腸出血性大腸桿菌O157：H7（EHEC O157：H7）基因工程多亞單位融合蛋白疫苗的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-08-18 21:16
【摘要】： 腸出血性大腸桿菌(Enterohemorrhagic Escherichia coli, EHEC ) O157:H7(本文簡(jiǎn)稱O157)是一種重要的人畜共患傳染病病原菌。自1982年被確認(rèn)為致病菌以來(lái)的20多年中,世界各地包括中國(guó)都有不同規(guī)模的暴發(fā)流行。EHEC O157:H7感染可使人患腹瀉、出血性結(jié)腸炎(hemorrhagic colitis,HC),還可在5~10%的病例中引發(fā)溶血性尿毒綜合征(hemolytic uremic syndrome,HUS)及血栓性血小板減少紫癜(thrombotic thrombocytopenic purpura,TTP)等嚴(yán)重并發(fā)癥,嚴(yán)重者可導(dǎo)致死亡。O157的感染因具有暴發(fā)流行趨勢(shì)、強(qiáng)烈的致病性與致死性和抗生素治療可加劇病情的危險(xiǎn)性等特點(diǎn),已經(jīng)成為全球性的公共衛(wèi)生問(wèn)題。然而目前對(duì)其感染仍缺乏有效的防治方法,研究證明:抗生素可促使O157菌釋放致死性志賀毒素(Stx),從而使患者并發(fā)HUS的危險(xiǎn)性增加。由于O157的暴發(fā)流行和治療上的困難,疫苗成為預(yù)防和控制O157感染最簡(jiǎn)單、經(jīng)濟(jì)、快捷的手段,發(fā)展有效的O157疫苗就顯得亟為緊迫。 由于O157菌體抗原成份復(fù)雜,含有多種致病因子,且與普通大腸桿菌有大量的共同抗原,易引發(fā)不良反應(yīng),使O157全菌疫苗研究和應(yīng)用受到限制。隨著基因工程技術(shù)的成熟,亞單位基因工程疫苗將成為O157疫苗研究的主攻方向。 Ⅲ型分泌蛋白A (E. coli secreted protein A,EspA)、緊密粘附素(Intimin)及志賀毒素ⅡB亞單位(Shiga like toxinⅡB subunit, Stx2B)為O157的重要保護(hù)性候選抗原,本課題在本實(shí)驗(yàn)室前期對(duì)它們研究的基礎(chǔ)上,構(gòu)建EspA、IntiminC端免疫保護(hù)性片段及Stx2B的融合基因;并通過(guò)原核表達(dá)系統(tǒng)制備多亞單位融合蛋白;純化目的蛋白后對(duì)Balb/c小鼠進(jìn)行免疫后攻毒保護(hù)實(shí)驗(yàn),評(píng)價(jià)該疫苗的免疫保護(hù)率,為多亞單位O157疫苗的研制提供實(shí)驗(yàn)依據(jù)。 本實(shí)驗(yàn)完成了以下幾個(gè)方面的工作: 1. espA -eaeC300(EI)融合基因的構(gòu)建、表達(dá)、純化及部分生物學(xué)性質(zhì)研究。 1.1采用PCR法自O(shè)157菌基因組擴(kuò)增EspA的編碼基因espA和IntiminC300的編碼基因eaeC300。通過(guò)基因重組技術(shù)構(gòu)建融合基因espA-eaeC300,酶切、測(cè)序證實(shí)與預(yù)期序列完全一致。陽(yáng)性重組子轉(zhuǎn)化大腸桿菌BL21(DE3)后,IPTG誘導(dǎo)表達(dá),目的蛋白表達(dá)率約40%,SDS-PAGE分析目的蛋白的分子量約54KD,超聲破菌后電泳證實(shí)目的蛋白以包涵體形式表達(dá)。Western blotting顯示融合蛋白EI具有與EspA和IntiminC300兔多抗血清的免疫反應(yīng)性。目的蛋白經(jīng)包涵體洗滌和親和層析純化后純度為90%,免疫家兔獲得了雙擴(kuò)效價(jià)為1:32的多抗血清。 1.2重組融合蛋白EI生物學(xué)作用研究: 1.2.1 EHEC O157體外粘附細(xì)胞模型建立。選用培養(yǎng)簡(jiǎn)單、生長(zhǎng)周期較短、貼壁生長(zhǎng)的HeLa細(xì)胞,將O157接種于HEPES-DMEM液體培養(yǎng)基內(nèi),培養(yǎng)至對(duì)數(shù)生長(zhǎng)期(OD600nm≈0.6),然后將貼壁生長(zhǎng)在蓋玻片上的HeLa細(xì)胞與細(xì)菌于37℃、5%CO2共同孵育4h,姬姆薩染色及電鏡觀察。