肺炎链球菌溶血素粘膜免疫保护效果的研究

发布时间：2018-04-22 16:04

本文选题：肺炎链球菌 + 疫苗　；参考：《重庆医科大学》2011年硕士论文

【摘要】：目的肺炎链球菌(streptococcus pneumoniae, S.pn)是引起严重侵袭性感染和上呼吸道感染最重要的病原体之一, WHO估计全球每年约有160万人死于肺炎链球菌引起的各种疾病,约100万病例为5岁以下儿童,而90%以上的死亡病例在发展中国家;随着耐药菌株的不断出现,肺炎链球菌感染的治疗面临越来越多的问题,有效的疫苗成为预防其感染的一个重要手段。目前,免疫途径的选择正在成为疫苗开发需要考虑的问题,与传统的肌肉注射免疫方式相比,粘膜免疫在产生局部免疫反应的同时,还可以刺激系统性免疫反应,对许多感染性疾病的防御都起重要作用,因此,WHO将粘膜免疫定为儿科疫苗发展的方向。溶血素(Pneumolysin, Ply)为肺炎链球菌的重要毒素之一,几乎存在于所有血清型的S.pn中,具有很好的保守性,最新研究报道显示,Ply在菌体表面存在表达。但是,由于Ply具有细胞毒性,必须对其优化。本课题组前期研究表明,第146位丙氨基酸缺失后可以导致Ply毒力和溶血活性完全丧失,并能诱导宿主产生保护性抗体,因此,突变的Ply是肺炎链球菌良好的疫苗候选蛋白,本课题拟研究ΔA146 Ply粘膜免疫对肺炎链球菌感染的保护作用。方法利用重组蛋白原核表达技术对第146位氨基酸缺失的Ply蛋白(ΔA146 Ply)进行体外表达,重组蛋白经粘膜途径免疫BALB/C小鼠,制备ΔA146 Ply特异性抗血清。SDS-PAGE及Western blot分析目的蛋白的表达及在六株常见致病性肺炎链球菌中的保守性,间接ELISA检测血清及唾液中ΔA146 Ply特异性抗体效价,并分析其产生IgG的亚型,同时,无菌操作获得免疫后小鼠脾细胞,检测其培养上清中相应的细胞因子水平。19F型S.pn鼻腔感染免疫后小鼠,取其鼻腔灌洗液和肺匀浆,铺板计数其中定植的细菌数量;致病性肺炎链球菌NCTC7466,CMCC(B)31436、CMCC(B)31207及CMCC(B)31614菌株经鼻腔感染免疫后小鼠,检测重组Ply蛋白对其在小鼠鼻咽部及肺部感染的保护作用并评价其主动免疫保护效果。进行体外抗黏附实验,检测ΔA146 Ply蛋白及其抗血清是否对肺炎链球菌R6黏附A549细胞具有抑制作用。结果包含肺炎链球菌ply(146位氨基酸突变)序列的重组工程菌BL21(DE3)经IPTG诱导得到大量以可溶形式表达的重组蛋白,经Ni-NTA亲和层析纯化,SDS-PAGE检测发现得到了纯度95%的纯化蛋白。Western blot检测发现,Ply在六株常见的致病性肺炎链球菌NCTC7466,TIGR4,CMCC(B)31436、CMCC(B)31207,CMCC(B)31614及CMCC(B)31693中具有很好的保守性。重组蛋白经粘膜免疫BALB/C小鼠,检测其血清及唾液中特异性抗体发现,血清中ΔA146Ply特异性IgG效价高达5.12×10~5,唾液中IgG及IgA效价分别达到4.0×10~3和4.8×10~3;利用Santa公司羊抗鼠IgG1,IgG2a,IgG2b,IgG3二抗亚型检测免疫后血清中IgG亚型,结果显示,重组蛋白ΔA146 Ply粘膜免疫BALB/C小鼠主要刺激宿主产生IgG1,和IgG2b为主,提示宿主产生的免疫反应主要为Th2型。检测免疫后小鼠脾细胞培养上清中相应细胞因子水平发现,ΔA146 Ply粘膜免疫后刺激宿主产生了高水平的IL-17A,剂量高达678.55±189(pg/ml),峰值出现于刺激后48小时。CMCC(B)31693抗定植实验显示,ΔA146 Ply粘膜免疫后肺炎链球菌在小鼠鼻咽部及肺部的定植降低10-20倍(p0.05)。致病性肺炎链球菌NCTC7466 , CMCC(B)31436、CMCC(B)31207及CMCC(B)31614菌株经鼻腔感染免疫后小鼠,检测其在小鼠鼻咽部及肺部的细菌数量发现,肺炎链球菌CMCC(B)31436及CMCC(B)31614在小鼠鼻咽部的定植降低20倍左右(p0.05),肺炎链球菌NCTC7466,CMCC(B)314367及CMCC(B)31614在小鼠肺内的定植降低20-30倍(p0.05)。主动免疫保护实验显示,重组蛋白ΔA146 Ply粘膜免疫可以有效延长小鼠生存时间,提高其生存率,其对肺炎链球菌NCTC7466,CMCC(B)31436、CMCC(B)31614及CMCC(B)31207的保护率分别为50%,41.7%,50%,41.7%。体外抗黏附实验结果显示,ΔA146 Ply特异性抗血清100倍、500倍、2500倍和10000倍稀释时,对肺炎链球菌R6对黏附A549细胞的抑制率分别为65%、60%、55%和35%;重组ΔA146 Ply蛋白在1μg/ml, 10μg/ml, 20μg/ml, 50μg/ml时对肺炎链球菌R6黏附A549细胞的抑制率分别为15%、20%、25%和 30%。结论上述结果提示,ΔA146 Ply蛋白经粘膜免疫BALB/C小鼠可有效降低肺炎链球菌在宿主鼻咽部和肺部的定植,其保护作用可能依赖于高水平的IgA抗体及IL-17A;同时ΔA146 Ply蛋白粘膜免疫能有效保护宿主抵御四种致病性血清型S.pn的致死性感染,其保护作用可能依赖于小鼠体液免疫激活而产生的特异性抗体。
[Abstract]:objective
Streptococcus pneumoniae (S.pn) is one of the most important pathogens causing severe invasive infection and upper respiratory tract infection. WHO estimates that about 1 million 600 thousand people worldwide die from various diseases caused by Streptococcus pneumoniae every year. About 1 million cases are children under 5 years of age, and more than 90% of the deaths are in developing countries. With the continuous emergence of strains, the treatment of Streptococcus pneumoniae is facing more and more problems. Effective vaccines have become an important means to prevent infection.
