大肠杆菌O157:H7糖缀合物疫苗的生物法合成及免疫学研究

发布时间：2018-05-24 23:41

本文选题：糖缀合物疫苗 + E.coli　；参考：《山东大学》2016年博士论文

【摘要】：大肠杆菌(E. coli) O157:H7是严重的肠道致病菌,通常引起腹泻、出血性结肠炎、溶血性尿毒症等。传统的抗生素治疗会引起肠道内E. coli 0157:H7细胞裂解并向肠道黏膜释放志贺样毒素等,有加重病情的风险。疫苗作为替代手段,对预防E. coli 0157:H7的感染具有重要意义。O-抗原或O-PS,作为脂多糖的重要组成部分,暴露在细菌外表面,是机体免疫系统识别的重要靶点。O-PS是不依赖于T细胞的抗原。O-PS免疫机体主要产生抗O-PS的IgM抗体,几乎不产生IgG抗体以及T细胞记忆,需要按期重复接种来维持免疫力,并且不能在免疫系统发育不完全的小于2周岁的婴幼儿、免疫力低下的老年人等人群中产生足够的免疫力。通过将O-PS连接到载体蛋白上,形成的糖缀合物是依赖于T细胞的抗原。糖缀合物免疫机体产生的抗O-PS抗体从IgM向IgG、IgA等转换,并产生T细胞记忆,不仅能在健康成年人体内产生持久的免疫力,而且针对婴幼儿、免疫力底下的老年人同样有效。糖缀合物疫苗,公认为是最有效和最安全的抗菌疫苗之一。为了有效预防E.coli 0157:H7的感染,本论文针对E. coli O157:H7的O-PS进行了糖缀合物疫苗的生物法合成及免疫学研究。本论文分为三部分研究内容。第一部分是生物法合成糖缀合物疫苗(第二章)；第二部分是糖缀合物疫苗O-PS-MBP的免疫学评价(第三章)；第三部分是载体蛋白及其不同位置多肽对糖缀合物疫苗抗多糖IgG滴度和亚型的影响(第四章)。第一部分：糖缀合物疫苗的合成通常采用化学法。但是化学法存在着生产成本高、质量难以控制、容易有批次间的差异等局限性。本部分我们采用基于空肠弯曲菌(C. jejuni)N-糖基化系统的体内酶法合成糖缀合物疫苗。糖基转移酶Pg1B是C.jejuni的关键酶,负责将糖链从类萜类脂载体连接到载体蛋白的氨基酸序列D/E-X-N-Y-S/T (X, Y≠P)的天冬酰胺(N)残基上。利用来自C. jejuni的N-糖基化系统和E. coli O-PS合成途径比较相似的特点,将C. jejuni N-糖基化系统的关键酶Pg1B的基因、E. coli O157:H7O-PS合成基因、载体蛋白(麦芽糖结合蛋白MBP、CRM197或AcrA)合成基因导入到敲除糖基转移酶基因waal的宿主菌E. coliW3110中,从而建立E. coli的N-糖基化系统。利用E.coli糖基化系统生产糖缀合物疫苗,具有构建方式灵活、一次发酵、纯化条件简单、成本低的特点,满足大规模制备的要求。同时,利用E.coli N-糖基化系统生产的糖缀合物疫苗糖基化位点、糖链长度等明确并可控,且生产批次之间没有差异,满足作为疫苗的质量要求。然后通过对重组菌的诱导、蛋白的表达和纯化,得到的糖缀合物经过定性分析(包括考马斯亮蓝染色检测western blot检测、MALDI-TOF检测、糖组成分析),得到了3个连有E. coli 0157:H7 O-PS的、载体蛋白不同的糖缀合物O-PS-MBP、O-PS-CRM、O-PS-AcrA。又通过定量分析得知,O-PS-MBP、O-PS-CRM、 O-PS-AcrA的得率约为0.5-1 mg/L(蛋白定量),且O-PS-MBP和O-PS-CRM的糖基化比例比O-PS-AcrA更高。利用E. coli N-糖基化系统生产糖缀合物O-PS-MBP、O-PS-CRM、O-PS-AcrA,开辟了一条合成E. coli O157:H7疫苗的新途径,具有广阔的应用前景。第二部分：适应性免疫应答分为体液免疫和细胞免疫。之前报道的抗E. coli 0151:H7的糖缀合物疫苗O-PS-rEPA和O-PS-Stx,更多关注的是其产生抗E. coli O157:H7多糖抗体的能力,即体液免疫。几乎没有文献报道糖缀合物疫苗刺激机体产生的细胞免疫,包括T细胞的增殖和分化、细胞因子的分泌等。现在,有越来越多的研究表明细胞免疫在抗菌感染的重要作用,而且其作用时间比抗体更加持久。目前的婴幼儿计划免疫中有很多疫苗是糖缀合物疫苗。然而,同时或先后免疫接种多种糖缀合物疫苗时,如果载体蛋白相同,可能会引起免疫抑制等负面作用。面对日益增多的新型的糖缀合物疫苗,除了广泛应用的载体蛋白外,急需寻找和开发新的载体蛋白。目前,探索新型载体蛋白主要集中在寻找含有T细胞表位的蛋白、有Toll样受体特性的蛋白等。最近,人们开始关注MBP在免疫学上的价值和应用。一些具有抗肿瘤、抗菌或抗寄生虫的活性蛋白在和MBP融合表达后活性明显提高。研究证明MBP有TLR4激动剂的性质,可促进Thl细胞的活化,并有激活树突状细胞和腹腔巨噬细胞、促进脾脏淋巴细胞的增殖等作用。然而,目前还没有文献报道MBP作为糖缀合物疫苗的载体蛋白以及MBP对糖缀合物疫苗的免疫增强作用。本部分,我们用以MBP作为载体蛋白的糖缀合物O-PS-MBP免疫BALB/c小鼠,检测O-PS-MBP激活的体液免疫和细胞免疫。体液免疫方面,O-PS-MBP激活小鼠产生了抗E. coli O157:H7多糖的血清IgG、IgM和肠道IgA。并且,O-PS-MBP免疫血清在补体环境中可以有效杀死E. coli O157:H7。细胞免疫方面,O-PS-MBP促进脾脏淋巴CD4+T细胞、CD8+T细胞的增殖。此外通过分析得出,O-PS-MBP免疫血清中IFN-γ、抗E. coli 0157:H7多糖IgG2a含量较高,说明O-PS-MBP刺激小鼠机体偏向Thl型免疫应答。进一步地,通过ELISPOT分析发现,MBP无论是单独免疫还是以糖缀合物的形式免疫,都能刺激机体产生IFN-y,抑制产生IL-4,说明载体蛋白MBP使O-PS-MBP偏向Thl型细胞免疫应答,有很好的免疫增强作用。以MBP作为糖缀合物疫苗的载体蛋白具有广阔的应用前景。第三部分：糖缀合物疫苗激活机体产生抗多糖抗体,并通过多糖抗体介导的调理素或补体作用吞噬病菌,达到清除病菌、保护机体的作用。糖缀合物疫苗产生的抗多糖IgG抗体是体液免疫应答的重要组成部分,IgG抗体滴度的高低直接决定着糖缀合物疫苗的免疫效果。IgG根据其结构特点,可分为不同的亚型,包括IgG1、 IgG2、IgG3等。不同的IgG亚型有不同的功能和特点。同时,IgG亚型分布反应了Th细胞免疫应答的方向(Thl或Th2)。研究糖缀合物疫苗引起的IgG亚型的分布或Th细胞分化的方向,对评价糖缀合物疫苗免疫机制和效果有重要意义。本部分,我们通过多肽免疫小鼠实验和不同载体蛋白的糖缀合物疫苗免疫小鼠实验,探讨了载体蛋白及其不同位置多肽对糖缀合物疫苗抗多糖IgG滴度和亚型的影响。首先是载体蛋白及其不同位置多肽对糖缀合物疫苗抗多糖IgG滴度的影响。通过ELISA检测3个载体蛋白不同的糖缀合物疫苗O-PS-MBP、O-PS-CRM、O-PS-AcrA分别免疫小鼠后的血清,结果发现载体蛋白不同的糖缀合物疫苗产生的抗多糖IgG滴度不同。此外,有文献报道,载体蛋白对糖缀合物疫苗抗多糖抗体有促进和抑制作用。然而,临床结果没有规律可循,也没有明确的机制去解释这种现象。通过以载体蛋白MBP上3个不同位置的多肽分别前免疫,以O-PS-MBP后免疫,检测多肽对O-PS-MBP抗多糖抗体的促进或抑制作用。结果发现载体蛋白MBP中靠近糖基化位点或者糖基化位点处的多肽,相比远离糖基化位点的多肽,对糖缀合物疫苗O-PS-MBP产生抗E. coli O157:H7多糖IgG有明显促进作用。