肺炎链球菌SPDO414对细菌毒力的影响及其作为疫苗候选蛋白的评价研究

发布时间：2018-04-23 14:24

本文选题：肺炎链球菌 + 毒力因子　；参考：《重庆医科大学》2012年硕士论文

【摘要】：【背景】肺炎链球菌作为一种常见的条件致病菌，可以引起多种感染性疾病。随着抗生素的滥用，耐药菌株的日益增多。而市面上的肺炎链球菌疫苗保护效果并不理想，导致对其防治形势严峻。新抗菌药和疫苗的开发需要深入了解其致病机制、寻找新的毒力因子。基因spd0414是本室用体内表达技术和差异荧光诱导技术筛选出的一个体内诱导基因，前期研究提示该基因在肺炎链球菌致病中可能发挥重要作用。本研究拟对其如何影响肺炎链球菌毒力进行探讨，阐明该基因在细菌感染致病中的作用，明确其编码蛋白SPD0414的亚细胞定位，并初步评价其作为肺炎链球菌蛋白疫苗候选蛋白的价值。【方法】首先在肺炎链球菌D39（2型），203（3型）中构建spd0414缺失的缺陷菌。野生菌D39，203和缺陷菌D39△spd0414,203△spd0414经鼻腔和腹腔两种途径感染小鼠，观察该基因的缺失对肺炎链球菌致病能力的影响。通过野生菌和缺陷菌的体内定植实验和体外粘附侵袭实验，，阐明该基因在细菌致病过程中的作用。再进一步通过蛋白抑制和抗血清封闭的方式，鉴定SPD0414能否直接通过与宿主的相互作用而影响细菌的粘附。在发现SPD0414可能是间接作用后，我们用荧光定量PCR的方法探讨了SPD0414蛋白对细菌其他毒力蛋白的影响。采用一种新颖的N端和C端融合表达GFP的方式对SPD0414进行亚细胞定位分析。并将SPD0414重组蛋白免疫小鼠，观察其对细菌感染的保护作用，初步评价了该蛋白作为肺炎链球菌蛋白疫苗候选蛋白的价值。【结果】基因spd0414缺失后，D39和203以及各自的缺陷菌在经腹腔感染时，小鼠死亡率无显著差别。在鼻腔感染途径中，D39与其缺陷菌的致病能力无显著差别，但203△spd0414毒力较野生菌显著下降(p0.05)。定植实验结果显示，spd0414缺失后D39和203菌株在小鼠鼻咽部和肺部的定植细菌数都显著降低(p0.05)。体外粘附实验显示，D39和203菌株的spd0414缺陷后，其对宿主细胞A549和CNE的粘附侵袭能力显著下降(p0.05)。但SPD0414截短蛋白或其抗血清都不能抑制野生菌对宿主细胞的粘附侵袭。进一步的研究显示，spd0414的缺失后，肺炎链球菌毒力因子溶血素（Ply）和自溶酶A（LytA）表达增高，肺炎球菌表面粘附蛋白A（PsaA）表达下调，而神经氨酸酶A（NanA）在D39和203菌株中的变化不一致。通过GFP融合表达亚细胞定位研究结果显示，转入SPD0414与GFP的N端、C端融合蛋白表达质粒后，细菌胞内都有荧光表达，提示该蛋白定位于胞质中，但不能确定是否有分泌。SPD0414截短蛋白免疫小鼠，可以产生特异性的抗体，对肺炎链球菌D39（血清2型）、CMCC31436（血清3型）、CMCC31614（血清14型）的鼻腔感染，分别产生大约50%、33.3%、41.7%的保护效果。【结论】spd0414的缺失，导致203鼻腔感染致病能力下降，但不影响D39的致病。spd0414在D39和203中都通过间接作用影响其粘附定植，这种作用可能与细菌粘附因子PsaA的表达相关。另外，spd0414的缺失，也影响了S.pn毒力因子LytA，Ply和NanA的mRNA表达。亚细胞定位研究显示SPD0414定位于胞质中，不能确定有无胞外分泌。而SPD0414免疫小鼠对肺炎链球菌的感染有一定的保护性，说明该蛋白可以作为肺炎链球菌蛋白疫苗候选蛋白。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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