheA基因敲除突變株(cheA-),采用PCR及測(cè)序?qū)heA突變株進(jìn)行鑒定。采用rCheA-IgG和rCheY-IgG錨定靶蛋白及蛋白磷酸化檢測(cè)試劑盒,測(cè)定cheA突變株與野生株CheA和CheY分子磷酸化水平。采用幽門(mén)螺桿菌趨化模型及BALB/c小鼠感染模型,比較cheA突變株與野生株體外趨化及體內(nèi)定植能力的差異。 結(jié)果PCR及測(cè)序結(jié)果證實(shí)cheA-突變株基因組中cheA基因被敲除。0.001～0.1 mol/L鹽酸作用10 min,野生株CheA和CheY磷酸化水平分別從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-突變株CheY磷酸化水平均較低且無(wú)明顯變化(P0.05)。cheA突變株對(duì)鹽酸、硫酸和乙酸趨化聚集環(huán)直徑(10～20±2.0～3.0 mm)明顯小于野生株(16～24±2.0～3.0 mm)(P0.05)。野生株感染小鼠胃黏膜標(biāo)本中幽門(mén)螺桿菌分離陽(yáng)性率(90%)明顯高于cheA突變株(40%)(P0.05),熒光定量PCR結(jié)果也顯示野生株感染小鼠胃黏膜標(biāo)本中幽門(mén)螺桿菌數(shù)量(6.3±2.1×103 copies/mg)也明顯高于突變株的(8.3±3.1×101 copies/mg) (P＜0.05) 結(jié)論cheA基因在幽門(mén)螺桿菌體外趨化和體內(nèi)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 鹵11.5 渭 mol and 55.5 鹵10.2 渭 mol to 10.8 鹵2.6 and 5.5 鹵1.2 渭 mol (P0.05), respectively, and the CheY phosphorylation level of cheA- mutant was decreased rapidly from 59.6 鹵11.5 渭 mol to 10.8 鹵2.6 渭 mol and 5.5 鹵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 鹵2.0 ~ 3.0 mm) was significantly smaller than that of wild plant (16 ~ 24 鹵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 鹵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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392
本文編號(hào)：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 鹵11.5 渭 mol and 55.5 鹵10.2 渭 mol to 10.8 鹵2.6 and 5.5 鹵1.2 渭 mol (P0.05), respectively, and the CheY phosphorylation level of cheA- mutant was decreased rapidly from 59.6 鹵11.5 渭 mol to 10.8 鹵2.6 渭 mol and 5.5 鹵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 鹵2.0 ~ 3.0 mm) was significantly smaller than that of wild plant (16 ~ 24 鹵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 鹵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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392
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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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