2型猪链球菌表面蛋白Hp0272参与逃逸宿主天然免疫的分子机制研究

发布时间：2018-01-08 18:28

  本文关键词：2型猪链球菌表面蛋白Hp0272参与逃逸宿主天然免疫的分子机制研究　出处：《华中农业大学》2012年硕士论文　论文类型：学位论文

  更多相关文章： 猪链球菌 Hp0272 hFH 抗吞噬机制 补体途径

【摘要】：猪链球菌(Streptococcus suis)属于兰氏(Lancefield)分类法的R群,能够感染人和猪,是重要的新发传染病病原体。35个血清型中以2型(Streptococcus suis serotype2, SS2)流行最广,致病性最强。SS2表面假说蛋白0272(hypothetical protein0272, Hp0272),作为粘附配体分子还具有强免疫原性,可作为候选的疫苗分子,该蛋白具有增强猪链球菌逃逸宿主天然免疫及抗吞噬杀伤的功能,但其分子机制还不清楚。 本研究建立了猪链球菌-人血清相互作用模型,利用流式细胞术(fluorescence activted cell sorter, FACS)分析确定了猪链球菌激活补体的途径,通过比较SS2强毒力株05ZYH33,突变株Δ0272和互补株CΔ0272表面补体的沉积,确定Hp0272在猪链球菌抑制替代补体途径激活中的作用,验证了Hp0272与人替代补体途径的负调节子H因子(human Factor H, hFH)的相互作用以及该相互作用对抑制人血清补体激活的影响；重组纯化了GST(glutathione S-transferase)标签Hp0272融合蛋白,通过GST-Pulldown技术初步研究了Hp0272与hFH的结合方式及其与其他血浆蛋白的结合。揭示了Hp0272在猪链球菌逃避宿主天然免疫反应,抗吞噬杀伤中的功能及分子机制。具体结果如下： 1.Hp0272对猪链球菌激活人血清补体的影响 通过流式细胞术分析比较了SS2强毒力株05ZYH33,突变株Δ0272和互补株CΔ0272表面补体(C3b/iC3b和iC3b)沉积,发现血清浓度为50%时,Δ0272表面沉积的C3b/iC3b(荧光指数(fluorescence index, FI)=(10.7±5.0)×107)显著高于05ZYH33(FI=(3.8±0.2)×107)和CΔ0272(FI=(4.6±2.1)×107),而Δ0272表面结合的iC3b(FI=(3.5±0.2)×106)显著低于05ZYH33(FI=(5.7±0.0)×106)和CΔ0272(FI=(4.2±0.0)×106)。提示Hp0272对猪链球菌激活人血清补体的过程中有抑制,Δ0272表面结合的iC3b显著低于05ZYH33和CΔ0272,提示Hp0272很可能与补体途径的负调节子有相互作用,将C3b降解为iC3b。 2.猪链球菌激活人血清补体的途径分析 构建FACS模型,并从抗体浓度方面优化了该模型,采用此模型通过经典(classical pathway, CP),替代(alternative pathway, AP)和凝集素(lectin pathway, LP)途径抑制剂进行抑制实验,发现经典途径和凝途径的抑制剂均不能起到抑制作用,而替代途径的抑制剂能抑制90%以上的C3沉积,说明猪链球菌主要通过替代途径激活补体,进一步推测Hp0272可能与hFH结合。 3.Hp0272与hFH相互作用的鉴定 采用配体印迹的方法鉴定到Hp0272与人血清中的hFH的特异性结合但不结合纯的hFH蛋白,提示Hp0272与hFH的结合可能是间接结合。通过FACS进行了菌株原位水平的验证,即结合到Δ0272表面的hFH(FI=(0.7±0.2)×106)显著低于05ZYH33(FI=(1.4±0.3)×106)和C△0272(FI=(2.4±0.6)×106);抗hFH抗体阻封实验发现05ZYH33和C△0272hFH封闭后补体结合量提高到2-3倍,而Δ0272无显著变化,说明Hp0272与hFH的结合抑制猪链球菌表面补体的激活。 4.Hp0272与hFH的结合方式及Hp0272与其他血浆蛋白配体的鉴定 构建立了pGEX-4T-1-0272的重组质粒,转化大肠杆菌Escherichia coli BL21,表达并纯化GST-Hp0272融合蛋白。采用此蛋白进行特异性强的GST-Pulldown,选取两条差异点进行质谱分析,结果发现其中一条为hFH与C3d的复合物,提示C3d介导了Hp0272与hFH的结合。另一条为人血浆激肽原,同时通过ELISA初步鉴定到Hp0272能与hIgG和hIgA结合,且结合位点位于Hp0272N端41-318aa区域内。
[Abstract]:Streptococcus suis (Streptococcus suis) belongs to gram (Lancefield) classification of the R group, and can infect pigs, is an important new infectious disease pathogens of.35 serotypes in type 2 (Streptococcus suis serotype2, SS2) the most popular, most pathogenic.SS2 surface protein 0272 (hypothetical hypothesis protein0272, Hp0272) as the adhesion ligands, with strong immunogenicity, can be used as vaccine candidate molecules, the protein of Streptococcus suis has enhanced the evasion of host innate immunity and anti phagocytosis, but its molecular mechanism is not clear.
This study established Streptococcus suis human serum interaction model, using flow cytometry (fluorescence activted cell sorter, FACS) analysis to determine the pathway of complement activation by Streptococcus suis, SS2 virulent strain 05ZYH33, the mutant strain C Delta 0272 and delta 0272 complementary deposition surface complement, Hp0272 inhibited the activation of the alternative complement way in the role of Streptococcus suis, verify the negative regulator factor H Hp0272 and one of the alternative complement pathway (human Factor, H, hFH) interaction and the interaction of human serum complement activation inhibitory effects; purification of recombinant GST (glutathione S-transferase) Hp0272 tag fusion protein was studied by GST-Pulldown technology the combination of Hp0272 and hFH and other plasma proteins. Hp0272 revealed to evade the host innate immune response in Streptococcus suis, anti phagocytic killing in power Energy and molecular mechanism. The specific results are as follows:
