【作者】 李志锋；

【导师】 严杰；

【作者基本信息】 浙江大学 ， 病原生物学， 2010， 硕士

【摘要】 背景和目的空肠弯曲菌（Campylobacter jejuni）经肠道感染,引起以腹泻为主的急性肠炎,也可导致反应性关节炎和Guillain-Barrre综合征等并发症,故该菌是重要的食源性病原菌。空肠弯曲菌依赖鞭毛的定向趋化运动,穿越粘膜表面粘液层到达空肠及回肠黏膜细胞表面定植,然后大量繁殖引起疾病。近年文献报道,不少细菌定植趋化运动受趋化（chemotaxis,che）相关二元信号系统（two-componentsystems,TCS）调控,通常呈MCPs→Ches→Flis/Mots三级调控模式。MCPs即接受甲基趋化蛋白（methyl-accepting chemotaxis proteins,MCPs）,是接受外界趋化信号的受体。已知空肠弯曲菌NCTC11168株基因组中含所有che相关TCS基因,但至今尚无相关研究报道。本研究中,我们克隆并构建了空肠弯曲菌NCTC11168株mcp1-mcp3基因并在大肠杆菌中表达,制备了重组表达产物rMCP1-MCP3抗血清及其IgG F（ab’）₂,参照Huqdahl等介绍的硬琼脂法（hard agar plus,HAP）建立了空肠弯曲菌体外趋化模型,并对脱氧胆酸钠（DOC）等12种物质诱导空肠弯曲菌体外趋化的作用进行了研究,同时构建了mcp1^—,mcp2^-—,mcp3^—和mcp1^—/mcp2^—变株,并利用HAP法检测突变株对DOC趋化能力的变化,最后用分别用野生株和突变株感染小鼠,观察其定植小鼠能力的变化。希望以上工作能为进一步阐明空肠弯曲菌che相关TCS信号传导及其调控机制提供一定的依据实验方法PCR扩增mcp1、mcp2和mcp3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rMCP1、rMCP2和rMCP3的表达情况,Ni-NTA亲和层析法提纯rMCPs。rMCPs免疫家兔获得抗血清,采用免疫扩散法测其效价。用饱和硫酸铵盐析法和DEAE-32离子交换法提纯IgG,经胃蛋白酶酶解和Sephadex G-100层析制备IgG F（ab’）₂。利用同源重组构建mcp1^—,mcp2^—,mcp3^—和mcp1^—/mcp2^—变株。建立HAP（hard-agarplus）法空肠弯曲菌体外趋化模型并检测8种物质的趋化诱导作用。采用基于IgGF（ab’）₂封闭的趋化阻断试验及mcp突变株与野生株趋化的差异性,确定不同MCPs的功能及其差异。建立空肠弯曲菌感染小鼠的模型,用银染及涂板计数的方法观察野生株和突变株定植小鼠能力的差异,以确定不同MCPs在定植小鼠时相关系。结果PCR扩增获得预期大小的mcp1、mcp2和mcp3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地表达各rMCP1s,其产量均约为细菌总蛋白的10%。rMCP1、rMCP2和rMCP3免疫家兔后能产生特异性抗体,其免疫扩散效价均为1:4。牛胆汁和脱氧胆酸钠（DOC）对空肠弯曲菌趋化有浓度依赖性诱导作用（P＜0.05）。MCP1和MCP2被其IgG F（ab’）₂封闭后,空肠弯曲菌对DOC的趋化能力明显减弱（P＜0.05）,MCP3被封闭后对空肠弯曲菌趋化能力无影响（P＞0.05）。mcp1^—,mcp2^—和mcp1^—/mcp2^—突变株对DOC的趋化环直径分别减小16%,22%和51%。mcp1^—,mcp2^—和mcp1^—/mcp2^—突变株定植小鼠的能力跟野生株相比也明显减弱。结论DOC为空肠弯曲菌较强的体外趋化引诱物质,MCP1和MCP2可能为空肠弯曲菌对DOC趋化过程中特异受体。更多还原