因此,载体蛋白的促进或抑制作用和糖缀合物疫苗的糖基化位点的多少有密切关系。后免疫的糖缀合物疫苗的糖基化位点越多,靠近糖基化位点的或糖基化位点处的多肽就越多,越会发生促进现象；相反,糖基化位点越少,靠近糖基化位点的或糖基化位点处的多肽就越少,越会发生抑制现象。其次是检测载体蛋白及其不同位置多肽对糖缀合物疫苗抗多糖抗体IgG亚型的影响。尽管糖缀合物疫苗的抗多糖IgG亚型分布与抗载体蛋白IgG亚型分布不一致,但是通过比较MBP和O-PS-MBP免疫血清中抗不同位置多肽的IgG亚型发现,与MBP相比,O-PS-MBP抗靠近糖基化位点的或糖基化位点处的多肽的IgG2a显著降低,而免疫O-PS-MBP产生的抗多糖抗体以IgG2a为主。由于靠近糖基化位点的或糖基化位点处的多肽包含有糖缀合物降解形成的寡糖-多肽中的部分多肽,由此,我们可以推断得出,寡糖-多肽中的抗多肽的IgG亚型分布和抗寡糖的IgG亚型分布是一致的。综上所述,寡糖-多肽的多肽可促进糖缀合物疫苗中抗多糖抗体IgG的产生并且影响抗多糖抗体的IgG亚型分布。因此,通过改变糖缀合物疫苗的糖基化位点的个数或改变糖基化位点的位置,可控制糖缀合物疫苗抗多糖IgG的滴度和亚型分布。总之,本论文通过建立E. coli N-糖基化系统为生产糖缀合物疫苗开辟了新的途径。合成的糖缀合物疫苗能够有效地激活机体产生适应性免疫应答,保护机体免受E. coli O157:H7感染。此外,本文着重分析了载体蛋白对糖缀合物疫苗免疫活性的影响,并阐述了MBP作为载体蛋白的免疫增强作用,为理性地选择载体蛋白和研究糖缀合物疫苗免疫机制有一定的意义。
[Abstract]:E. coli (E.) is a serious intestinal pathogenic bacteria, which usually causes diarrhea, hemorrhagic colitis, hemolytic uremia, etc. traditional antibiotic therapy can cause the lysis of E. coli 0157:H7 cells in the intestine and release Shiga like toxin to the intestinal mucosa, and the risk of adding serious illness. The vaccine is used as a substitute for the prevention of E. coli 015. The infection of 7:H7 is of great significance.O- antigen or O-PS. As an important component of lipopolysaccharide, it is exposed to the outer surface of the bacteria. It is an important target for the identification of the immune system..O-PS is an antigen that does not depend on the antigen of T cells to produce the anti O-PS IgM antibody, and does not produce the IgG antibody and T cell memory. It needs to be repeated on time. Inoculation to maintain immunity, and not to produce sufficient immunity in the immune system that is less than 2 years old, the aged people of less than 2 years old, the aged people with low immunity. The glycoconjugate formed by connecting to the carrier protein is an antigen dependent on the T cell. The anti O-PS antibody produced by the glycoconjugate immune body from the Ig M to IgG, IgA and so on, and produce T cell memory, not only can produce persistent immunity in healthy adults, but also be effective for infants and young people under immune system. Glycoconjugate vaccine is recognized as one of the most effective and safest antibacterial vaccines. This paper aims at the effective prevention of E.coli 0157:H7 infection. This paper aims at E. C. The biosynthesis and immunology of the glycoconjugate vaccine was carried out by the O-PS of Oli O157:H7. This paper is divided into three parts. The first part is the biosynthesis of conjugate vaccine (second chapters) by biological method; the second part is the immunological evaluation of the glycoconjugate vaccine O-PS-MBP (third chapter); the third part is the carrier protein and its different location. The effect of polypeptide on the anti polysaccharide IgG titer and subtype of glycoconjugate vaccine (fourth chapter). Part 1: synthesis of glycoconjugate vaccine is usually used chemical method. But chemical method has the limitation of high production cost, difficult quality control, easy to have difference between batch and so on. In this part, we use C. jejuni based N- glycosyl group. Glycosyltransferase Pg1B is the key enzyme of C.jejuni in vivo. Glycosyltransferase is the key enzyme to connect the sugar