肠出血性大肠杆菌O157：H7新型融合蛋白免疫原性分析及动物保护效果评价
发布时间：2018-09-04 20:07
【摘要】： 肠出血性大肠杆菌(enterohemorrhagic E.coli,EHEC)O157:H7于1982年首次被确认为人致病菌以来,世界各地包括中国都有不同规模的暴发流行。EHECO157:H7感染除可使人发生常规腹泻外,还可在5%-10%的病例中引发严重并发症,甚至死亡。该菌是重要的食源性致病菌,危害严重,缺乏有效的防治手段,而抗生素治疗可能会加剧溶血性尿毒综合症(haemolutic uraemic syndrome,HUS)。由于以上特点EHEC O157:H7成为世界公共卫生问题,引起微生物学家和公共卫生工作者的广泛关注。目前,临床针对EHEC O157:H7感染只是对症治疗和适当的抗菌治疗。疫苗被认为是应对EHEC O157:H7暴发流行和散发感染最简单,经济,快捷的手段。 EHEC O157:H7的致病因子主要有黏附相关因子、毒素和溶血素等,这些组分以及它们的全分子或部分结构是目前最重要的保护性抗原的筛选目标。 Intimin是引起肠上皮细胞黏附和擦拭性(attaching and effacing,A/E)损伤的重要因子。本论文选取Intmin具有免疫保护性的片段制备免疫血清,首先通过体外试验验证其免疫原性和免疫反应性,之后在细胞水平检测其抗黏附效果,最后在动物水平观察其保护效果。我们还针对导致病人出血性结肠炎(hemorrhagic colitis,HC),HUS等许多并发症的重要毒素因子Stx1和Stx2,选取其具有免疫保护性的B亚单位,为了提供针对EHEC O157:H7更广泛的保护,我们采用不同技术方案以保护性片段为元件构建融合蛋白,并展开相关生物学活性研究,具体包括: 第一部分Int281的表达与免疫原性分析 EHEC O157:H7 Intimin为引起A/E损伤的关键因子,本实验在基因工程菌中实现肠出血性大肠杆菌(EHEC)O157:H7紧密黏附素(intimin)保护性片段(Int281)的高效表达,并进行抗原性的初步分析。应用PCR从EHEC O157:H7基因组中钓取int281基因,插入pMD18-T克隆载体。克隆质粒测序鉴定后,采用NdeⅠ、NotⅠ限制性核酸内切酶双酶切pMD18-T-int281质粒获得int281基因,连接同样经过双酶切的pET-22b(+)。表达质粒测序鉴定后转化E.coli BL21(DE3),IPTG诱导表达,目的蛋白以包涵体形式表达,表达量约25%。变复性后经镍柱纯化后纯度达95%上。经SDS-PAGE和Western blot检测相对分子质量,重组Int281蛋白免疫BALB/c小鼠,ELISA检测鼠血清抗体效价。免疫印迹检测显示,重组Int281片段可以特异性地识别抗O157:H7多抗,具有良好的免疫反应性。免疫小鼠抗体效价达1:10~6,且此抗体水平在三个月后保持稳定,说明我们制备的Int281多肽具有良好的免疫原性。经反复冻存验证重组蛋白具有良好的物理稳定性。本试验为下一步制备抗-Intimin抗体及研究其对EHEC O157:H7黏附定植的被动保护效果奠定基础。 第二部分新型融合蛋白Stx2B-Stx1B的免疫原性分析及其在小鼠EHEC O157:H7致死模型上的保护效果评价 本室前期研究表明,克隆表达的Stx1B和Stx2B亚单位及其多肽片段无毒,且具有良好的免疫保护性,针对Stx1B和Stx2B的鸡多克隆抗体具有阻断毒素活性的作用。但以上抗体都无法提供针对双产毒株的更全面的保护效果,我们试图构建新型融合蛋白Stx2B-Stx1B(简称2S),以提供针对双产毒株更广泛的保护效果。 在本研究中,我们构建新型融合蛋白2S,即Stx2B和Stx1B用一柔性linker连接。在原核表达系统E.coli获得了高产量的重组蛋白。基于阴离子交换层析,建立了2S的简便纯化方法。采用抗-Stx1B和抗-Stx2B特异抗体,通过ELISA确定纯化的2S蛋白包含两种单独B亚单位相同的抗原表位。EHEC O157:H7感染会引发机体的系统免疫反应,因此我们试图通过采用弗氏佐剂,腹腔途径免疫的策略诱导产生体液免疫应答来抵抗EHEC O157:H7感染引发的毒效应。与Stx1B、Stx2B单独免疫组和Stx1B+Stx2B混合免疫组相比,经ELISA检测新型融合蛋白2S能提供高水平抗体效价,2S免疫小鼠倾向于引发Th2型免疫反应。体外检测到高水平中和抗体效价。2S免疫小鼠能抵抗高剂量EHEC O157:H7裂解菌攻击。以上结果使我们认为这种新型融合蛋白能同时提供针对两种毒素的保护,可以作为抗EHEC O157:H7感染性并发症的候选疫苗分子。 第三部分新型融合蛋白Stx2B-Stx1B-Int281的免疫原性分析及其在小鼠EHECO157:H7感染模型上的保护效果评价 我们构建的原核表达质粒pET-22b(+)-ssi经测序等鉴定与设计序列完全一致,重组Stx2B-Stx1B-Int281(即SSI),蛋白在E.coli BL21(DE3)得到高效表达,经SDS-PAGE和Western blot鉴定蛋白大小符合设计相对分子质量(46kDa)。Westernblot结果证明该融合蛋白能被鸡抗-O157:H7全菌蛋白多抗识别,初步说明其具有免疫反应性。ELISA测定新融合蛋白抗原表位结果显示,融合蛋白三种成份均能表现其各自免疫反应性,实验系统采用单体蛋白作为阳性对照,经抗体孵育,显色后比较其OD值高低。结果显示,SSI蛋白与Stx1B单体相比,没有得到单体那样高的检测值,我们推测可能与其在融合蛋白中的位置和构象有关,中间部位不利于其表位的充分暴露。表位未充分暴露的证据在后续试验结果也有体现,ELISA检测结果显示,抗-Stx1B的IgG抗体效价和针对Stx1的中和抗体效价均显著低于Stx1B单独免疫组产生的相应抗体水平。 通过动物免疫,我们获得了与Stx2B和Int281单独免疫相似的IgG抗体效价和中和效价。抗EHEC O157:H7对HEp-2细胞水平的黏附实验结果显示,小鼠抗-SSI血清效果优于抗-Int281血清。对免疫小鼠的攻毒结果也说明SSI能抵抗致死剂量EHEC O157:H7的攻击,至少对10~9CFU的EHEC O157:H7病原菌产生完全保护,病理分析结果显示:对比模型组出现的明显炎症性细胞浸润等感染损伤,SSI与Int281免疫均能对小鼠肠组织起到保护作用,对比模型组出现的明显肾小球、肾小管通透性增大和出血倾向,SSI能有效抑制这些症状的出现。 以上结果均说明SSI在抗EHEC O157:H7感染和防治其并发症方面是有效的,可作为候选疫苗分子进行后续研究。
