幽門螺桿菌cheA和cheY基因表達(dá)及其產(chǎn)物與細(xì)菌趨化的相關(guān)性
發(fā)布時(shí)間：2018-10-29 14:49
【摘要】： 背景和目的幽門螺桿菌(Helicobacterium pylori)感染與人胃炎、消化性潰瘍、胃腺癌等疾病的發(fā)病密切相關(guān)。在許多細(xì)菌致病過程中,首先對(duì)其合適的宿主寄生部位進(jìn)行趨化和定植,然后大量繁殖引起疾病。近年發(fā)現(xiàn),細(xì)菌趨化(chemotaxis,Che)受二元信號(hào)傳導(dǎo)系統(tǒng)(two-component signaling,TCS)調(diào)控。CheA/CheY是幽門螺桿菌趨化相關(guān)TCS,CheA和CheY突變株雖有動(dòng)力,但不能定植于小鼠胃粘膜,提示Che-TCS在細(xì)菌定植中起關(guān)鍵作用。然而,幽門螺桿菌Che-TCS的激活信號(hào)及其阻斷藥物至今未見報(bào)道,該Che-TCS的工作機(jī)制仍有待于深入研究。 本研究中,我們克隆了幽門螺桿菌NCTC11637株CheA和CheY基因并構(gòu)建了原核表達(dá)系統(tǒng),制備了目的重組表達(dá)產(chǎn)物rCheA和rCheY抗血清及其IgG,采用硬瓊脂法(hard agar plus,HAP)建立了幽門螺桿菌體外趨化模型,并對(duì)幽門螺桿菌體外趨化誘導(dǎo)物進(jìn)行了篩選,以期為進(jìn)一步闡明幽門螺桿菌Che-TCS信號(hào)傳導(dǎo)及其調(diào)控機(jī)制奠定基礎(chǔ)。 實(shí)驗(yàn)方法PCR擴(kuò)增幽門螺桿菌NCTC11637株全長cheA和cheY基因片段,T-A克隆后測(cè)序。構(gòu)建上述目的基因原核表達(dá)系統(tǒng),采用SDS-PAGE和BioRad凝膠成像分析系統(tǒng)檢查目的重組蛋白rCheA和rCheY的表達(dá)情況,Ni-NTA親和層析法提純r(jià)CheA和rCheY。rCheA和rCheY免疫家兔獲得抗血清,用飽和硫酸銨沉淀法和DEAE-32離子交換法提純IgG,用免疫擴(kuò)散法檢測(cè)抗血清及其IgG效價(jià)。采用硬瓊脂法建立幽門螺桿菌NCTC11637株體外趨化模型并檢測(cè)11種物質(zhì)的趨化誘導(dǎo)作用,同時(shí)分別觀察抗rCheA IgG(rCheA-IgG)和氯氰碘柳胺鈉對(duì)細(xì)菌趨化的抑制作用。 結(jié)果PCR擴(kuò)增獲得預(yù)期大小的cheA和cheY基因片段,其核苷酸和氨基酸序列與文獻(xiàn)報(bào)道完全相同。所構(gòu)建的原核表達(dá)系統(tǒng)能有效表達(dá)rCheA和rCheY。rCheA和rCheY兔抗血清及其IgG免疫擴(kuò)散效價(jià)均分別為1:4和1:2。鹽酸、硫酸和乙酸對(duì)該幽門螺桿菌均有趨化誘導(dǎo)作用,一定濃度的rCheA-IgG和氯氰碘柳胺鈉均對(duì)幽門螺桿菌趨化有抑制作用(P＜0.05)。 結(jié)論本研究成功地構(gòu)建了幽門螺桿菌NCTC11637株cheA和cheY基因原核表達(dá)系統(tǒng)。H~+是誘導(dǎo)幽門螺桿菌趨化的信號(hào)物質(zhì),rCheA-IgG和氯氰碘柳胺鈉均能抑制幽門螺桿菌對(duì)H~+的趨化。
[Abstract]:Background and objective Helicobacter pylori (Helicobacterium pylori) infection is closely related to human gastritis, peptic ulcer, gastric adenocarcinoma and other diseases. During the pathogenic process of many bacteria, the parasitic site of its suitable host is first chemotaxis and colonization, and then the disease is caused by mass propagation. It has been found that bacterial chemotaxis (chemotaxis,Che) is regulated by binary signal transduction system (two-component signaling,TCS). CheA/CheY is a mutant of Helicobacter pylori chemotaxis associated with TCS,CheA and CheY, but it can not be colonized in the gastric mucosa of mice. The results suggest that Che-TCS plays a key role in bacterial colonization. However, the activation signal of Helicobacter pylori (Che-TCS) and its blocking drugs have not been reported, and the mechanism of the Che-TCS remains to be further studied. In this study, we cloned the CheA and CheY genes of Helicobacter pylori NCTC11637 strain and constructed the prokaryotic expression system. We prepared the recombinant expression products rCheA and rCheY antiserum and their IgG, using hard Agar (hard agar plus,. The in vitro chemotaxis model of Helicobacter pylori was established by HAP, and the in vitro chemotaxis inducer of Helicobacter pylori was screened in order to lay a foundation for further elucidation of Che-TCS signal transduction and its regulatory mechanism of Helicobacter pylori. Methods the full-length cheA and cheY gene fragments of Helicobacter pylori NCTC11637 strain were amplified by PCR and sequenced after T-A cloning. The prokaryotic expression system of the target gene was constructed. The expression of rCheA and rCheY was detected by SDS-PAGE and BioRad gel imaging analysis system. The antiserum was obtained from rabbits immunized with rCheA, rCheY.rCheA and rCheY by Ni-NTA affinity chromatography. IgG, was purified by saturated ammonium sulfate precipitation method and DEAE-32 ion exchange method. The titers of antiserum and its IgG were detected by immunodiffusion method. In vitro chemotaxis model of Helicobacter pylori NCTC11637 strain was established by hard Agar method and the chemotaxis induced by 11 substances were detected. The inhibitory effects of anti-rCheA IgG (rCheA-IgG) and chlorocyanoiodothylamine sodium on bacterial chemotaxis were observed respectively. Results the expected size of cheA and cheY gene fragments were obtained by PCR amplification. The nucleotide and amino acid sequences were identical to those reported in the literature. The constructed prokaryotic expression system could effectively express rCheA, rCheY.rCheA and rCheY rabbit antiserum and their IgG immunodiffusion titers were 1:4 and 1: 2, respectively. Hydrochloric acid, sulfuric acid and acetic acid could induce the chemotaxis of Helicobacter pylori, and the chemotaxis of Helicobacter pylori was inhibited by certain concentrations of rCheA-IgG and sodium chloride iodidamine (P < 0. 05). Conclusion the prokaryotic expression system of cheA and cheY genes of Helicobacter pylori NCTC11637 strain was successfully constructed. H ~ is the signal material that induces the chemotaxis of Helicobacter pylori. Both rCheA-IgG and sodium chloride can inhibit the H ~ chemotaxis 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】：2010
