腸出血型大腸埃希菌O157:H7候選疫苗的構建及動物效果評價
發(fā)布時間：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EHEC O157:H7感染尚缺乏有效的防治措施,安全有效的疫苗是預防其感染的首選。本研究選擇EspA和Tir兩個關鍵性定植功能蛋白候選抗原,同時構建具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,并評價其保護效果。二、研究方法首先通過重疊延伸PCR方法擴增目的片段EspA-Tir-M,將EspA-Tir-M與原核質(zhì)粒pET28a(+)連接,構建大腸桿菌原核表達系統(tǒng),通過誘導蛋白表達和純化濃縮蛋白EspA-Tir-M。將制備EspA-Tir-M蛋白分別通過滴鼻和皮下注射兩種方式免疫小鼠,測定小鼠血清IgG、糞便SIgA,血清中細胞因子IFN-γ、IL-4和IL-10的表達水平,并進行攻毒實驗,觀察小鼠的死亡率、排菌量及組織病理切片,對比兩種免疫方式的保護效果;其次,再將EspA-Tir-M融合基因與pMG36e質(zhì)粒連接后電轉化至嗜酸乳桿菌感受態(tài),構建表達EspA-Tir-M的重組嗜酸乳桿菌,并進行多重PCR和測序鑒定重組嗜酸乳桿菌,進行SDS-PAGE及Western Blot分析重組嗜酸乳桿菌表達的蛋白情況;并通過體內(nèi)外實驗評價該重組嗜酸乳桿菌的保護效果,體外實驗通過競爭、排斥實驗和細胞骨架染色,觀察重組嗜酸乳桿菌抑制EHEC O157:H7黏附LoVo細胞及預防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,誘導小鼠產(chǎn)生特異性抗體和細胞因子的表達水平,以及EHEC O157:H7攻毒后對小鼠的保護效果。三、結果本實驗成功構建EspA-Tir-M原核表達系統(tǒng),且誘導表達的EspA-Tir-M主要以可溶性形式存在,純化后蛋白純度可達90%,將蛋白采取皮下和滴鼻兩種免疫方式,結果表明滴鼻免疫比皮下免疫可更好的誘導小鼠產(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的表達水平,而皮下免疫只能提高IL-10和IFN-γ的表達水平,此外,滴鼻免疫的保護率高達88.9%,病理切片也顯示滴鼻免疫可有效預防EHEC 0157:H7對腸道的損傷,而皮下免疫的保護效果則較差。同時首次成功構建了表達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可溶性蛋白;重組嗜酸乳桿菌體外實驗可競爭性排斥達60%EHEC O157:H7黏附LoVo細胞,而預防性排斥高達94%,并且可阻止EHEC O157:H7對LoVo細胞造成A/E損傷;體內(nèi)實驗表明重組嗜酸乳桿菌可誘導小鼠產(chǎn)生較高水平的IgG和SIgA,提高小鼠血清中IFN-γ、IL-4和IL-10的表達水平,對小鼠的保護率達77.8%,并有效縮短糞便排菌量和排菌時間,病理切片顯示有效減輕EHEC O157:H7引起的A/E損傷。四、結論本研究首次構建了預防EHEC O157:H7的新型雙價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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392
本文編號：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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392
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