腸出血性大腸桿菌O157：H7新型融合蛋白免疫原性分析及動(dòng)物保護(hù)效果評(píng)價(jià)
發(fā)布時(shí)間：2018-09-04 20:07
【摘要】： 腸出血性大腸桿菌(enterohemorrhagic E.coli,EHEC)O157:H7于1982年首次被確認(rèn)為人致病菌以來,世界各地包括中國都有不同規(guī)模的暴發(fā)流行。EHECO157:H7感染除可使人發(fā)生常規(guī)腹瀉外,還可在5%-10%的病例中引發(fā)嚴(yán)重并發(fā)癥,甚至死亡。該菌是重要的食源性致病菌,危害嚴(yán)重,缺乏有效的防治手段,而抗生素治療可能會(huì)加劇溶血性尿毒綜合癥(haemolutic uraemic syndrome,HUS)。由于以上特點(diǎn)EHEC O157:H7成為世界公共衛(wèi)生問題,引起微生物學(xué)家和公共衛(wèi)生工作者的廣泛關(guān)注。目前,臨床針對(duì)EHEC O157:H7感染只是對(duì)癥治療和適當(dāng)?shù)目咕委�。疫苗被認(rèn)為是應(yīng)對(duì)EHEC O157:H7暴發(fā)流行和散發(fā)感染最簡(jiǎn)單,經(jīng)濟(jì),快捷的手段。 EHEC O157:H7的致病因子主要有黏附相關(guān)因子、毒素和溶血素等,這些組分以及它們的全分子或部分結(jié)構(gòu)是目前最重要的保護(hù)性抗原的篩選目標(biāo)。 Intimin是引起腸上皮細(xì)胞黏附和擦拭性(attaching and effacing,A/E)損傷的重要因子。本論文選取Intmin具有免疫保護(hù)性的片段制備免疫血清,首先通過體外試驗(yàn)驗(yàn)證其免疫原性和免疫反應(yīng)性,之后在細(xì)胞水平檢測(cè)其抗黏附效果,最后在動(dòng)物水平觀察其保護(hù)效果。我們還針對(duì)導(dǎo)致病人出血性結(jié)腸炎(hemorrhagic colitis,HC),HUS等許多并發(fā)癥的重要毒素因子Stx1和Stx2,選取其具有免疫保護(hù)性的B亞單位,為了提供針對(duì)EHEC O157:H7更廣泛的保護(hù),我們采用不同技術(shù)方案以保護(hù)性片段為元件構(gòu)建融合蛋白,并展開相關(guān)生物學(xué)活性研究,具體包括: 第一部分Int281的表達(dá)與免疫原性分析 EHEC O157:H7 Intimin為引起A/E損傷的關(guān)鍵因子,本實(shí)驗(yàn)在基因工程菌中實(shí)現(xiàn)腸出血性大腸桿菌(EHEC)O157:H7緊密黏附素(intimin)保護(hù)性片段(Int281)的高效表達(dá),并進(jìn)行抗原性的初步分析。應(yīng)用PCR從EHEC O157:H7基因組中釣取int281基因,插入pMD18-T克隆載體�？寺≠|(zhì)粒測(cè)序鑒定后,采用NdeⅠ、NotⅠ限制性核酸內(nèi)切酶雙酶切pMD18-T-int281質(zhì)粒獲得int281基因,連接同樣經(jīng)過雙酶切的pET-22b(+)。表達(dá)質(zhì)粒測(cè)序鑒定后轉(zhuǎn)化E.coli BL21(DE3),IPTG誘導(dǎo)表達(dá),目的蛋白以包涵體形式表達(dá),表達(dá)量約25%。變復(fù)性后經(jīng)鎳柱純化后純度達(dá)95%上。經(jīng)SDS-PAGE和Western blot檢測(cè)相對(duì)分子質(zhì)量,重組Int281蛋白免疫BALB/c小鼠,ELISA檢測(cè)鼠血清抗體效價(jià)。免疫印跡檢測(cè)顯示,重組Int281片段可以特異性地識(shí)別抗O157:H7多抗,具有良好的免疫反應(yīng)性。免疫小鼠抗體效價(jià)達(dá)1:10~6,且此抗體水平在三個(gè)月后保持穩(wěn)定,說明我們制備的Int281多肽具有良好的免疫原性。經(jīng)反復(fù)凍存驗(yàn)證重組蛋白具有良好的物理穩(wěn)定性。本試驗(yàn)為下一步制備抗-Intimin抗體及研究其對(duì)EHEC O157:H7黏附定植的被動(dòng)保護(hù)效果奠定基礎(chǔ)。 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致死模型上的保護(hù)效果評(píng)價(jià) 本室前期研究表明,克隆表達(dá)的Stx1B和Stx2B亞單位及其多肽片段無毒,且具有良好的免疫保護(hù)性,針對(duì)Stx1B和Stx2B的雞多克隆抗體具有阻斷毒素活性的作用。但以上抗體都無法提供針對(duì)雙產(chǎn)毒株的更全面的保護(hù)效果,我們?cè)噲D構(gòu)建新型融合蛋白Stx2B-Stx1B(簡(jiǎn)稱2S),以提供針對(duì)雙產(chǎn)毒株更廣泛的保護(hù)效果。 