本文選題：肺炎鏈球菌 + 毒力因子　； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：【背景】肺炎鏈球菌作為一種常見的條件致病菌，可以引起多種感染性疾病。隨著抗生素的濫用，耐藥菌株的日益增多。而市面上的肺炎鏈球菌疫苗保護(hù)效果并不理想，導(dǎo)致對(duì)其防治形勢(shì)嚴(yán)峻。新抗菌藥和疫苗的開發(fā)需要深入了解其致病機(jī)制、尋找新的毒力因子。 基因spd0414是本室用體內(nèi)表達(dá)技術(shù)和差異熒光誘導(dǎo)技術(shù)篩選出的一個(gè)體內(nèi)誘導(dǎo)基因，前期研究提示該基因在肺炎鏈球菌致病中可能發(fā)揮重要作用。本研究擬對(duì)其如何影響肺炎鏈球菌毒力進(jìn)行探討，闡明該基因在細(xì)菌感染致病中的作用，明確其編碼蛋白SPD0414的亞細(xì)胞定位，并初步評(píng)價(jià)其作為肺炎鏈球菌蛋白疫苗候選蛋白的價(jià)值。 【方法】首先在肺炎鏈球菌D39（2型），203（3型）中構(gòu)建spd0414缺失的缺陷菌。野生菌D39，203和缺陷菌D39△spd0414,203△spd0414經(jīng)鼻腔和腹腔兩種途徑感染小鼠，觀察該基因的缺失對(duì)肺炎鏈球菌致病能力的影響。通過(guò)野生菌和缺陷菌的體內(nèi)定植實(shí)驗(yàn)和體外粘附侵襲實(shí)驗(yàn)，，闡明該基因在細(xì)菌致病過(guò)程中的作用。再進(jìn)一步通過(guò)蛋白抑制和抗血清封閉的方式，鑒定SPD0414能否直接通過(guò)與宿主的相互作用而影響細(xì)菌的粘附。在發(fā)現(xiàn)SPD0414可能是間接作用后，我們用熒光定量PCR的方法探討了SPD0414蛋白對(duì)細(xì)菌其他毒力蛋白的影響。采用一種新穎的N端和C端融合表達(dá)GFP的方式對(duì)SPD0414進(jìn)行亞細(xì)胞定位分析。并將SPD0414重組蛋白免疫小鼠，觀察其對(duì)細(xì)菌感染的保護(hù)作用，初步評(píng)價(jià)了該蛋白作為肺炎鏈球菌蛋白疫苗候選蛋白的價(jià)值。 【結(jié)果】基因spd0414缺失后，D39和203以及各自的缺陷菌在經(jīng)腹腔感染時(shí)，小鼠死亡率無(wú)顯著差別。在鼻腔感染途徑中，D39與其缺陷菌的致病能力無(wú)顯著差別，但203△spd0414毒力較野生菌顯著下降(p0.05)。定植實(shí)驗(yàn)結(jié)果顯示，spd0414缺失后D39和203菌株在小鼠鼻咽部和肺部的定植細(xì)菌數(shù)都顯著降低(p0.05)。體外粘附實(shí)驗(yàn)顯示，D39和203菌株的spd0414缺陷后，其對(duì)宿主細(xì)胞A549和CNE的粘附侵襲能力顯著下降(p0.05)。但SPD0414截短蛋白或其抗血清都不能抑制野生菌對(duì)宿主細(xì)胞的粘附侵襲。進(jìn)一步的研究顯示，spd0414的缺失后，肺炎鏈球菌毒力因子溶血素（Ply）和自溶酶A（LytA）表達(dá)增高，肺炎球菌表面粘附蛋白A（PsaA）表達(dá)下調(diào)，而神經(jīng)氨酸酶A（NanA）在D39和203菌株中的變化不一致。通過(guò)GFP融合表達(dá)亞細(xì)胞定位研究結(jié)果顯示，轉(zhuǎn)入SPD0414與GFP的N端、C端融合蛋白表達(dá)質(zhì)粒后，細(xì)菌胞內(nèi)都有熒光表達(dá)，提示該蛋白定位于胞質(zhì)中，但不能確定是否有分泌。SPD0414截短蛋白免疫小鼠，可以產(chǎn)生特異性的抗體，對(duì)肺炎鏈球菌D39（血清2型）、CMCC31436（血清3型）、CMCC31614（血清14型）的鼻腔感染，分別產(chǎn)生大約50%、33.3%、41.7%的保護(hù)效果。 【結(jié)論】spd0414的缺失，導(dǎo)致203鼻腔感染致病能力下降，但不影響D39的致病。spd0414在D39和203中都通過(guò)間接作用影響其粘附定植，這種作用可能與細(xì)菌粘附因子PsaA的表達(dá)相關(guān)。另外，spd0414的缺失，也影響了S.pn毒力因子LytA，Ply和NanA的mRNA表達(dá)。亞細(xì)胞定位研究顯示SPD0414定位于胞質(zhì)中，不能確定有無(wú)胞外分泌。而SPD0414免疫小鼠對(duì)肺炎鏈球菌的感染有一定的保護(hù)性，說(shuō)明該蛋白可以作為肺炎鏈球菌蛋白疫苗候選蛋白。
[Abstract]:[background] Streptococcus pneumoniae, as a common conditional pathogen, can cause a variety of infectious diseases. With the abuse of antibiotics, drug resistant strains are increasing. The protection effect of Streptococcus pneumoniae vaccine on the market is not ideal, leading to the severe prevention and control situation. The development of new antifungal drugs and vaccines needs to be deeply understood. The disease mechanism and the search for new virulence factors.
