【作者】 张雪寒；

【导师】 张书霞； 何孔旺；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2011， 博士

【摘要】 食源性病原微生物是当今世界上不断增多的食品安全和突发性公共卫生事件的主要诱因。其中,肠出血性大肠杆菌(enterohaemorrhagic Escherichia coli, EHEC)0157：H7可致人腹泻、出血性结肠炎、还可在5%-10%的病例中引发溶血性尿毒综合征(Hemolytic uremic syndrome, HUS)及血栓性血小板减少紫癜等严重并发症,严重者可致死亡。近20余年来,EHEC O157:H7引发的食物中毒在世界各地包括中国都有不同规模的暴发流行。在我们研究中选用EHEC O157:H7三个主要的毒力基因tir. stx2b和stx1b,运用原核表达系统在体外进行串联表达,获得重组蛋白。选择能够增强肠通透的霍乱弧菌封闭小带毒素zot基因进行串联,希望能够增强机体的粘膜免疫,尤其是肠道的粘膜免疫。选用链霉素处理的Balb/c小鼠作为评价动物,皮下和鼻腔两个途径免疫Stx2B-Tir-Stx1B-Zot蛋白,通过测定血清中的IgG滴度和IgA滴度、粪便中的IgA滴度以及粪便排菌来衡量Stx2B-Tir-Stx1B-Zot蛋白的免疫原性,为EHEC O157:H7亚单位疫苗的研制筛选良好的免疫原。1.肠出血性大肠杆菌0157：H7 tir基因重组蛋白的表达及鉴定克隆、表达肠出血性大肠杆菌(enterohaemorrhagic Escherichia coli, EHEC)0157：H7转位紧密素受体(tir)基因,并研究其免疫原性。选用pET28原核表达载体,构建pET28-tir重组质粒,并在大肠杆菌BL21中进行高效表达。选用家兔制备高滴度多克隆抗体,Western blot分析其免疫原性。结果显示成功获得高效表达的重组Tir蛋白,并制备了兔源Tir多克隆抗体,Western blot分析此抗体能与EHEC O157:H7(?)勺天然Tir蛋白发生特异性抗原抗体反应。我们在大肠杆菌中成功克隆表达了tir基因,所获重组Tir蛋白具有良好的反应原性。2.大肠杆菌O157:H7 Tir重组蛋白的生物学特性以HEp-2细胞为模型,评价重组Tir蛋白制备的抗体是否能够抑制(?)EHEC O157:H7的粘附和A/E损伤的形成。应用纯化后的重组Tir蛋白,皮下免疫Balb/c小鼠进行免疫保护性实验。两免断尾采血,间接ELISA测定血清特异性抗体效价。二免14d,10’。CFUEHEC O157:H7口服灌胃攻毒。每隔两天采集粪便检测排菌量。结果显示：表达的Tir蛋白具有天然Tir蛋白的功能特性,其抗体能够较大程度地抑制EHEC 0157：H7对HEp-2细胞的粘附和A/E损伤的形成。二免Balb/c小鼠存活率高达100%,排菌减少,8d后检测不到排菌,而对照组在第16d仍然能检测到排菌。因此,体外表达的重组Tir蛋白具有良好的抗原性,可作为EHEC O157:H7基因工程疫苗研制的首选抗原之一3.肠出血性大肠杆菌O157:H7 Stx2B-Tir-StxlB-Zot多价融合蛋白表达及鉴定EHEC O157:H7主要定植在肠道内,研制能够增强粘膜免疫基因工程疫苗具有很大优势。构建表达tir和stxb的融合基因,将tir基因中间295个氨基酸残基(Tir295)与stXB亚基基因72个氨基酸串联构建ppGEX-stx2b-tir-stx1b重组质粒,并与霍乱弧菌封闭小带毒素(Zot)靠近C端基因串联,构建重组质粒pGEX-stx2b-tir-stxl b-zot,转化于BL21(DE3),用IPTG进行诱导表达,经SDS-PAGE电泳检测,该融合蛋白获得了高效表达,主要以可溶性形式存在于菌体胞浆中。Western blot鉴定显示,Stx2B-Tir-Stx1B-Zot具有良好的反应原性。4.大肠杆菌O157:H7 Stx2B-Tir-StxlB-Zot重组蛋白的免疫原性Stx2B-Tir-Stx1B-Zot重组蛋白,分别皮下和鼻腔免疫Balb/c小鼠,二次免疫后测定血清中IgG和IgA滴度,粪便中IgA滴度。定期采集粪便,测定粪便排菌情况。结果显示：鼻腔免疫后Balb/c小鼠存活率100%,攻毒7d后检测不到排菌,并且最高排菌量只有3×103CFU/0.1g,血清中IgG滴度接近105,粪便中IgA接近102；皮下免疫后Balb/c小鼠存活率88%,攻毒9d后检测不到排菌,并且最高排菌量只有1.1×105CFU/0.1g,血清中IgG滴度也接近105,粪便中检测不到IgAStx2B-Tir-Stx1B-Zot鼻腔免疫能够发挥更好的免疫原性,可以作为EHEC 0157：H7亚单位疫苗研制的免疫原。更多还原