chain from the class terpene like carrier to the amino acid sequence of the carrier protein D/E-X-N-Y-S/T (X, Y P) on the asparagine (N) residue. It is similar to the N- glycosylation system from C. jejuni and the E. coli synthesis pathway. The gene of the key enzyme of the C. jejuni N- glycosylation system, the E. coli O157:H7O-PS synthesis gene, the carrier protein (maltose binding protein MBP, CRM197 or AcrA), was introduced into the host strain of the host bacterium that knockout glycosyltransferase gene, and the glycosylation system was built. The production of glycoconjugate vaccine has the characteristics of flexible construction, single fermentation, simple purification conditions and low cost, which meets the requirements of large scale preparation. At the same time, the glycosylation site of glycoconjugate vaccines produced by E.coli N- glycosylation system, sugar chain length and so on are clear and controllable, and there is no difference between production batches to meet the vaccine. Quality requirements. Then through the induction of recombinant bacteria, protein expression and purification, the glycoconjugate obtained by the qualitative analysis (including Coomassie brilliant blue staining detection Western blot detection, MALDI-TOF detection, sugar composition analysis), obtained 3 E. coli 0157:H7 O-PS, the carrier protein of different glycoconjugate O-PS-MBP, O-PS-CRM, O-PS-Acr. A. also found that the yield of O-PS-MBP, O-PS-CRM, and O-PS-AcrA was about 0.5-1 mg/L (protein quantitative), and that the ratio of glycosylation of O-PS-MBP and O-PS-CRM was higher than O-PS-AcrA. The new way to produce conjugate vaccines was opened with E. coli N- glycosylation system. The second part: the second part: the adaptive immune response is divided into humoral immunity and cell immunity. The previous reported glycoconjugate vaccines against E. coli 0151:H7, O-PS-rEPA and O-PS-Stx, are more concerned with the ability to produce anti E. coli O157:H7 polysaccharide antibodies, that is, humoral immunity. Few literature reports on the glycoconjugate vaccine stimulator The cell immunity produced by the body, including the proliferation and differentiation of T cells, the secretion of cytokines, and so on. Now, more and more studies have shown that cellular immunity plays an important role in antibacterial infection, and its action time is more lasting than antibody. When immunized with a variety of glycoconjugate vaccines, if the carrier protein is the same, it may cause negative effects, such as immunosuppression. Facing the increasing number of new glycoconjugate vaccines, in addition to the widely used carrier protein, it is urgent to find and develop new carrier proteins. At present, new carrier proteins are mainly focused on the search for T cells. Protein of the epitopes, proteins with Toll like receptor properties. Recently, people began to pay attention to the immunological value and application of MBP. Some active proteins with anti tumor, antibacterial, or anti parasites have been significantly improved after the fusion and expression of MBP. The study shows that MBP has the properties of TLR4 agonists, which can promote the activation of Thl cells