肠出血性大肠杆菌O157：H7（EHEC O157：H7）基因工程多亚单位融合蛋白疫苗的实验研究
发布时间：2018-08-18 21:16
【摘要】： 肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli, EHEC ) O157:H7(本文简称O157)是一种重要的人畜共患传染病病原菌。自1982年被确认为致病菌以来的20多年中,世界各地包括中国都有不同规模的暴发流行。EHEC O157:H7感染可使人患腹泻、出血性结肠炎(hemorrhagic colitis,HC),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(thrombotic thrombocytopenic purpura,TTP)等严重并发症,严重者可导致死亡。O157的感染因具有暴发流行趋势、强烈的致病性与致死性和抗生素治疗可加剧病情的危险性等特点,已经成为全球性的公共卫生问题。然而目前对其感染仍缺乏有效的防治方法,研究证明:抗生素可促使O157菌释放致死性志贺毒素(Stx),从而使患者并发HUS的危险性增加。由于O157的暴发流行和治疗上的困难,疫苗成为预防和控制O157感染最简单、经济、快捷的手段,发展有效的O157疫苗就显得亟为紧迫。 由于O157菌体抗原成份复杂,含有多种致病因子,且与普通大肠杆菌有大量的共同抗原,易引发不良反应,使O157全菌疫苗研究和应用受到限制。随着基因工程技术的成熟,亚单位基因工程疫苗将成为O157疫苗研究的主攻方向。 Ⅲ型分泌蛋白A (E. coli secreted protein A,EspA)、紧密粘附素(Intimin)及志贺毒素ⅡB亚单位(Shiga like toxinⅡB subunit, Stx2B)为O157的重要保护性候选抗原,本课题在本实验室前期对它们研究的基础上,构建EspA、IntiminC端免疫保护性片段及Stx2B的融合基因;并通过原核表达系统制备多亚单位融合蛋白;纯化目的蛋白后对Balb/c小鼠进行免疫后攻毒保护实验,评价该疫苗的免疫保护率,为多亚单位O157疫苗的研制提供实验依据。 本实验完成了以下几个方面的工作: 1. espA -eaeC300(EI)融合基因的构建、表达、纯化及部分生物学性质研究。 1.1采用PCR法自O157菌基因组扩增EspA的编码基因espA和IntiminC300的编码基因eaeC300。通过基因重组技术构建融合基因espA-eaeC300,酶切、测序证实与预期序列完全一致。阳性重组子转化大肠杆菌BL21(DE3)后,IPTG诱导表达,目的蛋白表达率约40%,SDS-PAGE分析目的蛋白的分子量约54KD,超声破菌后电泳证实目的蛋白以包涵体形式表达。Western blotting显示融合蛋白EI具有与EspA和IntiminC300兔多抗血清的免疫反应性。目的蛋白经包涵体洗涤和亲和层析纯化后纯度为90%,免疫家兔获得了双扩效价为1:32的多抗血清。 1.2重组融合蛋白EI生物学作用研究: 1.2.1 EHEC O157体外粘附细胞模型建立。选用培养简单、生长周期较短、贴壁生长的HeLa细胞,将O157接种于HEPES-DMEM液体培养基内,培养至对数生长期(OD600nm≈0.6),然后将贴壁生长在盖玻片上的HeLa细胞与细菌于37℃、5%CO2共同孵育4h,姬姆萨染色及电镜观察。结果显示O157菌与HeLa细胞呈聚集性粘附。 1.2.2采用EI兔抗血清与细菌预中和及细菌与抗体同时加入方法,与HeLa细胞粘附,用PBS洗去未粘附细菌,胰酶消化细胞,倍比稀释后接种LB平板,计数单菌落,统计学分析EI兔抗血清阻断粘附的效果。结果显示:抗体预先中和的O157菌与抗体同时加入的O157菌没有显著性差异(P0.05),表明EI多抗血清不能阻断细菌与细胞的粘附。 1.2.3用EI兔抗血清作为一抗,FITC标记的羊抗兔IgG作为二抗,检测O157菌与HeLa细胞的粘附。结果显示:EI兔抗血清可以与天然O157菌发生抗原抗体反应。 1.2.4 FAS(Fluorescent actin staining )试验检测EI兔抗血清阻止O157菌与HeLa细胞间粘附和擦拭性(attaching and effacing, A/E)损伤的效果。结果显示:EI兔抗血清可以影响O157菌对HeLa细胞造成的A/E损伤。 2. espA -eaeC300-stx2b (EIS)融合基因的构建、表达与纯化采用重叠延伸PCR的方法,EI为融合基因的前端,通过linker GSGGSG与stx2b相连接,从而获得融合基因EIS;将融合基因构建在原核表达载体pET-28a(+)中,通过酶切、测序证实与理论预测值一致性为99.8%(1781/1784)。阳性重组子转化大肠杆菌BL21 (DE3)后,IPTG诱导表达,目的蛋白表达率约30%,SDS-PAGE分析目的蛋白的分子量约64KD,超声破菌后电泳证实目的蛋白以包涵体形式表达。目的蛋白经包涵体洗涤和亲和层析纯化后纯度为90%。Western blotting显示融合蛋白EIS具有与EspA、Intimin C300兔多抗血清及兔抗O157:H7超声上清血清的免疫反应性,且表现出与Gb3的结合活性。 3.融合蛋白的动物免疫及攻毒保护试验。用纯化后的EI和EIS重组蛋白免疫Balb/c小鼠,ELISA结果显示血清特异性抗体的效价显著增高,表明重组蛋白具有很强的免疫原性。以野生型O157活菌攻毒免疫保护实验证明两组融合蛋白均能有效保护活菌攻毒引起的Balb/c小鼠死亡,其中融合蛋白EIS对O157的感染保护率为92%,是各免疫组中最高的。O157超声上清致死保护实验结果显示,融合蛋白EIS具有抗毒素致死的保护作用,致死保护率为67%,表明融合蛋白中Stx2B亚单位抗原刺激机体产生能够中和Stx毒素的抗体。 综上所述,本研究成功表达了EI和EIS融合蛋白,建立了O157体外粘附细胞模型;通过对融合蛋白部分生物学功能研究显示:EI和EIS均具有良好的免疫原性和免疫反应性;融合蛋白EI刺激机体产生的抗体具有一定的保护作用,其对于HeLa细胞的保护不是通过减少O157对细胞的粘附,而是通过阻断A/E损伤的形成来发挥作用;融合蛋白EIS增加了Stx2B亚单位抗原,刺激机体产生能够中和毒素的抗体,提高了疫苗的免疫保护率。本课题的实施为研制O157:H7基因工程多亚单位融和蛋白疫苗奠定了良好的基础。
