中文摘要：

目的克隆空肠弯曲菌（Campy1obacter jejuni）接受甲基趋化蛋白（MCP）编码基因mcp1、mcp2和mcp3并构建其原核表达系统，建立空肠弯曲菌体外趋化模型并确定趋化诱导物质，了解MCPs与趋化诱导物之间的关系。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，SDS—PAGE和Bio-Rad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，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。建立HAP（hard—agar plus）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IgG F（ab′）2封闭的趋化阻断试验，确定不同MCPs的功能及其差异。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rMCPs，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）。结论本研究成功地表达了空肠弯曲菌MCPs蛋白。牛胆汁和DOC是诱导空肠弯曲菌趋化的信号物质，MCP1和MCP2可能参与该菌对DOC的趋化过程。

英文摘要：

Objective To clone mcp1, mcp2 and mcp3 genes that encoding methyl-accepting chemotaxis proteins（MCP） of Campylobacter jejuni for construction of their prokaryotic expression systems, and to establish chemotactic model in vitro of the microbe for determining chemotaxis-inducing substances and to understand relationship among MCPs and cbemotactic 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 Bio-Rad 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 of 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′）2 were then prepared by pepsin enzymolysis and Sephadex G-100 chromatography. Chemotactie 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 determine the function and diversity of MCPs. Results The segments with expected sizes amplified from mep1, mep2 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