and have the activation tree. The protrusion and peritoneal macrophages promote the proliferation of splenic lymphocytes. However, there has not been a report of MBP as a carrier protein of glycoconjugate vaccine and the immune enhancement effect of MBP on glycoconjugate vaccine. In this part, we immunized BALB/c mice with the glycoconjugate of MBP as a carrier protein to detect O-P S-MBP activates humoral and cellular immunity. In humoral immunity, O-PS-MBP activates the serum IgG, IgM and intestinal IgA. against E. coli O157:H7 polysaccharide in mice, and O-PS-MBP immune sera can effectively kill E. coli O157:H7. cell immunity in the complement environment, promote the proliferation of spleen lymphoid cells and proliferate cells. In addition, the analysis showed that the content of IFN- gamma in O-PS-MBP immune sera and the IgG2a content of anti E. coli 0157:H7 polysaccharide was higher, which indicated that O-PS-MBP stimulated the Thl immune response in mice. Further, the ELISPOT analysis showed that MBP, either alone or in the form of glycoconjugate, could stimulate the organism to produce IFN-y and inhibit the production of IL. -4, which indicates that the carrier protein MBP makes O-PS-MBP biased toward Thl cell immune response and has a good immune enhancement. The carrier protein of MBP as a glycoconjugate vaccine has a broad application prospect. The third part: glycoconjugate vaccine activates the body to produce anti polysaccharide antibody, and phagocytosis through the action of the peptide or complement mediated by polysaccharide antibody. The anti polysaccharide IgG antibody produced by the glycoconjugate vaccine is an important part of the humoral immune response. The titer of IgG antibody directly determines the immune effect of the glycoconjugate vaccine.IgG, according to its structural characteristics, it can be divided into different subtypes, including IgG1, IgG2, IgG3 and so on. Different IgG subtypes. There are different functions and characteristics. At the same time, the distribution of IgG subtypes reacts with the direction of Th cell immune response (Thl or Th2). The study of the distribution of IgG subtypes caused by glycoconjugate vaccines or the direction of Th cell differentiation is of great significance for evaluating the immune mechanism and effect of glycoconjugate vaccines. The effect of carrier protein and its different position peptides on the titer and subtype of polysaccharide IgG in glycoconjugate vaccine was investigated with the glycoconjugate vaccine of the carrier protein. First, the effect of the carrier protein and its different position peptides on the anti polysaccharide IgG titer of the glycoconjugate vaccine. The different sugars of 3 carrier proteins were detected by ELISA. The conjugate vaccine O-PS-MBP, O-PS-CRM, and O-PS-AcrA immunized the serum of the mice respectively. The results showed that the anti polysaccharide IgG titer of the glycoconjugate vaccine was different. In addition, it was reported that the carrier protein could promote and inhibit the polysaccharide conjugate vaccine against polysaccharide antibody. However, the clinical results were not regularly followed, nor were the clinical