副結(jié)核分枝桿菌MAP3061c蛋白表面展示疫苗載體的構(gòu)建及其免疫效力研究
發(fā)布時(shí)間：2018-11-08 11:20
【摘要】：副結(jié)核分枝桿菌(Mycobacterium avium subsp.Paratuberculosis,MAP)是引發(fā)副結(jié)核病或者約內(nèi)氏病(Johne’s disease,JD)的病原體,它主要感染反芻動(dòng)物,造成慢性且無法治愈的肉芽腫腸炎,它的感染范圍較廣,包括家養(yǎng)的牛、羊及野生動(dòng)物鹿等。由于患病動(dòng)物的產(chǎn)奶能力和繁殖能力下降,對我國養(yǎng)殖業(yè)以及乳制品產(chǎn)業(yè)帶來巨大的影響,造成巨大的經(jīng)濟(jì)損失,因此副結(jié)核病的防治已經(jīng)成為我國養(yǎng)殖業(yè)所面臨的重大問題。因而需要開發(fā)一種高效,經(jīng)濟(jì),穩(wěn)定的疫苗對副結(jié)核病進(jìn)行控制。而表面展示疫苗在設(shè)計(jì)方面具有獨(dú)特的優(yōu)勢,它將抗原遞呈在細(xì)菌表面,這樣使呈現(xiàn)在細(xì)菌表面的多肽抗原更容易被免疫系統(tǒng)識別。并且細(xì)菌的外膜蛋白和分泌的毒素都具有較強(qiáng)免疫原性,可以作為所展示的外源蛋白的免疫佐劑。因此,在新型疫苗的研制方面具有廣闊的應(yīng)用前景。本研究以冰核蛋白(Ice Nucleation Protein)作為展示平臺,在大腸桿菌中構(gòu)建表面展示系統(tǒng)。將目的基因克隆于構(gòu)建的pET-INP表面展示載體,轉(zhuǎn)入大腸桿菌BL21(DE3)中,IPTG誘導(dǎo)表達(dá)。經(jīng)過Western-blot鑒定,亞細(xì)胞組分分離,蛋白酶K處理,斑點(diǎn)印跡鑒定,免疫熒光等實(shí)驗(yàn)方法,確定展示蛋白成功表達(dá),并且展示在大腸桿菌表面。用成功構(gòu)建的MAP3061c蛋白表面展示疫苗載體免疫Balb/C小鼠,定期采取血清、分離脾臟淋巴細(xì)胞,分別檢測抗體水平、細(xì)胞因子水平、CD4+和CD8+細(xì)胞數(shù)量,分析體液與細(xì)胞免疫水平。然后進(jìn)行攻毒實(shí)驗(yàn),通過檢測細(xì)胞因子變化,小鼠體重變化,以及小鼠結(jié)腸、肝、脾等病理變化評價(jià)免疫保護(hù)效果。結(jié)果表明,與對照組相比,重組菌免疫組抗體水平有較大程度提升,在三免后,其抗體水平為空載體對照組的4.2倍,顯示該表面展示疫苗載體能刺激機(jī)體產(chǎn)生針對所展示蛋白的高水平特異性抗體;展示疫苗組的CD4+和CD8+T細(xì)胞顯著增多,而且IFN-γ、IL-4、IL-23/IL-17等細(xì)胞因子水平顯著升高,說明重組菌INP-MAP3061c能夠介導(dǎo)機(jī)體產(chǎn)生較強(qiáng)的細(xì)胞免疫反應(yīng)。在攻毒后,表面展示疫苗免疫組小鼠在攻毒16周之前增重率為增長狀態(tài),在16-20周為緩慢下降狀態(tài),但依然為正值,說明小鼠體重一直處于增長狀態(tài),沒有消瘦。肝、脾等組織病理結(jié)果表明,MAP3061c蛋白表面展示疫苗可以降低病理損傷程度,減緩病程。綜上,本研究以副結(jié)核分枝桿菌強(qiáng)免疫原性蛋白MAP3061c為對象,借助細(xì)菌表面展示平臺,將其展示于大腸桿菌表面,所構(gòu)建的表面展示活疫苗載體能夠在小鼠中激發(fā)強(qiáng)烈的體液與細(xì)胞免疫,對副結(jié)核分枝桿菌感染具有較好的抵抗效果,為新型副結(jié)核疫苗的研制奠定基石,為副結(jié)核病的防控提供新思路。
[Abstract]:Mycobacterium paratuberculosis (Mycobacterium avium subsp.Paratuberculosis,MAP) is the pathogen that causes paratuberous or Johne's disease,JD disease. It mainly infects ruminants and causes chronic and incurable granulomatous enteritis. Including domestic cattle, sheep and wild animal deer and so on. Because the ability of producing milk and breeding of diseased animals has declined, it has brought great influence to the breeding and dairy industry of our country, and caused huge economic losses. Therefore, the prevention and treatment of paratuberous tuberculosis has become a major problem in the breeding industry of our country. Therefore, there is a need to develop an efficient, economical and stable vaccine for the control of paratuberculosis. The surface display vaccine has unique advantages in design. It presents the antigen on the bacterial surface, which makes it easier for the immune system to recognize the polypeptide antigen present on the bacterial surface. The outer membrane protein and toxin secreted by bacteria have strong immunogenicity and can be used as the immunoadjuvant of foreign proteins. Therefore, it has a broad application prospect in the development of new vaccines. In this study, the ice nucleoprotein (Ice Nucleation Protein) was used as the display platform to construct a surface display system in Escherichia coli. The target gene was cloned into the constructed pET-INP surface display vector and transferred into Escherichia coli BL21 (DE3) for IPTG induced expression. After Western-blot identification, subcellular component separation, protease K treatment, dot blot identification, immunofluorescence and other experimental methods, it was confirmed that the display protein was successfully expressed and displayed on the surface of