【分類號(hào)】：R392
本文編號(hào)：2298004
[Abstract]:Background and objective Helicobacter pylori (Helicobacterium pylori) infection is closely related to human gastritis, peptic ulcer, gastric adenocarcinoma and other diseases. During the pathogenic process of many bacteria, the parasitic site of its suitable host is first chemotaxis and colonization, and then the disease is caused by mass propagation. It has been found that bacterial chemotaxis (chemotaxis,Che) is regulated by binary signal transduction system (two-component signaling,TCS). CheA/CheY is a mutant of Helicobacter pylori chemotaxis associated with TCS,CheA and CheY, but it can not be colonized in the gastric mucosa of mice. The results suggest that Che-TCS plays a key role in bacterial colonization. However, the activation signal of Helicobacter pylori (Che-TCS) and its blocking drugs have not been reported, and the mechanism of the Che-TCS remains to be further studied. In this study, we cloned the CheA and CheY genes of Helicobacter pylori NCTC11637 strain and constructed the prokaryotic expression system. We prepared the recombinant expression products rCheA and rCheY antiserum and their IgG, using hard Agar (hard agar plus,. The in vitro chemotaxis model of Helicobacter pylori was established by HAP, and the in vitro chemotaxis inducer of Helicobacter pylori was screened in order to lay a foundation for further elucidation of Che-TCS signal transduction and its regulatory mechanism of Helicobacter pylori. Methods the full-length cheA and cheY gene fragments of Helicobacter pylori NCTC11637 strain were amplified by PCR and sequenced after T-A cloning. The prokaryotic expression system of the target gene was constructed. The expression of rCheA and rCheY was detected by SDS-PAGE and BioRad gel imaging analysis system. The antiserum was obtained from rabbits immunized with rCheA, rCheY.rCheA and rCheY by Ni-NTA affinity chromatography. IgG, was purified by saturated ammonium sulfate precipitation method and DEAE-32 ion exchange method. The titers of antiserum and its IgG were detected by immunodiffusion method. In vitro chemotaxis model of Helicobacter pylori NCTC11637 strain was established by hard Agar method and the chemotaxis induced by 11 substances were detected. The inhibitory effects of anti-rCheA IgG (rCheA-IgG) and chlorocyanoiodothylamine sodium on bacterial chemotaxis were observed respectively. Results the expected size of cheA and cheY gene fragments were obtained by PCR amplification. The nucleotide and amino acid sequences were identical to those reported in the literature. The constructed prokaryotic expression system could effectively express rCheA, rCheY.rCheA and rCheY rabbit antiserum and their IgG immunodiffusion titers were 1:4 and 1: 2, respectively. Hydrochloric acid, sulfuric acid and acetic acid could induce the chemotaxis of Helicobacter pylori, and the chemotaxis of Helicobacter pylori was inhibited by certain concentrations of rCheA-IgG and sodium chloride iodidamine (P < 0. 05). Conclusion the prokaryotic expression system of cheA and cheY genes of Helicobacter pylori NCTC11637 strain was successfully constructed. H ~ is the signal material that induces the chemotaxis of Helicobacter pylori. Both rCheA-IgG and sodium chloride can inhibit the H ~ chemotaxis 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】：2010
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 許文亮,劉永定,宋立榮;藍(lán)藻的二元信號(hào)傳導(dǎo)系統(tǒng)[J];微生物學(xué)雜志;2003年01期
，本文編號(hào)：2298004
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2298004.html
最近更新
教材專著