【Abstract】 Background and Objective Campylobacter jejuni is a major foodborne causative agent of human gastroenteritis throughout the world.Sequelae of the campylobacteriosis can be severe and include arthropathies and Guillain-Barre syndrome（GBS）,an autoimmune disease resulting in acute motor nerve paralysis.In becteria,two-component signiling system（TCS）,which usually composed of sensor protein and response regulator protein,has the functions to sense and response to enviromental signals.The TCS responsible for bacterial movement towards favorable locations in hosts,i.e.chemotaxis,is termed as Che-TCS.In a Che-TCS there are three major groups of functional proteins:MCPs to receive signals of attractants,histidine kinase to start intracellular signal transmission though phosphoryl group transfer and flagellar motor switch proteins to decide the rotation orientation of flagella.Up to now the Che-TCS of Campylobacter jejuni has not been well studied.In our study,we constructed prokaryotic expression systems of mcp1、mcp2 and mcp3 genes,and made antiserum and IgG F（ab’）₂ of rMCP1-MCP3,established chemotactic model in vitro of the microbe for determing chemotaxis-inducing substances.We constructed mcp1^—,mcp2^—,mcp3^—and mcp1^—/mcp2^—mutants.We detected the chemotactic ability of mcp mutants toward DOC.At last we determined the coloniozation of the mutants on jejunal mucosa of mice.All the work are expected to increas our understanding of the relationship among MCPs and chemotactic inducers.Methods The segments of mcp1,mcp2 and mcp3 genes were amplified by PCR and then sequenced after T-A cloning.Prokaryotic expression systems of the genes were subsequently constructed.SDS-PAGE plus BioRad Gel Image Analyzer were used to examine the expression of target recombinant proteins rMCP1,rMCP2 and rMCP3,and Ni-NTA affinity chromatography was performed to purify the rMCPs.Rabbits were immunized with each the three rMCPs to obtain antisera.Immunodiffusion assay was performed to measure the titers of antisera.IgG in each of the antisera were extracted by ammonium sulfate precipitation and DEAE-32 ion exchange chromatography,and IgG F（ab’）₂s were then prepared by pepsin enzymolysis and Sephadex G-100 chromatography.We constructed mcp1^—,mcp2^—,mcp3^—and mcp1^—/mcp2^—mutants with homologous recombination.Chemotactic model in vitro of C.jejuni based on HAP（hard-agar plus） method was established to determine the chemotaxis-inducing effect of eight candidate substances.Chemotaxis inhibition test based on IgG F（ab’）₂ blocking was applied to deterimne the function and diversity of MCPs.Colonization model in mouse was established to determine the role MCPs play in coloning mice.Results The segments with expected sizes amplified from mcp1,mcp2 and mcp3 genes were obtained by PCRs,and their nucleotide and putative amino acid sequences were completely same as the reported.The constructed prokaryotic systems could efficiently express rMCPs with the yields about 10%of the total bacterial proteins. Immunization with rMCP1,MCP2 and rMCP3 enables the rabbits to produce specific antibody.All the antisera had 1:4 immunodiffusion titers.Both bovine bile and sodium deoxycholate（DOC） were able to induce chemotactic movement of C.jejuni in a dosage-dependent manner（P＜0.05）.When MCP1 and MCP2 were blocken with their IgG F（ab’）₂s,the chemotactic ability of C.jejuni were remarkably decreased（P＜0.05）. However,MCP3 blocking did not affect the chemotaxis（P＞0.05）.Furthermore,mcp2, mcp3 and both the two gene knock-out mutants（mcp1^—,mcp2^—and mcp1^—/mcp2^—） presented 16%,22%and 51%in diameter of the DOC-induced chemotactic ring of wild-type strain（P＜0.05）.mcp1^—,mcp2^—and mcp1^—/mcp2^—mutants also displayed significantly attenuated colonization on jejunal mucosa of mice compared to wild-type strain（P＜0.05）.And the CFU counting number of mcp1^—/mcp2^—mutant in the murine jejunal contents was much lower than that of mcp1^—and mcp2^—mutants（P＜0.05）.Conclusion All the results of this study lead a conclusion that DOC is an efficient chemical chemotactic attractant of C.jejuni in vitro,and Mcp1 and Mcp2 are the DOC-specific sensor proteins to evoke the chemotactic movement of C.jejuni tawards DOC.更多还原