肺炎链球菌Gts、PotD及SrtA蛋白联合免疫对其感染的保护作用及肺炎链球菌外膜蛋白SPD1741和SPD0280的初

发布时间：2018-06-09 14:14

本文选题：肺炎链球菌 + 蛋白质　；参考：《重庆医科大学》2012年博士论文

【摘要】：目的：肺炎链球菌（Streptococcus pneumoniae，S.pn）是人类主要致病菌，在临床上广泛引起中耳炎、败血症、脑膜炎及致死性肺炎等多种疾病。全球每年大约有160万人死于肺炎链球菌引起的各种疾患。尽管抗生素治疗是控制肺炎链球菌感染的有效手段，但并不能有效降低肺炎链球菌感染最初48小时的死亡率。随着抗生素的滥用，临床上耐药菌株明显增加。因此，通过接种肺炎链球菌疫苗以预防其感染成为积极有效的手段。新一代的蛋白疫苗是预防肺炎链球菌感染的未来发展方向，但目前国际上对蛋白质疫苗尚处于临床前研究阶段，由于单一蛋白的保护效果并不理想，联合蛋白疫苗是目前研究的热点。采用多种肺炎链球菌毒力蛋白联合免疫，以扩大其对机体的保护效应是一种可行方式,因为蛋白抗原联合疫苗提供了多种毒力作用的潜在靶标。 Gts、PotD和SrtA蛋白都是肺炎链球菌的重要毒力因子，生物信息学分析显示其在肺炎链球菌中保守性高，且均在细菌表面有表达，但尚未有报道对其作为疫苗候选蛋白进行系统的免疫保护效果评价。本研究拟先比较肺炎链球菌Gts、PotD和SrtA单个蛋白抗原免疫小鼠后，对肺炎链球菌感染的保护作用；然后采用不同血清型的肺炎链球菌，检测上述蛋白两两联合、三种蛋白组合后免疫小鼠，与单个蛋白抗原组比较，以评价其对肺炎链球菌感染的保护效果。方法：本研究首先通过基因克隆、蛋白表达及纯化技术，成功制备了肺炎链球菌Gts、PotD及SrtA重组蛋白。重组蛋白经鼻腔免疫途径，以研究抗原特异性反应，并检测其体液和细胞调节反应。为了模拟肺炎链球菌感染的自然途径，以评价Gts、PotD及SrtA重组蛋白抗原对小鼠鼻腔感染后的保护效果，选择肺炎链球菌中致病能力最强的D39菌株进行鼻腔攻毒实验，以构建肺炎链球菌性肺炎的小鼠模型。为了更充分地评估Gts、PotD和SrtA重组蛋白不同组合对多种血清型肺炎链球菌感染的保护效果，本研究采用上述重组蛋白抗原经小鼠腹腔免疫，然后选择不同血清型肺炎链球菌进行腹腔攻毒实验研究。再次采用特异性抗血清免疫小鼠后，选择D39肺炎链球菌进行鼻腔攻毒实验，以鉴定重组蛋白抗原的保护性是否由其特异性抗体介导的。采用细胞粘附实验，以分析Gts、PotD和SrtA重组蛋白及其特异性抗血清对D39细胞粘附于A549细胞的影响；最后，为了进一步探讨人体经肺炎链球菌感染后，该细菌表面抗原Gts、PotD和SrtA刺激机体产生的天然抗体滴度变化，本研究收集正常人群及急性肺炎患者，拟采用ELISA法测定血清中anti-Gts、anti-PotD及anti-SrtA抗血清的滴度变化。结果：在成功克隆和表达Gts、PotD和SrtA重组蛋白基础上，通过Western blot分析证实，CMCC（B）31216(serotype9V)、CMCC (B)31436(serotype3)、CMCC(B)31507(serotype7F)等8种血清型的肺炎链球菌均能表达Gts、PotD和SrtA蛋白，且蛋白抗原性未发生变异。粘附抑制实验证实，单一Gts、PotD和SrtA蛋白都能抑制D39对A549细胞的粘附，且2-3个蛋白质抗原的联合使用，具有明显的累加效应。此外，抗-Gts、抗-PotD和抗-SrtA抗血清的单独使用或2-3个抗血清的联合使用，也具有和蛋白抗原类似的抑制效应。通过鼻腔滴注Gts、PotD或SrtA重组蛋白，小鼠脾细胞产生了高水平的Th1介导的细胞因子IFN-γ、Th2介导的细胞因子IL-4、Treg介导的IL-10因子和Th17介导的细胞因子IL-17A，诱导了小鼠对肺炎链球菌的粘膜免疫及系统免疫。在构建的肺炎球菌感染的小鼠模型中，Gts、PotD或SrtA重组蛋白重组通过粘膜途径免疫小鼠，均能有效减少19F型肺炎链球菌在鼻咽部及肺部的定植。与单个蛋白抗原比较，Gts+SrtA、PotD+Gts+SrtA均能显著提高肺炎链球菌对肺部定植的保护作用。3个蛋白抗原联合粘膜免疫后，对肺炎链球菌中致病能力最强的D39血清型鼻腔感染保护率可达83.3%。腹腔免疫结果与粘膜免疫类似，几乎所有蛋白抗原联合都较单个蛋白的保护作用更强。但与蛋白两两组合比较，Gts+PotD+SrtA的三联蛋白抗原的联合腹腔免疫，其保护作用并未见明显加强。被动免疫研究提示，抗-Gts、抗-PotD和抗-SrtA抗血清联合免疫小鼠，其对小鼠腹腔感染肺炎链球菌的保护作用与主动免疫的效果相似，提示Gts、PotD及SrtA蛋白抗原通过主动免疫激发的保护效应与其抗体的作用密切相关。结论：通过本研究的分析显示，Gts、PotD及SrtA在肺炎链球菌中的保守性高，三种蛋白联合抗原的两两组合及三种联合，通过粘膜免疫途径或系统性免疫途径免疫小鼠，均能诱导小鼠对不同血清型肺炎链球菌感染的显著保护效果，显示Gts、PotD及SrtA是一组有开发潜力的候选疫苗组合。目的：大量表达、纯化肺炎链球菌（Streptococcus pneumoniae, S.pn）外膜蛋白SPD1741及SPD0280，用于晶体生长、优化和三维结构解析。