[Abstract]:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was first identified as a human pathogen in 1982. Epidemics of various sizes have occurred worldwide, including in China. EHECO157:H7 infection can cause severe complications and even death in 5% - 10% of cases in addition to regular diarrhea. EHEC O157:H7 has become a worldwide public health problem, which has attracted widespread attention of microbiologists and public health workers. At present, EHEC O1 is clinically targeted at. 57:H7 infection is only symptomatic treatment and appropriate antimicrobial treatment. Vaccines are considered to be the simplest, economical and quickest means of dealing with outbreaks and sporadic infections of EHEC O157:H7.
The pathogenic factors of EHEC O157:H7 mainly include adhesion-related factors, toxins and hemolysin, etc. These components and their whole or partial structures are the most important screening targets for protective antigens.
Intimin is an important factor causing attaching and effacing (A/E) injury of intestinal epithelial cells. In this paper, the immunoprotective fragment of Intimin was selected to prepare immune serum. First, the immunogenicity and immunoreactivity of Intimin were tested in vitro, then its anti-adhesion effect was detected at the cellular level, and finally in animal water. In order to provide wider protection against EHEC O157:H7, we also selected the B subunit with immune protection against Stx1 and Stx2, which are important toxin factors leading to hemorrhagic colitis (HC), HUS and many other complications. The fusion protein was constructed and related biological activities were studied.
Part one: expression and immunogenicity of Int281
EHEC O157:H7 Intimin is the key factor causing A/E damage. In this study, we cloned the intestinal hemorrhagic Escherichia coli O157:H7 intimin protective fragment (Int281) from EHEC O157:H7 genome by PCR and cloned it into pMD18-T. Vector. After cloning and sequencing, the pMD18-T-int281 gene was obtained by Nde I and Not I restriction endonuclease double digestion and linked to pET-22b (+). The expression plasmid was identified by sequencing and transformed into E. coli BL21 (DE3) and induced by IPTG. The expression level of the target protein was about 25% in the form of inclusion body. The purity of the recombinant Int281 protein was determined by SDS-PAGE and Western blot. BaLB/c mice were immunized with the recombinant Int281 protein and the antibody titer was detected by ELISA. 10-6, and the antibody level remained stable after three months, indicating that the recombinant protein had good immunogenicity. Repeated cryopreservation proved that the recombinant protein had good physical stability.
