腸出血型大腸埃希菌O157:H7候選疫苗的構(gòu)建及動(dòng)物效果評(píng)價(jià)
發(fā)布時(shí)間：2019-03-24 16:31
【摘要】：一、研究目的腸出血型大腸埃希菌(Enterohaemorrhagic Escherichia coli,EHEC)O157:H7為人畜共患病,臨床上可引起出血性腸炎、溶血性尿毒綜合癥。目前對(duì)EHEC O157:H7感染尚缺乏有效的防治措施,安全有效的疫苗是預(yù)防其感染的首選。本研究選擇EspA和Tir兩個(gè)關(guān)鍵性定植功能蛋白候選抗原,同時(shí)構(gòu)建具EspA-Tir-M融合蛋白為抗原的亞單位候選疫苗和以嗜酸乳桿菌為抗原投遞系統(tǒng)的活載體候選疫苗菌株,并評(píng)價(jià)其保護(hù)效果。二、研究方法首先通過(guò)重疊延伸PCR方法擴(kuò)增目的片段EspA-Tir-M,將EspA-Tir-M與原核質(zhì)粒pET28a(+)連接,構(gòu)建大腸桿菌原核表達(dá)系統(tǒng),通過(guò)誘導(dǎo)蛋白表達(dá)和純化濃縮蛋白EspA-Tir-M。將制備EspA-Tir-M蛋白分別通過(guò)滴鼻和皮下注射兩種方式免疫小鼠,測(cè)定小鼠血清IgG、糞便SIgA,血清中細(xì)胞因子IFN-γ、IL-4和IL-10的表達(dá)水平,并進(jìn)行攻毒實(shí)驗(yàn),觀察小鼠的死亡率、排菌量及組織病理切片,對(duì)比兩種免疫方式的保護(hù)效果;其次,再將EspA-Tir-M融合基因與pMG36e質(zhì)粒連接后電轉(zhuǎn)化至嗜酸乳桿菌感受態(tài),構(gòu)建表達(dá)EspA-Tir-M的重組嗜酸乳桿菌,并進(jìn)行多重PCR和測(cè)序鑒定重組嗜酸乳桿菌,進(jìn)行SDS-PAGE及Western Blot分析重組嗜酸乳桿菌表達(dá)的蛋白情況;并通過(guò)體內(nèi)外實(shí)驗(yàn)評(píng)價(jià)該重組嗜酸乳桿菌的保護(hù)效果,體外實(shí)驗(yàn)通過(guò)競(jìng)爭(zhēng)、排斥實(shí)驗(yàn)和細(xì)胞骨架染色,觀察重組嗜酸乳桿菌抑制EHEC O157:H7黏附LoVo細(xì)胞及預(yù)防A/E損傷形成的效果,體內(nèi)考察重組嗜酸乳桿菌灌胃免疫小鼠后,誘導(dǎo)小鼠產(chǎn)生特異性抗體和細(xì)胞因子的表達(dá)水平,以及EHEC O157:H7攻毒后對(duì)小鼠的保護(hù)效果。三、結(jié)果本實(shí)驗(yàn)成功構(gòu)建EspA-Tir-M原核表達(dá)系統(tǒng),且誘導(dǎo)表達(dá)的EspA-Tir-M主要以可溶性形式存在,純化后蛋白純度可達(dá)90%,將蛋白采取皮下和滴鼻兩種免疫方式,結(jié)果表明滴鼻免疫比皮下免疫可更好的誘導(dǎo)小鼠產(chǎn)生較高水平的特異性IgG和SIgA,滴鼻免疫可提高IL-4、IL-10和IFN-γ的表達(dá)水平,而皮下免疫只能提高IL-10和IFN-γ的表達(dá)水平,此外,滴鼻免疫的保護(hù)率高達(dá)88.9%,病理切片也顯示滴鼻免疫可有效預(yù)防EHEC 0157:H7對(duì)腸道的損傷,而皮下免疫的保護(hù)效果則較差。同時(shí)首次成功構(gòu)建了表達(dá)EHEC O157:H7 EspA-Tir-M蛋白的重組嗜酸乳桿菌,且主要以外分泌表達(dá)可溶性蛋白;重組嗜酸乳桿菌體外實(shí)驗(yàn)可競(jìng)爭(zhēng)性排斥達(dá)60%EHEC O157:H7黏附LoVo細(xì)胞,而預(yù)防性排斥高達(dá)94%,并且可阻止EHEC O157:H7對(duì)LoVo細(xì)胞造成A/E損傷;體內(nèi)實(shí)驗(yàn)表明重組嗜酸乳桿菌可誘導(dǎo)小鼠產(chǎn)生較高水平的IgG和SIgA,提高小鼠血清中IFN-γ、IL-4和IL-10的表達(dá)水平,對(duì)小鼠的保護(hù)率達(dá)77.8%,并有效縮短糞便排菌量和排菌時(shí)間,病理切片顯示有效減輕EHEC O157:H7引起的A/E損傷。四、結(jié)論本研究首次構(gòu)建了預(yù)防EHEC O157:H7的新型雙價(jià)EspA-Tir-M融合亞單位疫苗和重組嗜酸乳桿菌活載體疫苗,兩者均具有良好的免疫效果及保護(hù)效果,可作為EHEC O157:H7的候選疫苗。
[Abstract]:1. The purpose of this study is that (Enterohaemorrhagic Escherichia coli,EHEC O157:H7 is a zoonotic disease, which can cause hemorrhagic enteritis and hemolytic uremic syndrome. At present, there are no effective measures to prevent and treat EHEC O157:H7 infection. Safe and effective vaccine is the first choice to prevent the infection. In this study, two key colonization functional protein candidate antigens (EspA and Tir) were selected, and the candidate vaccine strains with EspA-Tir-M fusion protein and Lactobacillus acidophilus as antigen delivery system were constructed at the same time, and the candidate vaccine strains with EspA-Tir-M fusion protein as antigen and Lactobacillus acidophilus as antigen delivery system were constructed at the same time. And evaluate its protective effect. Secondly, firstly, the target fragment EspA-Tir-M, was amplified by overlap extension PCR method, and the EspA-Tir-M was ligated with the prokaryotic plasmid pET28a () to construct the prokaryotic