重组噬菌体诱导针对球形孢子丝菌的保护作用研究

发布时间：2018-01-18 07:00

本文关键词：重组噬菌体诱导针对球形孢子丝菌的保护作用研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：孢子丝菌病是由孢子丝菌复合体引起的亚急性或慢性皮下真菌病。近年来,全球范围内,孢子丝菌病的发病率有明显增加趋势。孢子丝菌复合体至少至包括有六个菌型,分别为S.pallida,S.brasiliensis,S.globosa,S.luriei,S.mexicana和S.schenckii。目前为止,S.globosa是中国尤其是东北地区最常见的甚至是唯一的致病菌种,S.globosa感染后临床表现多样,可以表现为固定型、淋巴管型,严重时也会出现播型孢子丝菌病。严重的孢子丝菌病多发生于免疫功能抑制的病人身上,尤其HIV感染者。目前,对于深部孢子丝菌病,应用抗真菌药物治疗,是最常用的治疗方式,但是由于疗程相对长,容易出现呕吐、腹泻、头痛、腹痛、过敏、肝损伤和耐药等,抗真菌药物的疗效受到限制。体液免疫和细胞免疫被认为是机体抵抗孢子丝菌感染最重要的方式。针对孢子丝菌的单克隆抗体在实验室中已经取得了非常好的疗效。大量的备选疫苗从免疫原性、安全性、保护机体的有效性上进行评估。既往的研究已经表明,用孢子丝菌孢壁蛋白或全菌免疫小鼠,可以引起有效的保护性免疫反应。但是,临床上,仍然没有公认有效并广泛使用的疫苗。Gp70(70-KDa glycoprotein)是孢子丝菌胞膜表面一种分子量为70k Da的糖蛋白,与真菌的毒力有关,在与宿主接触的过程中,主要起到粘附的作用。同时,Gp70也具有交叉免疫原性,在多个菌种当中都有存在。Gp70蛋白合成后,因转录后修饰方式不同,在不同的菌种中有不同的分子量。已经有实验证据表明,针对Gp70的单克隆抗体可以有效的诱导针对孢子丝菌的免疫反应。我们应用抗原检测系统(Gen Script,Antigen Design Tool,Optimum Antigen)检测Gp70蛋白序列后,鉴定出四个可能的表位,分别为avyvtsntehnsvvaipiar,gptntvshvffsgdqetvfttvk,tvipgqdatcwvaicpathtafvtdir,kpvqhalltplgldr。噬菌体(bacteriophage,phage)是感染细菌、真菌、放线菌或螺旋体等微生物的病毒,本世纪初在葡萄球菌和志贺菌中首先发现。噬菌体具有病毒的一些特性:个体微小,可以通过滤菌器;没有完整的细胞结构,主要由蛋白质构成的衣壳和包含于其中的核酸组成;由于噬菌体结构简单、基因数少,是分子生物学与基因工程的良好实验工具。噬菌体分双链噬菌体和单链丝状噬菌体两大类。噬菌体展示技术是1985年由Smith G P创立,他将外源基因插入丝状噬菌体的基因Ⅲ,使目的多肽以融合蛋白的形式展示在噬菌体表面。单链丝状噬菌体展示系统中,主要有pⅢ展示系统和pⅧ展示系统,而用于丝状噬菌体展示的载体分为噬菌体载体和噬菌粒载体两种。噬菌体载体比较稳定,可以直接将待展示的蛋白基因插入噬菌体载体将肽段展示于噬菌体表面后,无须佐剂的辅助,可以有效的诱导体液免疫与细胞免疫,我们将四个预测表位分别展示于噬菌体之后,发现展示肽段“kpvqhalltplgldr”(KR)的重组噬菌体可以有效的减轻小鼠病理损伤,延长小鼠的生存时间,结果如下。1、重组噬菌体可以诱导小鼠产生针对Gp70的特异性抗体。将KR肽段展示于噬菌体表面,能够刺激产生Gp70特异性抗体,从而与Gp70特异性结合。2、重组噬菌体可以诱导小鼠产生Th1(Help T cells)、Th17混合反应。重组噬菌体组Th1细胞的比例较对照组(mock组和HK-SG组;p=0.041),(HK-SG,heat-killed S.globosa)明显升高,而热灭活组与空白质粒组无明显变化。Th17细胞在重组质粒组、空白质粒对照组以及热灭活菌组与对照组相比,均明显升高(p=0.013)。3、重组噬菌体可以有效减少播散型孢子丝菌病小鼠脏器的真菌负荷量。我们通过检测播散型孢子丝菌病小鼠体内脏器的真菌负荷量,来评估重组噬菌体对BALB/c鼠的保护作用。结果表明,重组噬菌体免疫的小鼠肾脏菌落数较PBS(phosphate buffer saline)免疫小鼠明显减少。而热灭活孢子丝菌组与重组噬菌体组相比,二者无明显区别。同时在肝脏的真菌负荷量实验也得到了同样的结果。4、重组噬菌体可以减少播散型孢子丝菌病小鼠脏器的病理损伤。重组噬菌体与热灭活孢子丝菌组免疫小鼠较PBS与野生噬菌体免疫小鼠相比,炎性细胞明显减少。同样,肝脏检测中,也发现了类似的结果,PBS与野生噬菌体免疫小鼠的炎性细胞与重组噬菌体与热灭活孢子丝菌免疫噬菌体相比,炎性细胞明显增多。5、重组噬菌体可以有效的延长小鼠的生存时间。将BALB/c小鼠分别经PBS、野生噬菌体、重组噬菌体以及热灭活孢子丝菌免疫后,通过尾静脉注射致死量(0.2 m L,1×l08 cells/mouse)球形孢子丝菌,制作播散型孢子丝菌病模型。随访14天。结果表明,重组噬菌体免疫组小鼠的存活率达到80%。而PBS组的存活率只有20%。而热灭活孢子丝菌免疫小鼠的存活率达到70%。结果表明,重组噬菌体免疫小鼠的存活率明显高于PBS组。结论:本实验研究的结果表明,重组噬菌体可以刺激机体产生有效的细胞免疫以及体液免疫,帮助机体抵抗S.globosa的感染。目前这种免疫反应的具体机制仍不十分清楚,但是,重组噬菌体可以产生保护性抗体,促进细胞免疫反应以及特异性抗体的生成,为重组噬菌体在预防孢子丝菌感染方面的临床应用提供了理论依据。
[Abstract]:Sporotrichosis is a subacute or chronic subcutaneous fungal disease caused by Sporothrix complex. In recent years, the global scope, sporotrichosis incidence has increasing trend. Sporothrix complex until at least comprises six strains, respectively S.pallida, S.brasiliensis, S.globosa, S.luriei, and S.mexicana S.schenckii. S.globosa is China now, especially in the northeast and even the most common pathogenic bacteria only after S.globosa infection, clinical manifestations, can be expressed as a fixed type, lymphatic type, serious will also be broadcast sporotrichosis. Sporotrichosis from more serious immunosuppression patients the body, especially HIV infection. At present, for deep sporotrichosis, application