本文關(guān)鍵詞：重組噬菌體誘導(dǎo)針對(duì)球形孢子絲菌的保護(hù)作用研究　出處：《吉林大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 申克孢子絲菌 噬菌體 免疫 球形孢子絲菌 體液免疫 細(xì)胞免疫

【摘要】：孢子絲菌病是由孢子絲菌復(fù)合體引起的亞急性或慢性皮下真菌病。近年來(lái),全球范圍內(nèi),孢子絲菌病的發(fā)病率有明顯增加趨勢(shì)。孢子絲菌復(fù)合體至少至包括有六個(gè)菌型,分別為S.pallida,S.brasiliensis,S.globosa,S.luriei,S.mexicana和S.schenckii。目前為止,S.globosa是中國(guó)尤其是東北地區(qū)最常見(jiàn)的甚至是唯一的致病菌種,S.globosa感染后臨床表現(xiàn)多樣,可以表現(xiàn)為固定型、淋巴管型,嚴(yán)重時(shí)也會(huì)出現(xiàn)播型孢子絲菌病。嚴(yán)重的孢子絲菌病多發(fā)生于免疫功能抑制的病人身上,尤其HIV感染者。目前,對(duì)于深部孢子絲菌病,應(yīng)用抗真菌藥物治療,是最常用的治療方式,但是由于療程相對(duì)長(zhǎng),容易出現(xiàn)嘔吐、腹瀉、頭痛、腹痛、過(guò)敏、肝損傷和耐藥等,抗真菌藥物的療效受到限制。體液免疫和細(xì)胞免疫被認(rèn)為是機(jī)體抵抗孢子絲菌感染最重要的方式。針對(duì)孢子絲菌的單克隆抗體在實(shí)驗(yàn)室中已經(jīng)取得了非常好的療效。大量的備選疫苗從免疫原性、安全性、保護(hù)機(jī)體的有效性上進(jìn)行評(píng)估。既往的研究已經(jīng)表明,用孢子絲菌孢壁蛋白或全菌免疫小鼠,可以引起有效的保護(hù)性免疫反應(yīng)。但是,臨床上,仍然沒(méi)有公認(rèn)有效并廣泛使用的疫苗。Gp70(70-KDa glycoprotein)是孢子絲菌胞膜表面一種分子量為70k Da的糖蛋白,與真菌的毒力有關(guān),在與宿主接觸的過(guò)程中,主要起到粘附的作用。同時(shí),Gp70也具有交叉免疫原性,在多個(gè)菌種當(dāng)中都有存在。Gp70蛋白合成后,因轉(zhuǎn)錄后修飾方式不同,在不同的菌種中有不同的分子量。已經(jīng)有實(shí)驗(yàn)證據(jù)表明,針對(duì)Gp70的單克隆抗體可以有效的誘導(dǎo)針對(duì)孢子絲菌的免疫反應(yīng)。我們應(yīng)用抗原檢測(cè)系統(tǒng)(Gen Script,Antigen Design Tool,Optimum Antigen)檢測(cè)Gp70蛋白序列后,鑒定出四個(gè)可能的表位,分別為avyvtsntehnsvvaipiar,gptntvshvffsgdqetvfttvk,tvipgqdatcwvaicpathtafvtdir,kpvqhalltplgldr。噬菌體(bacteriophage,phage)是感染細(xì)菌、真菌、放線菌或螺旋體等微生物的病毒,本世紀(jì)初在葡萄球菌和志賀菌中首先發(fā)現(xiàn)。噬菌體具有病毒的一些特性:個(gè)體微小,可以通過(guò)濾菌器;沒(méi)有完整的細(xì)胞結(jié)構(gòu),主要由蛋白質(zhì)構(gòu)成的衣殼和包含于其中的核酸組成;由于噬菌體結(jié)構(gòu)簡(jiǎn)單、基因數(shù)少,是分子生物學(xué)與基因工程的良好實(shí)驗(yàn)工具。噬菌體分雙鏈?zhǔn)删w和單鏈絲狀噬菌體兩大類(lèi)。噬菌體展示技術(shù)是1985年由Smith G P創(chuàng)立,他將外源基因插入絲狀噬菌體的基因Ⅲ,使目的多肽以融合蛋白的形式展示在噬菌體表面。