肠出血型大肠埃希菌O157:H7候选疫苗的构建及动物效果评价
发布时间：2019-03-24 16:31
【摘要】：一、研究目的肠出血型大肠埃希菌(Enterohaemorrhagic Escherichia coli,EHEC)O157:H7为人畜共患病,临床上可引起出血性肠炎、溶血性尿毒综合症。目前对EHEC O157:H7感染尚缺乏有效的防治措施,安全有效的疫苗是预防其感染的首选。本研究选择EspA和Tir两个关键性定植功能蛋白候选抗原,同时构建具EspA-Tir-M融合蛋白为抗原的亚单位候选疫苗和以嗜酸乳杆菌为抗原投递系统的活载体候选疫苗菌株,并评价其保护效果。二、研究方法首先通过重叠延伸PCR方法扩增目的片段EspA-Tir-M,将EspA-Tir-M与原核质粒pET28a(+)连接,构建大肠杆菌原核表达系统,通过诱导蛋白表达和纯化浓缩蛋白EspA-Tir-M。将制备EspA-Tir-M蛋白分别通过滴鼻和皮下注射两种方式免疫小鼠,测定小鼠血清IgG、粪便SIgA,血清中细胞因子IFN-γ、IL-4和IL-10的表达水平,并进行攻毒实验,观察小鼠的死亡率、排菌量及组织病理切片,对比两种免疫方式的保护效果;其次,再将EspA-Tir-M融合基因与pMG36e质粒连接后电转化至嗜酸乳杆菌感受态,构建表达EspA-Tir-M的重组嗜酸乳杆菌,并进行多重PCR和测序鉴定重组嗜酸乳杆菌,进行SDS-PAGE及Western Blot分析重组嗜酸乳杆菌表达的蛋白情况;并通过体内外实验评价该重组嗜酸乳杆菌的保护效果,体外实验通过竞争、排斥实验和细胞骨架染色,观察重组嗜酸乳杆菌抑制EHEC O157:H7黏附LoVo细胞及预防A/E损伤形成的效果,体内考察重组嗜酸乳杆菌灌胃免疫小鼠后,诱导小鼠产生特异性抗体和细胞因子的表达水平,以及EHEC O157:H7攻毒后对小鼠的保护效果。三、结果本实验成功构建EspA-Tir-M原核表达系统,且诱导表达的EspA-Tir-M主要以可溶性形式存在,纯化后蛋白纯度可达90%,将蛋白采取皮下和滴鼻两种免疫方式,结果表明滴鼻免疫比皮下免疫可更好的诱导小鼠产生较高水平的特异性IgG和SIgA,滴鼻免疫可提高IL-4、IL-10和IFN-γ的表达水平,而皮下免疫只能提高IL-10和IFN-γ的表达水平,此外,滴鼻免疫的保护率高达88.9%,病理切片也显示滴鼻免疫可有效预防EHEC 0157:H7对肠道的损伤,而皮下免疫的保护效果则较差。同时首次成功构建了表达EHEC O157:H7 EspA-Tir-M蛋白的重组嗜酸乳杆菌,且主要以外分泌表达可溶性蛋白;重组嗜酸乳杆菌体外实验可竞争性排斥达60%EHEC O157:H7黏附LoVo细胞,而预防性排斥高达94%,并且可阻止EHEC O157:H7对LoVo细胞造成A/E损伤;体内实验表明重组嗜酸乳杆菌可诱导小鼠产生较高水平的IgG和SIgA,提高小鼠血清中IFN-γ、IL-4和IL-10的表达水平,对小鼠的保护率达77.8%,并有效缩短粪便排菌量和排菌时间,病理切片显示有效减轻EHEC O157:H7引起的A/E损伤。四、结论本研究首次构建了预防EHEC O157:H7的新型双价EspA-Tir-M融合亚单位疫苗和重组嗜酸乳杆菌活载体疫苗,两者均具有良好的免疫效果及保护效果,可作为EHEC O157:H7的候选疫苗。
[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- 纬, 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- 纬. However, subcutaneous immunization could only increase the expression of IL-10 and IFN- 纬. 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- 纬, 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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
本文编号：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- 纬, 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- 纬. However, subcutaneous immunization could only increase the expression of IL-10 and IFN- 纬. 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- 纬, 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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
【参考文献】
相关期刊论文 前7条
1 林如琴;吴娴波;张益多;范宏英;;肠出血型大肠埃希菌O157∶H7基因工程疫苗研究进展[J];现代预防医学;2016年01期
2 王明祥;姚明霞;郭静;杨洋;;黔南州2011年部分食品中食源性致病菌监测分析[J];现代预防医学;2014年07期
3 王春娟;王增国;吴首芝;王戬;魏晓光;刘继峰;李广帅;;西安市O157出血性大肠杆菌监测研究[J];现代预防医学;2013年11期
4 崔强;周志江;刘建青;肖华志;;肠出血性大肠杆菌粘附机理的研究进展[J];中国人兽共患病学报;2012年08期
5 李永红;;中国部分地区人群和动物中肠出血性大肠埃希菌O157∶H7分布特征[J];疾病监测;2008年07期
6 汪华,景怀琦,李红卫,倪大新,赵广法,顾玲,杨晋川,史智扬,刘光中,胡晓抒,徐建国;江苏省淮北地区肠出血性大肠埃希菌O157:H7感染性腹泻并发急性肾衰的研究[J];中华流行病学杂志;2004年11期
7 孟冬梅,孙建荣,许珂,张燕琴,潘劲草;浙江省首例肠出血性大肠杆菌O157∶H7菌株的分离和鉴定[J];中国公共卫生;1999年04期
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