Effect of 1.Hp0272 on serum complement of Streptococcus suis activator
SS2 strong virulence strain 05ZYH33 were compared by flow cytometry, Delta 0272 mutant and complementary strain C a 0272 surface complement (C3b/iC3b and iC3b) deposition, found that serum concentration of 50%, a 0272 surface deposition of C3b/iC3b (fluorescence index (fluorescence index, FI) = (10.7 + 5) * 107) was significantly higher than that of 05ZYH33 (FI= (3.8 + 0.2) * 107) and C (FI= ^ 0272 (4.6 + 2.1) * 107), and combined with the delta 0272 surface iC3b (FI= (3.5 + 0.2) * 106 (FI=) was significantly lower than that of 05ZYH33 (5.7 + 0) * 106 (FI=) and C a 0272 (4.2 + 0) * 106). Prompt inhibition of Hp0272 activation of human complement of Streptococcus suis, a 0272 surface bound iC3b was significantly lower than that of 05ZYH33 and C was 0272, suggesting that Hp0272 may be a negative regulator of the complement pathway interactions, C3b degradation is iC3b.
Analysis of the pathway of human serum complement activated by 2. Streptococcus suis
To construct the FACS model, and from the aspects of antibody concentration to optimize the model, using this model through classic (classical pathway, CP (alternative), pathway, AP) alternative and lectin (lectin pathway LP) pathway inhibitor inhibition and found that inhibitors of the classical pathway and the coagulation pathway can inhibit, and inhibitors the alternative pathway can inhibit the deposition of C3 90% and above, that mainly through the alternative pathway of complement activation of Streptococcus suis, further suggesting that Hp0272 may bind to hFH.
Identification of the interaction between 3.Hp0272 and hFH
By using the method of identification of ligand blotting to specific Hp0272 and hFH in human serum binding but not with pure hFH protein, suggesting that Hp0272 combined with hFH may be indirect combination by FACS. Verify that the in situ strain level, according to a 0272 surface of the hFH (FI= (0.7 + 0.2) * 106 (FI=) was significantly lower than that of 05ZYH33 (1.4 + 0.3) * 106 (FI=) and C Delta 0272 (2.4 + 0.6) * 106); anti hFH antibody blocking experiments showed that the 05ZYH33 and C letter Delta 0272hFH closed after the complement fixation increased to 2-3 times, and a 0272 no significant change, suggest that the activation of the combination of Hp0272 and hFH the inhibition of Streptococcus suis surface complement.
The combination of 4.Hp0272 and hFH and identification of Hp0272 and other plasma protein ligands
The building of a recombinant plasmid pGEX-4T-1-0272 and transformed into Escherichia coli Escherichia coli BL21, expression and purification of GST-Hp0272 fusion protein. The specificity of GST-Pulldown with this protein, selecting two different spots by mass spectrometry analysis, one of which is hFH and C3d complexes were found, suggesting that C3d mediated by the combination of Hp0272 and hFH. And another is the human plasma kininogen, at the same time through the identification of ELISA to Hp0272 can combine with hIgG and hIgA, and the binding sites located in the Hp0272N terminal region of 41-318aa.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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