【Abstract】 Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic enteric pathogen of worldwide importance, and is one of Shiga toxin-producing Escherichia coli (STEC). The type III secretion system of E. coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E.coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXcp of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery.In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-StxlB-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST’Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Our aim is to determine whether antibodies specific for Stx2B-Tir-Stx1B-Zot could prevent O157:H7 colonization, to determine the role of Zot in the blocking colonization as well as to develop the subunit vaccine for EHEC O157:H7.1. Construction and Expression of Recombinant Protein Tir from Escherichia coli O157:H7The objective of the experiment is to clone, express and study the antigenicity of translocation intimin receptor(tir) gene from EHEC O157:H7. The vector pET28 and BL21(DE3) were used to construct and overexpress the recombinant protein of tir gene by prokaryotic expression. The recombinant Tir protein was used to immunize rabbits to obtain high-titer polyclonal antibodies,which was used to analyze the antigenicity of Tir by Western blot. We obtain successfully high-level expression of recombinant Tir protein and the molecular weight is 48kD.The polyclonal antibody against Tir protein have good a reactive fragment with the native Tir from Escherichia coli O157:H7. In conclusion, tir gene was cloned and expressed successfully in Escherichia coli, and recombinant Tir protein has a good antigenicity.2. Biological Characterization of Recombinant Tir Protein from Escherichia coli O157:H7HEp-2 cell and the polyclonal anti-Tir were used to evaluate adherence, adherence inhibition and attaching and effacing lesion (A/E) of EHEC O157:H7. Balb/c mice were inoculated with purified 100μg Tir protein subcutaneously and the protection rate and fecal shedding were analyzed after orally challenged by 1010CFU EHEC O157:H7. Anti-Tir IgG titers were detected by indirect ELISA. Balb/c mice immunized twice showed survival rate up to 100% compared to the 50% survival rate. Moreover, no EHEC O157:H7 in the fecal samples was detected after 8d challenged with O157:H7,but non-immunized mice still sheded the organism on 16th day.The above-mentioned results indicated that Tir protein has a good antigenicity and could be used for EHEC 0157 genetic engineering vaccines.3. Expression Stx2B-Tir-StxlB-Zot Multivalent fusion protein from EHEC O157:H7EHEC O157:H7 mainly colonize the intestine tissue, therefore, developing the genetic engineering vaccine enhancing the mucosal immunity has a significant dominance. In this study, we amplified tir, stx1b and stx2b genes and contructed the recombinant plasmid of pGEX-stx2b-tir-stx1b. The positive pGEX-stx2b-tir-stx1b were digested with Xho I, dephosphorylated with alkaline phosphatase and ligated with the zot gene to construct the pGEX-stx2b-tir-stx1b-zot. BL21(pGEX-stx2b-tir-stx1b-zot) was induced with IPTG and analysize with SDS-PAGE. All the results indicated that the recombinant Stx2B-Tir-Stx1B-Zot was successfully expressed, the molecular weight is 107kD and the anti-Stx2B-Tir-Stx1B-Zot antibody are able to react with the native Stx2B,Tir and Stx1B from EHEC 0157:H7. 4. Immunogenicity Analysis of Stx2B-Tir-StxlB-Zot from EHEC O157:H7Balb/c mice were immunized subcutaneously and intranasally with Stx2B-Tir-StxlB-Zot protein, respectively at week 0,3. After the second immunization, the blood from the immunized and naive mice were collected to prepare sera for detecting the IgG and IgA titers. The feces form the immunized and naive mice were collected to detect the secretory IgA titers. Balb/c mice immunized intranasally have 100% survival rate, no detectable bacteia on the 7th after challenged, the highest shedding of 3×103CFU/0.1g and 105 IgG titers in sera and aroud102IgA titers in feces. Balb/c mice immunized subcutanously have 88% survival rate, no detectable bacteia on the 9th after challenged, the highest shedding of 1.1×105CFU/0.1g and 105 IgG titers in sera and no IgA titers in feces. Stx2B-Tir-Stx1B-Zot immunized intranasally is capable to play better immogenicity and able to be a preferred antigen to develop the subunit vaccine for EHEC O157:H7.更多还原