[Abstract]:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 (hereinafter referred to as O157) is an important zoonotic pathogen. Since it was identified as a pathogen in 1982, outbreaks of different sizes have occurred worldwide, including in China. EHEC O157:H7 infection can cause diarrhea and hemorrhage. Hemorrhagic colitis (HC) can also cause severe complications such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) in 5 to 10% of cases. O157 infection can lead to death in severe cases. O157 infection has an outbreak epidemic trend and is highly pathogenic. It has become a global public health problem with the characteristics of lethal and antibiotic treatment that can exacerbate the risk of HUS. However, there is still no effective way to prevent and control the infection. Studies have shown that antibiotics can promote the release of lethal Shiga toxin (Stx) from O157 bacteria, thus increasing the risk of HUS in patients with O157 outbreaks. Because of the difficulties in epidemic and treatment, vaccines have become the simplest, economical and fast means to prevent and control O157 infection. It is urgent to develop an effective O157 vaccine.
O157 bacterium antigen has complex components, contains many pathogenic factors, and has a large number of common antigens with E. coli, which is easy to cause adverse reactions, so the research and application of O157 vaccine is limited. With the maturity of genetic engineering technology, subunit genetic engineering vaccine will become the main research direction of O157 vaccine.
E. coli secreted protein A (EspA), Intimin and Shiga toxin II B subunit (Stx2B) are important protective candidate antigens of O157. On the basis of their previous studies in our laboratory, we constructed the fusion of EspA, Intimin C-terminal immunoprotective fragment and Stx2B. Gene; Prokaryotic expression system was used to prepare the fusion protein of multiple subunits; after purifying the target protein, the immune protection of Balb/c mice was carried out, and the immune protection rate of the vaccine was evaluated, which provided experimental basis for the development of the vaccine of multiple subunits O157.
This experiment has completed the following aspects:
Construction, expression, purification and biological characterization of 1. espA -eaeC300 (EI) fusion gene.
1.1 The gene encoding EspA and Intimin C300 was amplified by PCR from O157 strain genome. The fusion gene espA-eaeC300 was constructed by gene recombination technology. The sequence was confirmed to be identical with the expected sequence. After the positive recombinant was transformed into E. coli BL21 (DE3), IPTG was induced and the expression rate of the target protein was about 40%, SDS-PA was detected. Western blotting showed that the fusion protein EI had immunoreactivity with rabbit multi-antibody sera of EspA and Intimin C300. The purity of the fusion protein was 90% after inclusion body washing and affinity chromatography. The price is 1:32 antiserum.