expression system of E. coli. Expression and purification of concentrated protein EspA-Tir-M. by inducing protein expression The mice were immunized with EspA-Tir-M protein by nasal drip and subcutaneous injection respectively. The expression levels of cytokines IFN- 緯, IL-4 and IL-10 in serum IgG, fecal SIgA, serum of mice were measured, and the virulence test was carried out. The mortality, bacterial excretion and histopathological sections of mice were observed to compare the protective effects of the two immune methods. Secondly, EspA-Tir-M fusion gene was ligated with pMG36e plasmid and transformed into Lactobacillus acidophilus to construct recombinant Lactobacillus acidophilus expressing EspA-Tir-M. The recombinant Lactobacillus acidophilus was identified by multiplex PCR and sequencing. The expression of recombinant Lactobacillus acidophilus was analyzed by SDS-PAGE and Western Blot. The protective effect of the recombinant Lactobacillus acidophilus was evaluated in vitro and in vivo. In vitro, competition, exclusion and cytoskeleton staining were used to evaluate the protective effect of Lactobacillus acidophilus. To observe the inhibitory effect of Lactobacillus acidophilus on EHEC O157:H7 adhesion to LoVo cells and to prevent the formation of LoVo cells, and to observe the expression level of specific antibodies and cytokines in mice immunized by Lactobacillus acidophilus intragastrically in vivo. And the protective effect of EHEC O157:H7 on mice. 3. Results the prokaryotic expression system of EspA-Tir-M was successfully constructed, and the induced expression of EspA-Tir-M was mainly in soluble form, the purity of the purified protein was up to 90%, and the protein was immunized subcutaneously and intranasally. The results showed that intranasal immunization could induce higher level of specific IgG and SIgA, intranasal immunization than subcutaneous immunization could increase the expression level of IL-4,IL-10 and IFN- 緯. However, subcutaneous immunization could only increase the expression of IL-10 and IFN- 緯. In addition, the protective rate of nasal immunization was as high as 88.9%. Pathological sections also showed that nasal immunization could effectively prevent the injury of intestinal tract caused by EHEC 0157:H7. The protective effect of subcutaneous immunity is poor. At the same time, the recombinant Lactobacillus acidophilus expressing EHEC O157:H7 EspA-Tir-M protein was successfully constructed for the first time, and the soluble protein was expressed mainly in exocrine. In vitro, recombinant Lactobacillus acidophilus could competitively reject 60%EHEC O157:H7 to adhere to LoVo cells, while prophylactic rejection was as high as 94%, and it could prevent EHEC O157:H7 from causing damage to LoVo cells. In vivo experiments showed that recombinant Lactobacillus acidophilus could induce high levels of IgG and SIgA, to increase the expression of IFN- 緯, IL-4 and IL-10 in serum of mice, and the protective rate of Lactobacillus acidophilus on mice was 77.8%. The amount of excretory bacteria and the time of excreting bacteria were shortened effectively. The pathological sections showed that the damage of EHEC O157:H7-induced A + E was effectively alleviated. 