在本研究中,我們構(gòu)建新型融合蛋白2S,即Stx2B和Stx1B用一柔性linker連接。在原核表達(dá)系統(tǒng)E.coli獲得了高產(chǎn)量的重組蛋白�；陉庪x子交換層析,建立了2S的簡(jiǎn)便純化方法。采用抗-Stx1B和抗-Stx2B特異抗體,通過ELISA確定純化的2S蛋白包含兩種單獨(dú)B亞單位相同的抗原表位。EHEC O157:H7感染會(huì)引發(fā)機(jī)體的系統(tǒng)免疫反應(yīng),因此我們?cè)噲D通過采用弗氏佐劑,腹腔途徑免疫的策略誘導(dǎo)產(chǎn)生體液免疫應(yīng)答來抵抗EHEC O157:H7感染引發(fā)的毒效應(yīng)。與Stx1B、Stx2B單獨(dú)免疫組和Stx1B+Stx2B混合免疫組相比,經(jīng)ELISA檢測(cè)新型融合蛋白2S能提供高水平抗體效價(jià),2S免疫小鼠傾向于引發(fā)Th2型免疫反應(yīng)。體外檢測(cè)到高水平中和抗體效價(jià)。2S免疫小鼠能抵抗高劑量EHEC O157:H7裂解菌攻擊。以上結(jié)果使我們認(rèn)為這種新型融合蛋白能同時(shí)提供針對(duì)兩種毒素的保護(hù),可以作為抗EHEC O157:H7感染性并發(fā)癥的候選疫苗分子。 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感染模型上的保護(hù)效果評(píng)價(jià) 我們構(gòu)建的原核表達(dá)質(zhì)粒pET-22b(+)-ssi經(jīng)測(cè)序等鑒定與設(shè)計(jì)序列完全一致,重組Stx2B-Stx1B-Int281(即SSI),蛋白在E.coli BL21(DE3)得到高效表達(dá),經(jīng)SDS-PAGE和Western blot鑒定蛋白大小符合設(shè)計(jì)相對(duì)分子質(zhì)量(46kDa)。Westernblot結(jié)果證明該融合蛋白能被雞抗-O157:H7全菌蛋白多抗識(shí)別,初步說明其具有免疫反應(yīng)性。ELISA測(cè)定新融合蛋白抗原表位結(jié)果顯示,融合蛋白三種成份均能表現(xiàn)其各自免疫反應(yīng)性,實(shí)驗(yàn)系統(tǒng)采用單體蛋白作為陽性對(duì)照,經(jīng)抗體孵育,顯色后比較其OD值高低。結(jié)果顯示,SSI蛋白與Stx1B單體相比,沒有得到單體那樣高的檢測(cè)值,我們推測(cè)可能與其在融合蛋白中的位置和構(gòu)象有關(guān),中間部位不利于其表位的充分暴露。表位未充分暴露的證據(jù)在后續(xù)試驗(yàn)結(jié)果也有體現(xiàn),ELISA檢測(cè)結(jié)果顯示,抗-Stx1B的IgG抗體效價(jià)和針對(duì)Stx1的中和抗體效價(jià)均顯著低于Stx1B單獨(dú)免疫組產(chǎn)生的相應(yīng)抗體水平。 通過動(dòng)物免疫,我們獲得了與Stx2B和Int281單獨(dú)免疫相似的IgG抗體效價(jià)和中和效價(jià)�？笶HEC O157:H7對(duì)HEp-2細(xì)胞水平的黏附實(shí)驗(yàn)結(jié)果顯示,小鼠抗-SSI血清效果優(yōu)于抗-Int281血清。對(duì)免疫小鼠的攻毒結(jié)果也說明SSI能抵抗致死劑量EHEC O157:H7的攻擊,至少對(duì)10~9CFU的EHEC O157:H7病原菌產(chǎn)生完全保護(hù),病理分析結(jié)果顯示:對(duì)比模型組出現(xiàn)的明顯炎癥性細(xì)胞浸潤等感染損傷,SSI與Int281免疫均能對(duì)小鼠腸組織起到保護(hù)作用,對(duì)比模型組出現(xiàn)的明顯腎小球、腎小管通透性增大和出血傾向,SSI能有效抑制這些癥狀的出現(xiàn)。 以上結(jié)果均說明SSI在抗EHEC O157:H7感染和防治其并發(fā)癥方面是有效的,可作為候選疫苗分子進(jìn)行后續(xù)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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
，
本文編號(hào)：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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
，
本文編號(hào)：2223197
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