鼻腔、腹腔免疫DnaJ蛋白对小鼠肺炎链球菌感染的保护效果及机制的研究

发布时间：2018-04-23 12:25

本文选题：肺炎链球菌 + 蛋白疫苗　；参考：《重庆医科大学》2010年硕士论文

【摘要】： 目的肺炎链球菌(streptococcus pneumoniae, S.pn)是严重侵袭性感染和上呼吸道感染最重要的病原体之一,使用有效的疫苗预防其感染是一个重要手段,WHO建议将S.pn结合疫苗纳入国家免疫计划,目前S.pn疫苗为全球最急需的前3位疫苗之一。肺炎链球菌DnaJ(Hsp40)是热休克蛋白40家族的成员之一,重组蛋白DnaJ腹腔免疫BALB/C小鼠后,可抵御血清型3型肺炎链球菌致死剂量的鼻腔感染,具有保护效果[1]。然而尚不清楚,重组蛋白DnaJ作为免疫原是否对不同血清型的肺炎链球菌均具有保护作用,其经鼻腔粘膜途径免疫是否具有免疫原性、是否能够保护宿主抵御S.pn致死性感染及其发挥作用的免疫学机制。本研究拟对DnaJ的相关免疫保护作用及机制进行研究。方法原核表达并纯化DnaJ全长蛋白,重组蛋白腹腔免疫BALB/C小鼠,制备DnaJ特异性抗血清。菌落PCR及Western blot分析DnaJ在肺炎链球菌不同血清型菌株的表达及保守性。重组蛋白DnaJ经腹腔和粘膜两种途径免疫BALB/C小鼠,建立相应的S.pn感染模型,评价其主动免疫保护效果;间接ELISA分别检测免疫后血清中抗体亚型、脾细胞培养上清的细胞因子水平;19F型S.pn经鼻腔感染粘膜免疫的小鼠,取鼻腔灌洗液和肺匀浆,铺板计数其中细菌数。采用R6型肺炎链球菌吸附抗体实验、被动免疫保护实验,评价DnaJ特异性抗体的效果;通过黏附A549细胞实验检测抗血清对细菌黏附的抑制作用。结果肺炎链球菌DnaJ片段被正确克隆到pET-32(a)载体中。重组工程菌经IPTG诱导得到可溶形式表达的重组蛋白,Ni-NTA亲和层析纯化后,获得纯度90%的纯化蛋白。菌落PCR及Western blot检测发现,DnaJ在S.pn血清型2型,3型,6B型,14型,19F型及R6菌株中均有表达,保守性好。重组蛋白经腹腔及鼻粘膜两种途径免疫BALB/C小鼠均可以抵御致死性S.pn的感染,其中,腹腔免疫对血清型2型,3型,6B型及14型的保护效率分别为50%,50%,58.3%,41.7%,而粘膜免疫对血清型2型,3型,6B型及14型的保护效率分别为58.3%,66.7%,66.7%,50%;DnaJ蛋白经腹腔免疫途径主要刺激宿主产生IgG1,抗体滴度达4.27×106;经粘膜免疫途径主要刺激宿主IgG2b,抗体滴度达2.13×106。同时,粘膜免疫途径还刺激宿主高水平的细胞因子IL-10、IFN-γ和IL-17A,剂量分别为239.11±168.77,55.86±39.25,765.68±159.11(pg/ml)。抗定植实验结果显示,特异性抗血清能够显著减少小鼠鼻咽部及肺部定植的细菌数量(P0.05)。DnaJ特异性抗血清被动免疫BALB/C小鼠,可显著延长小鼠生存时间,提高小鼠生存率,其保护效率达50%;抗血清与R6型S.pn孵育后,血清中DnaJ特异性抗体效价显著下降,小鼠生存时间明显缩短,保护作用消失(P0.05)。同时,抗血清可以有效抑制肺炎链球菌黏附A549上皮细胞。结论上述结果显示,DnaJ蛋白在多种血清型S.pn中均有表达,具有较好的保守性。DnaJ蛋白通过腹腔免疫途径可以刺激宿主产生体液免疫反应,能有效抵御四种致病性血清型S.pn的致死性感染,其保护作用依赖于其特异性抗体;DnaJ蛋白通过粘膜免疫途径不仅可以刺激宿主产生体液免疫反应,还有效激发了细胞免疫反应,能有效抵御四种致病性血清型肺炎链球菌的鼻腔感染,具有较好的保护作用;同时,DnaJ蛋白通过粘膜免疫还可刺激宿主产生高水平的IL-17A,能有效抵御S.pn在鼻咽部的定植。
[Abstract]:objective
Streptococcus pneumoniae (S.pn) is one of the most important pathogens of severe invasive and upper respiratory tract infections. The use of effective vaccines to prevent infection is an important means. WHO recommends the integration of the S.pn vaccine into the national immunization program, and the S.pn vaccine is currently one of the most urgent top 3 vaccines in the world.
Streptococcus pneumoniae DnaJ (Hsp40) is one of the members of the heat shock protein 40 family. The recombinant protein DnaJ is immune to the lethal dose of serotype 3 of Streptococcus pneumoniae, which can protect the nasal cavity from the lethal dose of the serotype 3. However, it is not clear that the recombinant protein DnaJ is a immuno immunization with the different serotypes of Streptococcus pneumoniae. It has protective effect, whether it is immune to the nasal mucosa and whether it can protect the host against S.pn lethal infection and its immunological mechanism. This study is to study the protective effect and mechanism of DnaJ.
Method
Prokaryotic expression and purification of DnaJ full length protein, recombinant protein immunized with BALB/C mice, DnaJ specific antiserum was prepared. Colony PCR and Western blot were used to analyze the expression and conservatism of DnaJ in different serotype strains of Streptococcus pneumoniae. The recombinant protein DnaJ was immune to BALB/C mice through two ways of abdominal cavity and mucous membrane, and a corresponding S.pn infection model was established. The effect of active immunization protection, indirect ELISA detection of antibody subtype in serum after immunization, cytokine level of splenocytes culture supernatant, 19F type S.pn through nasal cavity infected mice, nasal lavage fluid and lung homogenate, pads count bacteria count, R6 type Streptococcus pneumoniae adsorption antibody experiment, passive immunization protection The effect of DnaJ specific antibody was evaluated, and the inhibitory effect of antiserum on bacterial adhesion was detected by adherent A549 cell experiment.