方法：分别将S.pn D39菌株的spd1741、spd0280基因克隆至原核表达载体pET32a(+)中，通过菌液PCR和重组质粒测序鉴定，再转化E. coli BL21(DE3)获得表达菌株；该菌株经IPTG诱导、高效表达出带His标签的可溶性融合蛋白；经亲和层析(Ni-NTA)、离子交换层析（DEAE）和分子筛层析后,获得高纯度的目的蛋白以进行晶体生长及优化；最后收集该晶体X射线衍射数据。结果：经纯化后的SPD1741及SPD0280重组蛋白纯度均达90%以上，采用悬滴气相扩散法培养的晶体，其X射线衍射能力分别达4.0和3.5。结论：本研究成功制备了高纯度的SPD1741及SPD0280重组蛋白,获得2种蛋白质母体晶体，，并收到X射线衍射数据。本研究为SPD1741及SPD0280的三维结构解析及其生物学功能研究奠定了基础。
[Abstract]:Objective: Streptococcus pneumoniae (S.pn) is the main cause of human pathogenic bacteria. It is clinically widely caused by many diseases such as otitis media, septicemia, meningitis and fatal pneumonia. About 1 million 600 thousand people die from various diseases caused by Streptococcus pneumoniae every year around the world. But it does not effectively reduce the first 48 hours mortality of Streptococcus pneumoniae infection. With the abuse of antibiotics, clinically resistant strains obviously increase. Therefore, it is a positive and effective way to prevent the infection by inoculating Streptococcus pneumoniae vaccine.
The new generation of protein vaccine is the future development direction for the prevention of Streptococcus pneumoniae infection, but at present, the international protein vaccine is still in the pre clinical stage, because the protection effect of single protein is not ideal, combined protein vaccine is a hot spot at present. The protective effect of the body is a feasible way, because the protein antigen combined vaccine provides many potential targets for virulence.
Gts, PotD and SrtA proteins are all important virulence factors of Streptococcus pneumoniae. Bioinformatics analysis shows that they are conserved in Streptococcus pneumoniae and are expressed on the surface of bacteria. However, there are no reports on the systematic immune protection effect on the vaccine candidate proteins. This study is to compare Streptococcus pneumoniae, Gts, PotD and SrtA. The protective effect of single protein antigen immunized mice on Streptococcus pneumoniae infection; then using different serotypes of Streptococcus pneumoniae to detect the combination of the above protein 22 and the three protein combinations to immunize mice and compare with the single protein antigen group to evaluate the protective effect on the infection of Streptococcus pneumoniae.