of antifungal treatment, is the most commonly used method of treatment, but the treatment period is relatively long, prone to vomiting, diarrhea, headache, abdominal Pain, allergy, liver injury and drug resistance, the antifungal efficacy is limited. Humoral and cellular immunity is thought to be the most important way of the body against Sporothrix schenckii infection. Monoclonal antibody against Sporothrix has achieved very good curative effect in the laboratory. A large number of candidate vaccine from immunogenicity. The safety and effectiveness of protection on the body for evaluation. Previous studies have shown that with Sporothrix spore wall proteins or whole cell immunity in mice, can induce protective immune response effectively. However, clinically,.Gp70 vaccine is still not effective and widely used (70-KDa glycoprotein) is a kind of molecular. Wire of the cell membrane surface glycoprotein 70K for Da, related to the fungal virulence, and host in the process of contact, mainly to adhesion. At the same time, Gp70 also have cross immunogenicity in multiple strains among There are.Gp70 protein synthesis, due to post-translational modification in different ways, with different molecular weight in different species. There have been experimental evidence suggests that monoclonal antibodies against Gp70 can effectively induce the immune response against Sporothrix. We used antigen detection system (Gen Script, Antigen Design Tool, Optimum Antigen) to detect the Gp70 protein sequences, identified four possible epitope, respectively avyvtsntehnsvvaipiar, gptntvshvffsgdqetvfttvk, tvipgqdatcwvaicpathtafvtdir, kpvqhalltplgldr. (bacteriophage, phage) is a phage infection of bacteria, fungi, actinomycetes or put microbial spirochetes of the virus, the beginning of this century in Staphylococcus aureus and Shigella first discovered some characteristics. Bacteriophages are viruses: individual small, can pass through the filter; incomplete cell structure, mainly by the protein composition and capsid contained in The nucleic acids; because bacteriophage has the advantages of simple structure, less number of genes, is a good experimental tool for molecular biology and gene engineering. Double stranded and single stranded phage phage phage two categories. Phage display technology was founded in 1985 by Smith G P, he will insert genes of filamentous phage III, the polypeptide of interest to in the form of a fusion protein displayed on the phage surface. ScFv phage display system, display system and main p III P VIII display system, and for the carrier of phage display into phage vector and phagemid vector two. Phage vector is stable and can be directly inserted into the phage protein gene to display vector peptide displayed on the phage surface, without adjuvant, can induce humoral and cellular immunity effectively, we will be the four epitopes were demonstrated On the phage display peptide found after the period of "kpvqhalltplgldr" (KR) of the recombinant phage can effectively reduce the pathological injury in mice, prolong the survival time of mice, the results were as