Result
DnaJ fragment of Streptococcus pneumoniae was correctly cloned into pET-32 (a) vector. Recombinant recombinant protein was induced by IPTG to obtain a soluble form of recombinant protein. Purified protein was purified by Ni-NTA affinity chromatography. The purity of purified protein was 90%. Colony PCR and Western blot detected DnaJ in S.pn serotype 2, type 3, 6B type, 14 type, 19F type and bacterial strain. BALB/C mice immunized with two kinds of intraperitoneal and nasal mucosa were immune to fatal S.pn infection. The protective efficiency of peritoneal immunity to serotype 2, type 3, 6B type and type 14 were 50%, 50%, 58.3%, 41.7% respectively, while the protective efficiency of mucous immunization against blood type 2, 3, 6B and 14 was 58.3%, 66., 66., 66.. 7%, 66.7%, 50%; DnaJ protein mainly stimulated the host to produce IgG1 by intraperitoneal immunization, and the antibody titer was 4.27 x 106. The mucosal immunization pathway mainly stimulated the host IgG2b, the antibody titer was 2.13 * 106., and the mucosal immune pathway also stimulated the host high level cytokine IL-10, IFN- gamma and IL-17A, and the dose was 239.11 + 168.77,55.86 + 39.25,76, respectively. 5.68 + 159.11 (pg/ml). The results of anti colonization showed that specific antiserum could significantly reduce the number of bacteria colonized in the nasopharynx and lung (P0.05).DnaJ specific antisera passively immune BALB/C mice. It could prolong the survival time of mice and increase the survival rate of mice, and the protection efficiency was 50%. After incubating with antiserum and R6 S.pn, serum The titer of DnaJ specific antibody decreased significantly, the survival time of mice was significantly shortened and the protective effect disappeared (P0.05). At the same time, antiserum could effectively inhibit the adhesion of Streptococcus pneumoniae to A549 epithelial cells.
conclusion
The above results show that DnaJ protein is expressed in a variety of serotype S.pn. The good conserved.DnaJ protein can stimulate the host's humoral immune response through the peritoneal immune pathway, and can effectively resist the fatal infection of four pathogenetic serotype S.pn, and its protective effect depends on its specific antibody; DnaJ protein is free from the mucous membrane. The epidemic method not only stimulates the host's humoral immune response, but also effectively stimulates the cellular immune response, effectively resisting the nasal infection of four pathogenic serotype Streptococcus pneumoniae, and has a good protective effect. At the same time, the DnaJ protein can stimulate the host to produce a high level of IL-17A through mucosal immunization, and can effectively resist S.pn in the nasopharynx. The colonization of the Ministry.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【共引文献】