Escherichia coli. Balb/C mice were immunized with successfully constructed MAP3061c protein surface display vaccine vector. Serum was used regularly to isolate spleen lymphocytes, antibody level, cytokine level, number of CD4 and CD8 cells were detected, humoral and cellular immunity levels were analyzed. The immune protective effect was evaluated by detecting the changes of cytokines, body weight, colon, liver, spleen and other pathological changes of mice. The results showed that, compared with the control group, the antibody level of the recombinant bacteria immunized group was increased to a great extent, and the antibody level of the recombinant bacteria immunized group was 4.2 times as high as that of the empty carrier control group after three immunizations. It is shown that the surface display vaccine vector can stimulate the body to produce a high level specific antibody against the displayed protein. The levels of CD4 and CD8 T cells and cytokines such as IFN- 緯 and IL-4,IL-23/IL-17 were significantly increased in the vaccine group, indicating that the recombinant strain INP-MAP3061c could induce a strong cellular immune response. After inoculation, the weight gain rate of the vaccinated mice was increased 16 weeks before the attack, and decreased slowly at 16-20 weeks, but it was still positive, indicating that the weight of the mice had been growing and no weight loss. The pathological results of liver and spleen showed that the surface display vaccine of MAP3061c protein could reduce the degree of pathological injury and slow down the course of disease. In this study, the strong immunogenicity protein (MAP3061c) of Mycobacterium paratuberculosis was used to display it on the surface of Escherichia coli with the help of bacterial surface display platform. The constructed surface display live vaccine vector can stimulate strong humoral and cellular immunity in mice and has good resistance to Mycobacterium paratuberculosis infection. To provide a new idea for the prevention and control of accessory tuberculosis.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.4
本文編號：2318337
[Abstract]:Mycobacterium paratuberculosis (Mycobacterium avium subsp.Paratuberculosis,MAP) is the pathogen that causes paratuberous or Johne's disease,JD disease. It mainly infects ruminants and causes chronic and incurable granulomatous enteritis. Including domestic cattle, sheep and wild animal deer and so on. Because the ability of producing milk and breeding of diseased animals has declined, it has brought great influence to the breeding and dairy industry of our country, and caused huge economic losses. Therefore, the prevention and treatment of paratuberous tuberculosis has become a major problem in the breeding industry of our country. Therefore, there is a need to develop an efficient, economical and stable vaccine for the control of paratuberculosis. The surface display vaccine has unique advantages in design. It presents the antigen on the bacterial surface, which makes it easier for the immune system to recognize the polypeptide antigen present on the bacterial surface. The outer membrane protein and toxin secreted by bacteria have strong immunogenicity and can be used as the immunoadjuvant of foreign proteins. Therefore, it has a broad application prospect in the development of new vaccines. In this study, the ice nucleoprotein (Ice Nucleation Protein) was used as the display platform to construct a