Methods: in this study, the recombinant protein of Streptococcus pneumoniae Gts, PotD and SrtA was successfully prepared by gene cloning, protein expression and purification. The recombinant protein was immunized by the nasal cavity to study the antigen specific reaction, and to detect the body fluid and cell regulation response. In order to evaluate the natural pathway of Streptococcus pneumoniae infection, Gts, Pot was evaluated. The protective effect of D and SrtA recombinant protein antigen on nasal infection in mice was selected, and the D39 strains with the strongest pathogenicity in Streptococcus pneumoniae were selected to carry out the nasal attack experiment in order to construct a mouse model of Streptococcus pneumoniae pneumonia. In order to assess more fully the Gts, PotD and SrtA recombinant proteins were different to a variety of serotype Streptococcus pneumoniae infection In this study, the recombinant protein antigen was immunized in mice by abdominal cavity, and then different serotypes of Streptococcus pneumoniae were selected to study the intraperitoneal attack. After the specific antiserum was used to immunize mice, D39 Streptococcus pneumoniae was selected to carry out the nasal attack test to identify whether the protection of the recombinant protein antigen was specific. The effect of Gts, PotD and SrtA recombinant protein and its specific antiserum on the adhesion of D39 cells to A549 cells; finally, in order to further explore the change of the titer of natural antibody produced by the surface antigen of the bacterial surface antigen, Gts, PotD and SrtA, in order to further investigate the human body through Streptococcus pneumoniae infection, the study was conducted. To collect normal population and acute pneumonia patients, the titers of anti-Gts, anti-PotD and anti-SrtA antiserum in serum were determined by ELISA.
Results: on the basis of successful cloning and expression of Gts, PotD and SrtA recombinant protein, it was confirmed by Western blot analysis that CMCC (B) 31216 (serotype9V), CMCC (B) 31436 (serotype3), CMCC (31507) and other serotypes of Streptococcus pneumoniae, and protein antigenicity, were not mutated. Adhesion inhibition experimental evidence In fact, a single Gts, PotD and SrtA protein can inhibit the adhesion of D39 to A549 cells, and the combined use of 2-3 protein antigens has obvious cumulative effects. In addition, the combination of anti -Gts, anti -PotD and anti -SrtA antiserum alone or combined with 2-3 antisera also has a similar inhibitory effect with the protein antigen. Gts, Po is dripped through the nasal cavity. TD or SrtA recombinant protein, mouse splenocytes produced a high level of Th1 mediated cytokine IFN- gamma, Th2 mediated cytokine IL-4, Treg mediated IL-10 factor and Th17 mediated cytokine IL-17A, inducing mucosal immunization and systemic immunity to Streptococcus pneumoniae in mice. In the mice model of pneumococcal infection, Gts, PotD The recombinant protein of SrtA or recombinant protein can effectively reduce the colonization of 19F type Streptococcus pneumoniae in the nasopharynx and lungs. Compared with single protein antigen, Gts+SrtA, PotD+Gts+SrtA can significantly improve the protective effect of Streptococcus pneumoniae on lung colonization,.3 egg white antigen combined with mucosal immunization, to Streptococcus pneumoniae. The most effective D39 serotype nasal infection rate is 83.3%.
The results of intraperitoneal immunization were similar to that of mucosal immune. Almost all protein antigens were more protective than single proteins. But compared with protein 22, the combination of Gts+PotD+SrtA's triplet antigen was not significantly enhanced. Passive immunization was suggested to resist -Gts, anti -PotD and anti -SrtA antiserum. The protective effect of CO immunized mice on Streptococcus pneumoniae in mice is similar to that of active immunization, suggesting that the protective effect of Gts, PotD and SrtA protein antigen through active immunization is closely related to the action of the antibody.
Conclusion: the analysis of this study showed that Gts, PotD and SrtA were conserved in Streptococcus pneumoniae, 22 combination of three protein combined antigens and three combinations, and immunized by mucosal immunization or systemic immunization could induce significant protective effects on different serotype Streptococcus pneumoniae infection in mice, showing Gts, Po TD and SrtA are a group of candidate vaccine combinations with potential development.
Objective: to express and purify Streptococcus pneumoniae (S.pn) outer membrane protein SPD1741 and SPD0280 for crystal growth, optimization and three-dimensional structure analysis. Methods: the spd1741, spd0280 gene of S.pn D39 strain was cloned to the prokaryotic expression vector pET32a (+), and was identified by bacterial liquid PCR and recombinant plasmid, and then transformed. The strain E. coli BL21 (DE3) was obtained. The strain was induced by IPTG to efficiently express the soluble fusion protein with His label. After affinity chromatography (Ni-NTA), ion exchange chromatography (DEAE) and molecular sieve chromatography, the high purity target protein was obtained for crystal growth and optimization. Finally, the crystal X ray diffraction data was collected. The purity of the purified SPD1741 and SPD0280 recombinant protein was more than 90%. The X ray diffraction ability of the crystal was 4 and 3.5. respectively. The high purity SPD1741 and SPD0280 recombinant protein were prepared and 2 kinds of protein matrix were obtained. This study received the X ray diffraction data. It laid a foundation for the analysis of three-dimensional structure and biological function of SPD1741 and SPD0280.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378

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