1.2 biological function of recombinant fusion protein EI:
1.2.1 The adherent cell model of EHEC O157 was established in vitro. The adherent HeLa cells were cultured in HEPES-DMEM liquid medium. O157 was inoculated into logarithmic growth phase (OD600nm 0.6). Then the adherent HeLa cells were incubated with bacteria at 37 C for 4 hours at 5% CO2 for Giemsa staining. The results showed that O157 and HeLa cells showed aggregated adhesion.
1.2.2 Antiserum and bacterial pre-neutralization of EI rabbits and the addition of both bacteria and antibodies were used to adhere to HeLa cells. The non-adherent bacteria were washed out with PBS and the trypsin digested cells were inoculated into LB plate after doubling dilution. Single colony was counted and the effect of blocking adhesion of EI rabbit anti-serum was statistically analyzed. There was no significant difference (P 0.05) between O157 and EI polyantiserum, indicating that EI polyantiserum could not block the adhesion of bacteria to cells.
1.2.3 Using EI rabbit antiserum as an antibody and FITC labeled sheep anti-rabbit IgG as a second antibody, the adhesion of O157 to HeLa cells was detected.
1.2.4 FAS (Fluorescent actin staining) assay was used to detect the effect of EI rabbit antiserum on preventing adhesion and effacing (A/E) injury between O157 and HeLa cells.
2. The fusion gene of espA-eaeC300-stx2b (EIS) was constructed, expressed and purified by overlapping extension PCR. EI was the front end of the fusion gene and linked with stx2b by linker GSGGSG. The fusion gene was constructed in prokaryotic expression vector pET-28a (+), and the sequence was confirmed to be consistent with the theoretical predicted value by restriction enzyme digestion. 99.8% (1781/1784). After transforming into E. coli BL21 (DE3), IPTG was induced to express. The expression rate of the target protein was about 30%. The molecular weight of the target protein was about 64 KD by SDS-PAGE analysis. The expression of the target protein in the form of inclusion body was confirmed by electrophoresis after ultrasonic breakage. The purity of the target protein was 90% after inclusion body washing and affinity chromatography. Ing showed that the fusion protein EIS had immunoreactivity with EspA, Intimin C300 rabbit polyantibody and O157:H7 supernatant, and showed binding activity with Gb3.
3. Animal immunization and protective test of fusion protein. Balb/c mice were immunized with purified EI and EIS recombinant protein. ELISA results showed that the titer of serum specific antibody was significantly increased, indicating that the recombinant protein had strong immunogenicity. The protective test of wild type O157 living bacteria showed that both fusion proteins could effectively protect Balb/c mice. The protective rate of fusion protein EIS against O157 infection was 92%, which was the highest among all immune groups. The results of ultrasonographic supernatant of O157 showed that the fusion protein EIS had the protective effect against lethal toxicity. The protective rate of death was 67%, indicating that Stx2B subunit antigen in fusion protein stimulated the production of organism. It can neutralize antibodies against Stx toxin.
To sum up, the fusion proteins of EI and EIS were successfully expressed, and the O157 cell adhesion model was established in vitro. The biological functions of the fusion proteins showed that both EI and EIS had good immunogenicity and immunoreactivity, and the antibodies produced by the fusion protein EI had protective effects on HeLa cells. The protection is not by reducing the adhesion of O157 to cells, but by blocking the formation of A/E damage; the fusion protein EIS increases the Stx2B subunit antigen, stimulates the body to produce antibodies capable of neutralizing toxins, and improves the immune protection rate of the vaccine. Miao laid a good foundation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
，
本文编号：2190700
[Abstract]:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 (hereinafter referred to as O157) is an important zoonotic pathogen. Since it was identified as a pathogen in 1982, outbreaks of different sizes have occurred worldwide, including in China. EHEC O157:H7 infection can cause diarrhea and hemorrhage. Hemorrhagic colitis (HC) can also cause severe complications such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) in 5 to 10% of cases. O157 infection can lead to death in severe cases. O157 infection has an outbreak epidemic trend and is highly pathogenic. It has become a global public health problem with the characteristics of lethal and antibiotic treatment that can exacerbate the risk of HUS. However, there is still no effective way to prevent and control the infection. Studies have shown that antibiotics can promote the release of lethal Shiga toxin (Stx) from O157 bacteria, thus increasing the risk of HUS in patients with O157 outbreaks. Because of the difficulties in epidemic and treatment, vaccines have become the simplest, economical and fast means to prevent and control O157 infection. It is urgent to develop an effective O157 vaccine.
O157 bacterium antigen has complex components, contains many pathogenic factors, and has a large number of common antigens with E. coli, which is easy to cause adverse reactions, so the research and application of O157 vaccine is limited. With the maturity of genetic engineering technology, subunit genetic engineering vaccine will become the main research direction of O157 vaccine.