單鏈絲狀噬菌體展示系統(tǒng)中,主要有pⅢ展示系統(tǒng)和pⅧ展示系統(tǒng),而用于絲狀噬菌體展示的載體分為噬菌體載體和噬菌粒載體兩種。噬菌體載體比較穩(wěn)定,可以直接將待展示的蛋白基因插入噬菌體載體將肽段展示于噬菌體表面后,無(wú)須佐劑的輔助,可以有效的誘導(dǎo)體液免疫與細(xì)胞免疫,我們將四個(gè)預(yù)測(cè)表位分別展示于噬菌體之后,發(fā)現(xiàn)展示肽段“kpvqhalltplgldr”(KR)的重組噬菌體可以有效的減輕小鼠病理?yè)p傷,延長(zhǎng)小鼠的生存時(shí)間,結(jié)果如下。1、重組噬菌體可以誘導(dǎo)小鼠產(chǎn)生針對(duì)Gp70的特異性抗體。將KR肽段展示于噬菌體表面,能夠刺激產(chǎn)生Gp70特異性抗體,從而與Gp70特異性結(jié)合。2、重組噬菌體可以誘導(dǎo)小鼠產(chǎn)生Th1(Help T cells)、Th17混合反應(yīng)。重組噬菌體組Th1細(xì)胞的比例較對(duì)照組(mock組和HK-SG組;p=0.041),(HK-SG,heat-killed S.globosa)明顯升高,而熱滅活組與空白質(zhì)粒組無(wú)明顯變化。Th17細(xì)胞在重組質(zhì)粒組、空白質(zhì)粒對(duì)照組以及熱滅活菌組與對(duì)照組相比,均明顯升高(p=0.013)。3、重組噬菌體可以有效減少播散型孢子絲菌病小鼠臟器的真菌負(fù)荷量。我們通過(guò)檢測(cè)播散型孢子絲菌病小鼠體內(nèi)臟器的真菌負(fù)荷量,來(lái)評(píng)估重組噬菌體對(duì)BALB/c鼠的保護(hù)作用。結(jié)果表明,重組噬菌體免疫的小鼠腎臟菌落數(shù)較PBS(phosphate buffer saline)免疫小鼠明顯減少。而熱滅活孢子絲菌組與重組噬菌體組相比,二者無(wú)明顯區(qū)別。同時(shí)在肝臟的真菌負(fù)荷量實(shí)驗(yàn)也得到了同樣的結(jié)果。4、重組噬菌體可以減少播散型孢子絲菌病小鼠臟器的病理?yè)p傷。重組噬菌體與熱滅活孢子絲菌組免疫小鼠較PBS與野生噬菌體免疫小鼠相比,炎性細(xì)胞明顯減少。同樣,肝臟檢測(cè)中,也發(fā)現(xiàn)了類(lèi)似的結(jié)果,PBS與野生噬菌體免疫小鼠的炎性細(xì)胞與重組噬菌體與熱滅活孢子絲菌免疫噬菌體相比,炎性細(xì)胞明顯增多。5、重組噬菌體可以有效的延長(zhǎng)小鼠的生存時(shí)間。將BALB/c小鼠分別經(jīng)PBS、野生噬菌體、重組噬菌體以及熱滅活孢子絲菌免疫后,通過(guò)尾靜脈注射致死量(0.2 m L,1×l08 cells/mouse)球形孢子絲菌,制作播散型孢子絲菌病模型。隨訪14天。結(jié)果表明,重組噬菌體免疫組小鼠的存活率達(dá)到80%。而PBS組的存活率只有20%。而熱滅活孢子絲菌免疫小鼠的存活率達(dá)到70%。結(jié)果表明,重組噬菌體免疫小鼠的存活率明顯高于PBS組。結(jié)論:本實(shí)驗(yàn)研究的結(jié)果表明,重組噬菌體可以刺激機(jī)體產(chǎn)生有效的細(xì)胞免疫以及體液免疫,幫助機(jī)體抵抗S.globosa的感染。目前這種免疫反應(yīng)的具體機(jī)制仍不十分清楚,但是,重組噬菌體可以產(chǎn)生保護(hù)性抗體,促進(jìn)細(xì)胞免疫反應(yīng)以及特異性抗體的生成,為重組噬菌體在預(yù)防孢子絲菌感染方面的臨床應(yīng)用提供了理論依據(jù)。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R756

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本文編號(hào)：1439908