At present, the selection of immune pathways is becoming a problem to be considered in the development of vaccine. Compared with the traditional intramuscular injection immunization, mucosal immunity can stimulate systemic immune response and play a important role in the defense of many infectious diseases. Therefore, WHO makes mucosal immunization a paediatric vaccine. The direction of development.
Pneumolysin (Ply) is one of the most important toxin of Streptococcus pneumoniae. It almost exists in all serotypes of S.pn and has good conservatism. The latest research report shows that Ply is expressed on the surface of the fungus. But, because Ply has cytotoxicity, it must be optimized. The previous study in this group showed that 146th bits of amino acid were deficient. After loss, the Ply toxicity and hemolytic activity can be completely lost and the host can induce protective antibodies. Therefore, the mutant Ply is a good candidate protein for Streptococcus pneumoniae. This topic is to study the protective effect of delta A146 Ply mucosal immunization on Streptococcus pneumoniae infection.
Method
The recombinant protein prokaryotic expression technique was used to express the 146th amino acid missing Ply protein (delta A146 Ply) in vitro. The recombinant protein was immunized with the mucosal pathway to immunization of BALB/C mice. The expression of delta A146 Ply specific antiserum.SDS-PAGE and Western blot analysis target protein and the conservatism in Six Common Pathogenic Streptococcus pneumoniae were prepared. The titer of the specific antibody of delta A146 Ply in serum and saliva was detected by ELISA, and the subtype of IgG was analyzed. At the same time, the spleen cells of mice after immunization were obtained by aseptic operation, and the corresponding cytokine level of.19F S.pn nasal infection in the culture supernatant was detected, and the nasal cavity lavage fluid and lung homogenate were obtained. The number of bacteria, Pathogenic Streptococcus pneumoniae NCTC7466, CMCC (B) 31436, CMCC (B) 31207 and CMCC (B) 31614 were immunized by nasal infection in mice. The protective effect of recombinant Ply protein on its nasopharynx and lung infection in mice was detected and its active immunity protection was evaluated. The anti adhesion test in vitro was conducted to detect the delta A146 Ply protein and its antiserum. Whether it has inhibitory effect on A549 cells adhered to Streptococcus pneumoniae R6.