4. Conclusion the new bivalent EspA-Tir-M fusion subunit vaccine and recombinant Lactobacillus acidophilus live vector vaccine for the prevention of EHEC O157:H7 were constructed for the first time in this study, both of which had good immune and protective effects. It can be used as a candidate vaccine for EHEC O157:H7.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R392
本文編號(hào)：2446492
[Abstract]:1. The purpose of this study is that (Enterohaemorrhagic Escherichia coli,EHEC O157:H7 is a zoonotic disease, which can cause hemorrhagic enteritis and hemolytic uremic syndrome. At present, there are no effective measures to prevent and treat EHEC O157:H7 infection. Safe and effective vaccine is the first choice to prevent the infection. In this study, two key colonization functional protein candidate antigens (EspA and Tir) were selected, and the candidate vaccine strains with EspA-Tir-M fusion protein and Lactobacillus acidophilus as antigen delivery system were constructed at the same time, and the candidate vaccine strains with EspA-Tir-M fusion protein as antigen and Lactobacillus acidophilus as antigen delivery system were constructed at the same time. And evaluate its protective effect. Secondly, firstly, the target fragment EspA-Tir-M, was amplified by overlap extension PCR method, and the EspA-Tir-M was ligated with the prokaryotic plasmid pET28a () to construct the prokaryotic expression system of E. coli. Expression and purification of concentrated protein EspA-Tir-M. by inducing protein expression The mice were immunized with EspA-Tir-M protein by nasal drip and subcutaneous injection respectively. The expression levels of cytokines IFN- 緯, IL-4 and IL-10 in serum IgG, fecal SIgA, serum of mice were measured, and the virulence test was carried out. The mortality, bacterial excretion and histopathological sections of mice were observed to compare the protective effects of the two immune methods. Secondly, EspA-Tir-M fusion gene was ligated with pMG36e plasmid and transformed into Lactobacillus acidophilus to construct recombinant Lactobacillus acidophilus expressing EspA-Tir-M. The recombinant Lactobacillus acidophilus was identified by multiplex PCR and sequencing. The expression of recombinant Lactobacillus acidophilus was analyzed by SDS-PAGE and Western Blot. The protective effect of the recombinant Lactobacillus acidophilus was evaluated in vitro and in vivo. In vitro, competition, exclusion and cytoskeleton staining were used to evaluate the protective effect of Lactobacillus acidophilus. To observe the inhibitory effect of Lactobacillus acidophilus on EHEC O157:H7 adhesion to LoVo cells and to prevent the formation of LoVo cells, and to observe the expression level of specific