結(jié)果顯示O157菌與HeLa細(xì)胞呈聚集性粘附。 1.2.2采用EI兔抗血清與細(xì)菌預(yù)中和及細(xì)菌與抗體同時(shí)加入方法,與HeLa細(xì)胞粘附,用PBS洗去未粘附細(xì)菌,胰酶消化細(xì)胞,倍比稀釋后接種LB平板,計(jì)數(shù)單菌落,統(tǒng)計(jì)學(xué)分析EI兔抗血清阻斷粘附的效果。結(jié)果顯示:抗體預(yù)先中和的O157菌與抗體同時(shí)加入的O157菌沒(méi)有顯著性差異(P0.05),表明EI多抗血清不能阻斷細(xì)菌與細(xì)胞的粘附。 1.2.3用EI兔抗血清作為一抗,FITC標(biāo)記的羊抗兔IgG作為二抗,檢測(cè)O157菌與HeLa細(xì)胞的粘附。結(jié)果顯示:EI兔抗血清可以與天然O157菌發(fā)生抗原抗體反應(yīng)。 1.2.4 FAS(Fluorescent actin staining )試驗(yàn)檢測(cè)EI兔抗血清阻止O157菌與HeLa細(xì)胞間粘附和擦拭性(attaching and effacing, A/E)損傷的效果。結(jié)果顯示:EI兔抗血清可以影響O157菌對(duì)HeLa細(xì)胞造成的A/E損傷。 2. espA -eaeC300-stx2b (EIS)融合基因的構(gòu)建、表達(dá)與純化采用重疊延伸PCR的方法,EI為融合基因的前端,通過(guò)linker GSGGSG與stx2b相連接,從而獲得融合基因EIS;將融合基因構(gòu)建在原核表達(dá)載體pET-28a(+)中,通過(guò)酶切、測(cè)序證實(shí)與理論預(yù)測(cè)值一致性為99.8%(1781/1784)。陽(yáng)性重組子轉(zhuǎn)化大腸桿菌BL21 (DE3)后,IPTG誘導(dǎo)表達(dá),目的蛋白表達(dá)率約30%,SDS-PAGE分析目的蛋白的分子量約64KD,超聲破菌后電泳證實(shí)目的蛋白以包涵體形式表達(dá)。目的蛋白經(jīng)包涵體洗滌和親和層析純化后純度為90%。Western blotting顯示融合蛋白EIS具有與EspA、Intimin C300兔多抗血清及兔抗O157:H7超聲上清血清的免疫反應(yīng)性,且表現(xiàn)出與Gb3的結(jié)合活性。 3.融合蛋白的動(dòng)物免疫及攻毒保護(hù)試驗(yàn)。用純化后的EI和EIS重組蛋白免疫Balb/c小鼠,ELISA結(jié)果顯示血清特異性抗體的效價(jià)顯著增高,表明重組蛋白具有很強(qiáng)的免疫原性。以野生型O157活菌攻毒免疫保護(hù)實(shí)驗(yàn)證明兩組融合蛋白均能有效保護(hù)活菌攻毒引起的Balb/c小鼠死亡,其中融合蛋白EIS對(duì)O157的感染保護(hù)率為92%,是各免疫組中最高的。O157超聲上清致死保護(hù)實(shí)驗(yàn)結(jié)果顯示,融合蛋白EIS具有抗毒素致死的保護(hù)作用,致死保護(hù)率為67%,表明融合蛋白中Stx2B亞單位抗原刺激機(jī)體產(chǎn)生能夠中和Stx毒素的抗體。 綜上所述,本研究成功表達(dá)了EI和EIS融合蛋白,建立了O157體外粘附細(xì)胞模型;通過(guò)對(duì)融合蛋白部分生物學(xué)功能研究顯示:EI和EIS均具有良好的免疫原性和免疫反應(yīng)性;融合蛋白EI刺激機(jī)體產(chǎn)生的抗體具有一定的保護(hù)作用,其對(duì)于HeLa細(xì)胞的保護(hù)不是通過(guò)減少O157對(duì)細(xì)胞的粘附,而是通過(guò)阻斷A/E損傷的形成來(lái)發(fā)揮作用;融合蛋白EIS增加了Stx2B亞單位抗原,刺激機(jī)體產(chǎn)生能夠中和毒素的抗體,提高了疫苗的免疫保護(hù)率。本課題的實(shí)施為研制O157:H7基因工程多亞單位融和蛋白疫苗奠定了良好的基礎(chǔ)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
，
本文編號(hào)：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
，
本文編號(hào)：2190700
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