The gene spd0414 is an in vivo inducible gene screened by the body expression technology and differential fluorescence induction technology in this room. The previous study suggests that the gene may play an important role in the pathogenesis of Streptococcus pneumoniae. This study is to discuss how it affects the virulence of Streptococcus pneumoniae and elucidate the role of the gene in the pathogenic bacteria infection. The subcellular localization of its encoded protein SPD0414 was identified, and its value as a candidate protein for pneumococcal protein vaccine was preliminarily evaluated.
[Methods] first, spd0414 deficient bacteria were constructed in the Streptococcus pneumoniae D39 (type 2) and 203 (type 3). The wild bacteria D39203 and the defective bacteria D39 Delta spd0414203 Delta spd0414 were infected by the nasal cavity and the abdominal cavity, and the effect of the deletion of the gene on the pathogenicity of Streptococcus pneumoniae was observed. The effect of the gene in the pathogenic process of bacteria was elucidated and the effect of the gene in the pathogenic process of bacteria was elucidated. Further, by means of protein inhibition and antiserum closure, SPD0414 could be identified directly by the interaction of the host to the adhesion of the bacteria. After the discovery of the possible indirect effect of SPD0414, we explored the method of fluorescence quantitative PCR. The effect of SPD0414 protein on other bacterial virulence proteins. A novel subcellular localization analysis of SPD0414 was carried out with a novel N end and C end fusion expression of GFP. The recombinant protein of SPD0414 was immunized to mice to observe the protective effect of the protein on bacterial infection. The protein was preliminarily evaluated as a candidate protein for Streptococcus pneumoniae protein vaccine. Value.
[results] there was no significant difference in the mortality rate between D39 and 203 and their respective defective bacteria in the abdominal infection. In the pathway of nasal cavity infection, there was no significant difference in the pathogenicity of D39 and the defective bacteria, but the toxicity of 203 Delta spd0414 was significantly lower than that of the wild bacteria (P0.05). The results of colonization test showed that D39 and 203 after spd0414 deletion were D39 and 203. The number of colonized bacteria in the nasopharynx and lungs of the mice decreased significantly (P0.05). In vitro adhesion experiments showed that the adhesion and invasion ability of D39 and 203 strains to A549 and CNE of host cells decreased significantly (P0.05), but the SPD0414 truncated protein or its anti blood purity could not inhibit the adhesion and invasion of the wild bacteria to the host cells. One step of the study showed that after the deletion of spd0414, the expression of Ply and A (LytA) of Streptococcus pneumoniae increased, and the expression of A (PsaA) of the pneumococcal surface adhesion protein (PsaA) was down, and the changes of the neuraminidase A (NanA) in D39 and 203 were not consistent. The results of subcellular localization through GFP fusion expression showed that it was turned into SPD0414. With the N terminal of GFP, C terminal fusion protein expression plasmid, the bacterial cell has fluorescence expression, suggesting that the protein is located in the cytoplasm, but it can not determine whether there is a.SPD0414 truncated protein immune mouse, can produce specific antibodies, Streptococcus pneumoniae D39 (serotype 2), CMCC31436 (serum type 3), CMCC31614 (serum type 14 type) nasal infection, The protective effects of about 50%, 33.3%, and 41.7% are produced respectively.
[Conclusion] the absence of spd0414 leads to a decrease in the pathogenicity of 203 nasal infection, but it does not affect D39's pathogenic.Spd0414 in D39 and 203 by indirect effect on its adhesion and colonization. This effect may be related to the expression of bacterial adhesion factor PsaA. In addition, the deletion of spd0414 also affects the S.pn virulence factor LytA, Ply and NanA mRNA expression. The subcellular localization study showed that SPD0414 was located in the cytoplasm and could not determine whether there was exocytosis, but the SPD0414 immunized mice had some protective effect on Streptococcus pneumoniae infection, indicating that the protein could be used as a candidate protein for Streptococcus pneumoniae protein vaccine.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 龔藝;崔亞利;牛司強(qiáng);張雪梅;胥文春;何於娟;王虹;;肺炎鏈球菌假想蛋白SPD0414的表達(dá)純化及保守性分析[J];中國(guó)免疫學(xué)雜志;2010年09期

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