E. coli secreted protein A (EspA), Intimin and Shiga toxin II B subunit (Stx2B) are important protective candidate antigens of O157. On the basis of their previous studies in our laboratory, we constructed the fusion of EspA, Intimin C-terminal immunoprotective fragment and Stx2B. Gene; Prokaryotic expression system was used to prepare the fusion protein of multiple subunits; after purifying the target protein, the immune protection of Balb/c mice was carried out, and the immune protection rate of the vaccine was evaluated, which provided experimental basis for the development of the vaccine of multiple subunits O157.
This experiment has completed the following aspects:
Construction, expression, purification and biological characterization of 1. espA -eaeC300 (EI) fusion gene.
1.1 The gene encoding EspA and Intimin C300 was amplified by PCR from O157 strain genome. The fusion gene espA-eaeC300 was constructed by gene recombination technology. The sequence was confirmed to be identical with the expected sequence. After the positive recombinant was transformed into E. coli BL21 (DE3), IPTG was induced and the expression rate of the target protein was about 40%, SDS-PA was detected. Western blotting showed that the fusion protein EI had immunoreactivity with rabbit multi-antibody sera of EspA and Intimin C300. The purity of the fusion protein was 90% after inclusion body washing and affinity chromatography. The price is 1:32 antiserum.
1.2 biological function of recombinant fusion protein EI:
1.2.1 The adherent cell model of EHEC O157 was established in vitro. The adherent HeLa cells were cultured in HEPES-DMEM liquid medium. O157 was inoculated into logarithmic growth phase (OD600nm 0.6). Then the adherent HeLa cells were incubated with bacteria at 37 C for 4 hours at 5% CO2 for Giemsa staining. The results showed that O157 and HeLa cells showed aggregated adhesion.
1.2.2 Antiserum and bacterial pre-neutralization of EI rabbits and the addition of both bacteria and antibodies were used to adhere to HeLa cells. The non-adherent bacteria were washed out with PBS and the trypsin digested cells were inoculated into LB plate after doubling dilution. Single colony was counted and the effect of blocking adhesion of EI rabbit anti-serum was statistically analyzed. There was no significant difference (P 0.05) between O157 and EI polyantiserum, indicating that EI polyantiserum could not block the adhesion of bacteria to cells.
1.2.3 Using EI rabbit antiserum as an antibody and FITC labeled sheep anti-rabbit IgG as a second antibody, the adhesion of O157 to HeLa cells was detected.
1.2.4 FAS (Fluorescent actin staining) assay was used to detect the effect of EI rabbit antiserum on preventing adhesion and effacing (A/E) injury between O157 and HeLa cells.
2. The fusion gene of espA-eaeC300-stx2b (EIS) was constructed, expressed and purified by overlapping extension PCR. EI was the front end of the fusion gene and linked with stx2b by linker GSGGSG. The fusion gene was constructed in prokaryotic expression vector pET-28a (+), and the sequence was confirmed to be consistent with the theoretical predicted value by restriction enzyme digestion. 99.8% (1781/1784). After transforming into E. coli BL21 (DE3), IPTG was induced to express. The expression rate of the target protein was about 30%. The molecular weight of the target protein was about 64 KD by SDS-PAGE analysis. The expression of the target protein in the form of inclusion body was confirmed by electrophoresis after ultrasonic breakage. The purity of the target protein was 90% after inclusion body washing and affinity chromatography. Ing showed that the fusion protein EIS had immunoreactivity with EspA, Intimin C300 rabbit polyantibody and O157:H7 supernatant, and showed binding activity with Gb3.
3. Animal immunization and protective test of fusion protein. Balb/c mice were immunized with purified EI and EIS recombinant protein. ELISA results showed that the titer of serum specific antibody was significantly increased, indicating that the recombinant protein had strong immunogenicity. The protective test of wild type O157 living bacteria showed that both fusion proteins could effectively protect Balb/c mice. The protective rate of fusion protein EIS against O157 infection was 92%, which was the highest among all immune groups. The results of ultrasonographic supernatant of O157 showed that the fusion protein EIS had the protective effect against lethal toxicity. The protective rate of death was 67%, indicating that Stx2B subunit antigen in fusion protein stimulated the production of organism. It can neutralize antibodies against Stx toxin.
To sum up, the fusion proteins of EI and EIS were successfully expressed, and the O157 cell adhesion model was established in vitro. The biological functions of the fusion proteins showed that both EI and EIS had good immunogenicity and immunoreactivity, and the antibodies produced by the fusion protein EI had protective effects on HeLa cells. The protection is not by reducing the adhesion of O157 to cells, but by blocking the formation of A/E damage; the fusion protein EIS increases the Stx2B subunit antigen, stimulates the body to produce antibodies capable of neutralizing toxins, and improves the immune protection rate of the vaccine. Miao laid a good foundation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
，
本文编号：2190700
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