results. There is a clear mechanism to explain this phenomenon. By immunization with 3 different locations on the carrier protein MBP and after O-PS-MBP immunization, the peptide's promoting or inhibiting effect on O-PS-MBP anti polysaccharide antibody is detected. The results show that the polypeptide in the carrier protein MBP near the glycosylation site or glycosylation site is far from the glycosylation. The polypeptide of the site has a significant effect on the production of anti E. coli O157:H7 polysaccharide IgG by the glycoconjugate vaccine O-PS-MBP. Therefore, the promotion or inhibition of the carrier protein is closely related to the glycosylation site of the glycoconjugate vaccine. The more glycosylation sites in the immunized glycoconjugate vaccines are near the glycosylation site or glycosylation site. The more peptides at the site, the more promoting phenomenon; on the contrary, the less the glycosylation site, the less the polypeptide near the glycosylation site or the glycosylation site, the more inhibition occurs. Secondly, the effect of the carrier protein and its different position peptides on the glycoconjugate vaccine against the polysaccharide antibody IgG subtype of the glycoconjugate vaccine. The distribution of the anti polysaccharide IgG subtype of the vaccine was not consistent with the distribution of the anti carrier protein IgG subtype, but by comparing the IgG subtypes of the MBP and O-PS-MBP immunized serum against different positions of polypeptide, the IgG2a of O-PS-MBP near the glycosylation site or the glycosylation site decreased significantly compared with MBP, and the anti polysaccharide produced by immune O-PS-MBP was produced. The antibody is dominated by IgG2a. Since the peptide near the glycosylation site or at the glycosylation site contains some polypeptides from oligosaccharides and peptides formed by glycoconjugate degradation, we can deduce that the distribution of IgG subtypes of anti peptides in oligosaccharide peptides and the distribution of anti oligosaccharide IgG subtypes are consistent. To sum up, oligosaccharide polypeptide Polypeptide can promote the production of anti polysaccharide antibody IgG in glycoconjugate vaccine and influence the distribution of IgG subtype of anti polysaccharide antibody. Therefore, by changing the number of glycosylation sites or changing the location of glycosylation sites in glycoconjugate vaccines, the titer and subtype distribution of polysaccharide conjugate vaccine against polysaccharide IgG can be controlled. The E. coli N- glycosylation system opens a new way for the production of glycoconjugate vaccines. The synthesized glycoconjugate vaccine can effectively activate the organism to produce adaptive immune response and protect the body from E. coli O157:H7 infection. In addition, the effect of the carrier protein on the immune activity of glycoconjugate vaccine is analyzed in this paper, and MBP is described in this paper. It is of great significance to rationally select carrier protein and study the immune mechanism of glycoconjugate vaccine for the enhancement of immune function of the carrier protein.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

【相似文献】