The second part: Immunogenicity analysis of a novel fusion protein Stx2B-Stx1B and evaluation of its protective effect on the lethal model of mouse EHEC O157:H7
Previous studies in our laboratory showed that the cloned Stx1B and Stx2B subunits and their polypeptide fragments were non-toxic and had good immune protection. Chicken polyclonal antibodies against Stx1B and Stx2B had the effect of blocking the toxin activity. However, none of the above antibodies could provide a more comprehensive protection effect against the double-producing strains. We tried to construct a new type of fusion. The recombinant protein Stx2B-Stx1B (2S) provides a more extensive protective effect against two strains.
In this study, we constructed a novel fusion protein 2S, Stx2B and Stx1B, which were linked by a flexible linker. High yield of recombinant protein was obtained in E.coli prokaryotic expression system. Based on anion exchange chromatography, a simple method for purification of 2S was established. EHEC O157:H7 infection can induce systemic immune responses, so we tried to induce humoral immune responses to resist the toxic effects of EHEC O157:H7 infection by using Freund's adjuvant and intraperitoneal immunization strategy. Compared with ELISA, the new fusion protein 2S could provide high antibody titer, and the mice immunized with 2S tended to induce Th2 type immune response. The high neutralizing antibody titer was detected in vitro. The mice immunized with 2S could resist the attack of high dose EHEC O157:H7 lysis bacteria. These results suggest that the new fusion protein can provide both specific antibodies. The protection of the toxin can be used as a candidate vaccine molecule against EHEC O157:H7 infectious complications.
Part III Immunogenicity Analysis of a Novel Fusion Protein Stx2B-Stx1B-Int281 and Its Protective Effect on Mice Infected with EHECO157:H7
The recombinant Stx2B-Stx1B-Int281 (SSI) protein was highly expressed in E.coli BL21 (DE3). The size of the fusion protein conformed to the designed relative molecular weight (46kDa) by SDS-PAGE and Western blot. Anti-O157:H7 whole bacterial protein polyclonal antibody recognition, preliminary showed that it has immunoreactivity. ELISA assay of the antigen epitope of the new fusion protein showed that the three components of the fusion protein can show their respective immune reactivity. The experimental system used monoclonal protein as a positive control, incubated with antibodies, and compared their OD values after color. Compared with Stx1B, White did not get the same high detection value as Stx1B. We speculate that it may be related to its position and conformation in the fusion protein, and the intermediate site is not conducive to the full exposure of its epitopes. The neutralizing antibody titers of Stx1 were significantly lower than those of Stx1B alone.
We obtained IgG antibody titers and neutralization titers similar to those of Stx2B and Int281 immunized alone. Anti-EHEC O157:H7 adherence to HEp-2 cells showed that mouse anti-SSI serum was more effective than anti-Int281 serum. Pathological analysis showed that SSI and Int281 immunization could protect the intestinal tissues of mice compared with the obvious inflammatory cell infiltration and other infection damage in the model group. Compared with the obvious glomerular, tubular permeability and bleeding tendency in the model group, SSI could protect the intestinal tissues of mice. Effective suppression of these symptoms.
These results indicate that SSI is effective in the prevention and treatment of EHEC O157:H7 infection and its complications, and can be used as a candidate vaccine molecule for further study.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
，
本文编号：2223197
[Abstract]:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was first identified as a human pathogen in 1982. Epidemics of various sizes have occurred worldwide, including in China. EHECO157:H7 infection can cause severe complications and even death in 5% - 10% of cases in addition to regular diarrhea. EHEC O157:H7 has become a worldwide public health problem, which has attracted widespread attention of microbiologists and public health workers. At present, EHEC O1 is clinically targeted at. 57:H7 infection is only symptomatic treatment and appropriate antimicrobial treatment. Vaccines are considered to be the simplest, economical and quickest means of dealing with outbreaks and sporadic infections of EHEC O157:H7.
The pathogenic factors of EHEC O157:H7 mainly include adhesion-related factors, toxins and hemolysin, etc. These components and their whole or partial structures are the most important screening targets for protective antigens.
