幽门螺杆菌cheA和cheY基因表达及其产物与细菌趋化的相关性
发布时间：2018-10-29 14:49
【摘要】： 背景和目的幽门螺杆菌(Helicobacterium pylori)感染与人胃炎、消化性溃疡、胃腺癌等疾病的发病密切相关。在许多细菌致病过程中,首先对其合适的宿主寄生部位进行趋化和定植,然后大量繁殖引起疾病。近年发现,细菌趋化(chemotaxis,Che)受二元信号传导系统(two-component signaling,TCS)调控。CheA/CheY是幽门螺杆菌趋化相关TCS,CheA和CheY突变株虽有动力,但不能定植于小鼠胃粘膜,提示Che-TCS在细菌定植中起关键作用。然而,幽门螺杆菌Che-TCS的激活信号及其阻断药物至今未见报道,该Che-TCS的工作机制仍有待于深入研究。 本研究中,我们克隆了幽门螺杆菌NCTC11637株CheA和CheY基因并构建了原核表达系统,制备了目的重组表达产物rCheA和rCheY抗血清及其IgG,采用硬琼脂法(hard agar plus,HAP)建立了幽门螺杆菌体外趋化模型,并对幽门螺杆菌体外趋化诱导物进行了筛选,以期为进一步阐明幽门螺杆菌Che-TCS信号传导及其调控机制奠定基础。 实验方法PCR扩增幽门螺杆菌NCTC11637株全长cheA和cheY基因片段,T-A克隆后测序。构建上述目的基因原核表达系统,采用SDS-PAGE和BioRad凝胶成像分析系统检查目的重组蛋白rCheA和rCheY的表达情况,Ni-NTA亲和层析法提纯rCheA和rCheY。rCheA和rCheY免疫家兔获得抗血清,用饱和硫酸铵沉淀法和DEAE-32离子交换法提纯IgG,用免疫扩散法检测抗血清及其IgG效价。采用硬琼脂法建立幽门螺杆菌NCTC11637株体外趋化模型并检测11种物质的趋化诱导作用,同时分别观察抗rCheA IgG(rCheA-IgG)和氯氰碘柳胺钠对细菌趋化的抑制作用。 结果PCR扩增获得预期大小的cheA和cheY基因片段,其核苷酸和氨基酸序列与文献报道完全相同。所构建的原核表达系统能有效表达rCheA和rCheY。rCheA和rCheY兔抗血清及其IgG免疫扩散效价均分别为1:4和1:2。盐酸、硫酸和乙酸对该幽门螺杆菌均有趋化诱导作用,一定浓度的rCheA-IgG和氯氰碘柳胺钠均对幽门螺杆菌趋化有抑制作用(P＜0.05)。 结论本研究成功地构建了幽门螺杆菌NCTC11637株cheA和cheY基因原核表达系统。H~+是诱导幽门螺杆菌趋化的信号物质,rCheA-IgG和氯氰碘柳胺钠均能抑制幽门螺杆菌对H~+的趋化。
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 许文亮,刘永定,宋立荣;蓝藻的二元信号传导系统[J];微生物学杂志;2003年01期
，本文编号：2298004
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