surface display system in Escherichia coli. The target gene was cloned into the constructed pET-INP surface display vector and transferred into Escherichia coli BL21 (DE3) for IPTG induced expression. After Western-blot identification, subcellular component separation, protease K treatment, dot blot identification, immunofluorescence and other experimental methods, it was confirmed that the display protein was successfully expressed and displayed on the surface of Escherichia coli. Balb/C mice were immunized with successfully constructed MAP3061c protein surface display vaccine vector. Serum was used regularly to isolate spleen lymphocytes, antibody level, cytokine level, number of CD4 and CD8 cells were detected, humoral and cellular immunity levels were analyzed. The immune protective effect was evaluated by detecting the changes of cytokines, body weight, colon, liver, spleen and other pathological changes of mice. The results showed that, compared with the control group, the antibody level of the recombinant bacteria immunized group was increased to a great extent, and the antibody level of the recombinant bacteria immunized group was 4.2 times as high as that of the empty carrier control group after three immunizations. It is shown that the surface display vaccine vector can stimulate the body to produce a high level specific antibody against the displayed protein. The levels of CD4 and CD8 T cells and cytokines such as IFN- 緯 and IL-4,IL-23/IL-17 were significantly increased in the vaccine group, indicating that the recombinant strain INP-MAP3061c could induce a strong cellular immune response. After inoculation, the weight gain rate of the vaccinated mice was increased 16 weeks before the attack, and decreased slowly at 16-20 weeks, but it was still positive, indicating that the weight of the mice had been growing and no weight loss. The pathological results of liver and spleen showed that the surface display vaccine of MAP3061c protein could reduce the degree of pathological injury and slow down the course of disease. In this study, the strong immunogenicity protein (MAP3061c) of Mycobacterium paratuberculosis was used to display it on the surface of Escherichia coli with the help of bacterial surface display platform. The constructed surface display live vaccine vector can stimulate strong humoral and cellular immunity in mice and has good resistance to Mycobacterium paratuberculosis infection. To provide a new idea for the prevention and control of accessory tuberculosis.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.4
【參考文獻(xiàn)】
相關(guān)期刊論文 前8條
1 王恒生;刁治民;陳克龍;魏波;;冰核微生物的研究動(dòng)態(tài)及開發(fā)應(yīng)用前景[J];江蘇農(nóng)業(yè)科學(xué);2014年07期
2 徐麗香;王作鎮(zhèn);舒正玉;武海龍;劉艷如;李欣;黃建忠;;細(xì)菌脂肪酶在大腸桿菌細(xì)胞表面的功能性展示[J];生物技術(shù)通報(bào);2014年06期
3 張家禾;左偉勇;洪偉鳴;管遠(yuǎn)紅;孟婷;劉莉;周作紅;;噬菌體展示技術(shù)及其在抗體制備中應(yīng)用的研究進(jìn)展[J];南昌大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2014年02期
4 張秀艷;侯曉彥;陳福生;王小紅;;酵母細(xì)胞表面展示載體的構(gòu)建及功能驗(yàn)證[J];生物技術(shù);2014年01期
5 楊曉;孫爽;王海鳳;杭海英;;兩種主流載體蛋白在大腸桿菌表面展示抗體應(yīng)用中的系統(tǒng)分析(英文)[J];生物化學(xué)與生物物理進(jìn)展;2013年12期
6 張秋香;侯慧麗;蘆穎;陳衛(wèi);鐘瑾;;基于冰核蛋白的乳酸菌表面展示系統(tǒng)的構(gòu)建[J];微生物學(xué)報(bào);2013年04期
7 徐榮榮;鄧開野;丁力行;;冰核活性細(xì)菌成冰的分子生物學(xué)基礎(chǔ)及其在食品冷凍中的應(yīng)用[J];仲愷農(nóng)業(yè)工程學(xué)院學(xué)報(bào);2012年03期
8 陶站華;張搏;;利用冰核蛋白N末端結(jié)構(gòu)域在大腸桿菌表面展示粘質(zhì)沙雷氏菌脂肪酶[J];微生物學(xué)通報(bào);2012年03期
，本文編號：2318337
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