Intimin is an important factor causing attaching and effacing (A/E) injury of intestinal epithelial cells. In this paper, the immunoprotective fragment of Intimin was selected to prepare immune serum. First, the immunogenicity and immunoreactivity of Intimin were tested in vitro, then its anti-adhesion effect was detected at the cellular level, and finally in animal water. In order to provide wider protection against EHEC O157:H7, we also selected the B subunit with immune protection against Stx1 and Stx2, which are important toxin factors leading to hemorrhagic colitis (HC), HUS and many other complications. The fusion protein was constructed and related biological activities were studied.
Part one: expression and immunogenicity of Int281
EHEC O157:H7 Intimin is the key factor causing A/E damage. In this study, we cloned the intestinal hemorrhagic Escherichia coli O157:H7 intimin protective fragment (Int281) from EHEC O157:H7 genome by PCR and cloned it into pMD18-T. Vector. After cloning and sequencing, the pMD18-T-int281 gene was obtained by Nde I and Not I restriction endonuclease double digestion and linked to pET-22b (+). The expression plasmid was identified by sequencing and transformed into E. coli BL21 (DE3) and induced by IPTG. The expression level of the target protein was about 25% in the form of inclusion body. The purity of the recombinant Int281 protein was determined by SDS-PAGE and Western blot. BaLB/c mice were immunized with the recombinant Int281 protein and the antibody titer was detected by ELISA. 10-6, and the antibody level remained stable after three months, indicating that the recombinant protein had good immunogenicity. Repeated cryopreservation proved that the recombinant protein had good physical stability.
The second part: Immunogenicity analysis of a novel fusion protein Stx2B-Stx1B and evaluation of its protective effect on the lethal model of mouse EHEC O157:H7
Previous studies in our laboratory showed that the cloned Stx1B and Stx2B subunits and their polypeptide fragments were non-toxic and had good immune protection. Chicken polyclonal antibodies against Stx1B and Stx2B had the effect of blocking the toxin activity. However, none of the above antibodies could provide a more comprehensive protection effect against the double-producing strains. We tried to construct a new type of fusion. The recombinant protein Stx2B-Stx1B (2S) provides a more extensive protective effect against two strains.
In this study, we constructed a novel fusion protein 2S, Stx2B and Stx1B, which were linked by a flexible linker. High yield of recombinant protein was obtained in E.coli prokaryotic expression system. Based on anion exchange chromatography, a simple method for purification of 2S was established. EHEC O157:H7 infection can induce systemic immune responses, so we tried to induce humoral immune responses to resist the toxic effects of EHEC O157:H7 infection by using Freund's adjuvant and intraperitoneal immunization strategy. Compared with ELISA, the new fusion protein 2S could provide high antibody titer, and the mice immunized with 2S tended to induce Th2 type immune response. The high neutralizing antibody titer was detected in vitro. The mice immunized with 2S could resist the attack of high dose EHEC O157:H7 lysis bacteria. These results suggest that the new fusion protein can provide both specific antibodies. The protection of the toxin can be used as a candidate vaccine molecule against EHEC O157:H7 infectious complications.
Part III Immunogenicity Analysis of a Novel Fusion Protein Stx2B-Stx1B-Int281 and Its Protective Effect on Mice Infected with EHECO157:H7
The recombinant Stx2B-Stx1B-Int281 (SSI) protein was highly expressed in E.coli BL21 (DE3). The size of the fusion protein conformed to the designed relative molecular weight (46kDa) by SDS-PAGE and Western blot. Anti-O157:H7 whole bacterial protein polyclonal antibody recognition, preliminary showed that it has immunoreactivity. ELISA assay of the antigen epitope of the new fusion protein showed that the three components of the fusion protein can show their respective immune reactivity. The experimental system used monoclonal protein as a positive control, incubated with antibodies, and compared their OD values after color. Compared with Stx1B, White did not get the same high detection value as Stx1B. We speculate that it may be related to its position and conformation in the fusion protein, and the intermediate site is not conducive to the full exposure of its epitopes. The neutralizing antibody titers of Stx1 were significantly lower than those of Stx1B alone.
We obtained IgG antibody titers and neutralization titers similar to those of Stx2B and Int281 immunized alone. Anti-EHEC O157:H7 adherence to HEp-2 cells showed that mouse anti-SSI serum was more effective than anti-Int281 serum. Pathological analysis showed that SSI and Int281 immunization could protect the intestinal tissues of mice compared with the obvious inflammatory cell infiltration and other infection damage in the model group. Compared with the obvious glomerular, tubular permeability and bleeding tendency in the model group, SSI could protect the intestinal tissues of mice. Effective suppression of these symptoms.
These results indicate that SSI is effective in the prevention and treatment of EHEC O157:H7 infection and its complications, and can be used as a candidate vaccine molecule for further study.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
，
本文编号：2223197
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