antibodies and cytokines in mice immunized by Lactobacillus acidophilus intragastrically in vivo. And the protective effect of EHEC O157:H7 on mice. 3. Results the prokaryotic expression system of EspA-Tir-M was successfully constructed, and the induced expression of EspA-Tir-M was mainly in soluble form, the purity of the purified protein was up to 90%, and the protein was immunized subcutaneously and intranasally. The results showed that intranasal immunization could induce higher level of specific IgG and SIgA, intranasal immunization than subcutaneous immunization could increase the expression level of IL-4,IL-10 and IFN- 緯. However, subcutaneous immunization could only increase the expression of IL-10 and IFN- 緯. In addition, the protective rate of nasal immunization was as high as 88.9%. Pathological sections also showed that nasal immunization could effectively prevent the injury of intestinal tract caused by EHEC 0157:H7. The protective effect of subcutaneous immunity is poor. At the same time, the recombinant Lactobacillus acidophilus expressing EHEC O157:H7 EspA-Tir-M protein was successfully constructed for the first time, and the soluble protein was expressed mainly in exocrine. In vitro, recombinant Lactobacillus acidophilus could competitively reject 60%EHEC O157:H7 to adhere to LoVo cells, while prophylactic rejection was as high as 94%, and it could prevent EHEC O157:H7 from causing damage to LoVo cells. In vivo experiments showed that recombinant Lactobacillus acidophilus could induce high levels of IgG and SIgA, to increase the expression of IFN- 緯, IL-4 and IL-10 in serum of mice, and the protective rate of Lactobacillus acidophilus on mice was 77.8%. The amount of excretory bacteria and the time of excreting bacteria were shortened effectively. The pathological sections showed that the damage of EHEC O157:H7-induced A + E was effectively alleviated. 4. Conclusion the new bivalent EspA-Tir-M fusion subunit vaccine and recombinant Lactobacillus acidophilus live vector vaccine for the prevention of EHEC O157:H7 were constructed for the first time in this study, both of which had good immune and protective effects. It can be used as a candidate vaccine for EHEC O157:H7.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前7條
1 林如琴;吳嫻波;張益多;范宏英;;腸出血型大腸埃希菌O157∶H7基因工程疫苗研究進(jìn)展[J];現(xiàn)代預(yù)防醫(yī)學(xué);2016年01期
2 王明祥;姚明霞;郭靜;楊洋;;黔南州2011年部分食品中食源性致病菌監(jiān)測(cè)分析[J];現(xiàn)代預(yù)防醫(yī)學(xué);2014年07期
3 王春娟;王增國(guó);吳首芝;王戩;魏曉光;劉繼峰;李廣帥;;西安市O157出血性大腸桿菌監(jiān)測(cè)研究[J];現(xiàn)代預(yù)防醫(yī)學(xué);2013年11期
4 崔強(qiáng);周志江;劉建青;肖華志;;腸出血性大腸桿菌粘附機(jī)理的研究進(jìn)展[J];中國(guó)人獸共患病學(xué)報(bào);2012年08期
5 李永紅;;中國(guó)部分地區(qū)人群和動(dòng)物中腸出血性大腸埃希菌O157∶H7分布特征[J];疾病監(jiān)測(cè);2008年07期
6 汪華,景懷琦,李紅衛(wèi),倪大新,趙廣法,顧玲,楊晉川,史智揚(yáng),劉光中,胡曉抒,徐建國(guó);江蘇省淮北地區(qū)腸出血性大腸埃希菌O157:H7感染性腹瀉并發(fā)急性腎衰的研究[J];中華流行病學(xué)雜志;2004年11期
7 孟冬梅,孫建榮,許珂,張燕琴,潘勁草;浙江省首例腸出血性大腸桿菌O157∶H7菌株的分離和鑒定[J];中國(guó)公共衛(wèi)生;1999年04期
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