follows:.1, the recombinant phage can induce the specific antibody against Gp70. The KR peptide displayed on the phage surface, can stimulate the production of specific antibody of Gp70 thus, the combination of.2 and Gp70 specific recombinant phage can induce Th1 (Help T cells), Th17 mixed reaction. The recombinant phage group the proportion of Th1 cells compared with the control group (mock group and HK-SG group; p= 0.041 (HK-SG), heat-killed, S.globosa) was significantly increased, and the heat inactivated group and blank plasmid group had no obvious change of.Th17 cells in recombinant plasmid group, blank plasmid group and heat inactivated bacteria group compared with the control group, were significantly higher (p=0.013).3, recombinant phage can effectively reduce disseminated sporotrichosis Fungal disease load of mouse organs. We through the amount of disseminated sporotrichosis in mice organs fungal load detection, to evaluate the protective effect of recombinant phage of BALB/c rats. The results showed that the number of mouse kidney colony recombinant phage immune PBS (phosphate buffer saline) immunized mice was significantly reduced. The heat inactivated spores silk bacterium group and recombinant phage group compared with no significant difference between the two. At the same time also got the same results in.4 fungal load experiment of the liver, the recombinant phage can reduce disseminated sporotrichosis in mice organs injury. Recombinant phage and heat inactivated Sporothrix group immunized mice than PBS and wild phage immune mice, inflammatory cells decreased significantly. Similarly, in the detection of liver, also found similar results, inflammatory cells and recombinant phage and phage PBS heat and wild mice Inactivation of s.schenckii immune phage compared to inflammatory cells significantly increased.5, the recombinant phage can effectively prolong the survival time of mice. BALB/c mice were treated with PBS, wild type phage, phage and inactivated sporotrichosis after immunization by intravenous injection of lethal dose (0.2 m L, 1 * l08 cells/mouse) spherical sporotrichosis, making disseminated sporotrichosis model. Followed up for 14 days. The results showed that the survival rate of 80%. and survival rate of PBS group is only 20%. and heat inactivated Sporothrix mice survival rate reached 70%. the results showed that the recombinant phage immune mice, the survival rate of mice immunized with recombinant phage compared with PBS group. Conclusion: the experimental results showed that the recombinant phage can induce effective immune cells and humoral immunity, help the body to resist S.globosa infection. At present this kind of immune response The specific mechanism is still not very clear. However, recombinant phage can produce protective antibodies, promote cellular immune response and specific antibody production, and provide a theoretical basis for the clinical application of recombinant bacteriophages in the prevention of sporidium infection.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R756

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