Result
Recombinant recombinant strain BL21 (DE3) containing Streptococcus pneumoniae ply (146 amino acid mutation) sequence was induced by IPTG to obtain a large number of recombinant proteins expressed in soluble form. Purified by Ni-NTA affinity chromatography, SDS-PAGE detection found that the purity of purified protein.Western blot was detected by SDS-PAGE, and Ply was found in six common Pathogenic Streptococcus pneumoniae NCTC7. 466, TIGR4, CMCC (B) 31436, CMCC (B) 31207, CMCC (B) 31614 and CMCC (B) 31693 have good conservatism. The recombinant protein was immunized with the mucous membrane of the mucous membrane to detect the specific antibodies in the serum and saliva. The delta A146Ply specificity IgG titer of the serum was up to 5.12, and the potency of the saliva was 4 * and 4.8, respectively. NTA Sheep anti rat IgG1, IgG2a, IgG2b, IgG3 two anti subtypes were used to detect the IgG subtypes in the immune sera. The results showed that the recombinant protein Delta A146 Ply mucosal immune BALB/C mice mainly stimulated the host to produce IgG1, and IgG2b, suggesting that the host immune response was mainly Th2 type. The corresponding cell causes in the splenocytes culture supernatant of mice after immunization were detected. At the sub level, a high level of IL-17A was produced by the immune stimulation of the delta A146 Ply mucosa. The dose was up to 678.55 + 189 (pg/ml). The peak appeared at 48 hours after the stimulation, and.CMCC (B) 31693 anti colonization test showed that the colonization of Streptococcus pneumoniae in the nasopharynx and lungs of the mice decreased by 10-20 times (P0.05). The pathogenic pneumonic chain of Streptococcus pneumoniae was reduced by A146 Ply. The bacteria NCTC7466, CMCC (B) 31436, CMCC (B) 31207 and CMCC (B) 31614 were immunized by nasal infection, and the number of bacteria in the nasopharynx and lungs of mice was detected. The colonization of Streptococcus pneumoniae CMCC (B) 31436 and CMCC (B) 31614 in the nasopharynx of mice decreased by 20 times (P0.05), 314367 and 31614 were 31614 in the nasopharynx. The colonization of lung in mice decreased by 20-30 times (P0.05). The active immune protection experiment showed that the recombinant protein Delta A146 Ply mucosal immune can effectively prolong the survival time of mice and improve its survival rate. The protection rate of Streptococcus pneumoniae NCTC7466, CMCC (B) 31436, CMCC (B) 31614 and CMCC (B) 31207 is 50%, 41.7%, 50%, and 41.7%. in vitro adherence experimental knot, respectively. The results showed that the inhibition rates of Streptococcus pneumoniae R6 against adhesion A549 cells were 65%, 60%, 55% and 35% respectively when the delta A146 Ply specific antiserum was diluted 100 times, 500 times, 2500 times and 10000 times, and the inhibition rates of the recombinant Delta A146 Ply protein at 1 mu g/ml, 10 um g/ml, 20 g/ml and 50 mu g/ml respectively were 15%, 20%, and A549 cells respectively.
30%.
conclusion
These results suggest that A146 Ply protein can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lungs of the host by mucous membrane immunization, and its protective effect may depend on the high level of IgA antibody and IL-17A, while the delta A146 Ply protein mucosal immunization can effectively protect the host from the lethal infection of the four pathogenic serotype S.pn. The protective effect may depend on the specific antibody produced by humoral immune activation in mice.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【参考文献】