本文關鍵詞：2型豬鏈球菌表面蛋白Hp0272參與逃逸宿主天然免疫的分子機制研究　出處：《華中農(nóng)業(yè)大學》2012年碩士論文　論文類型：學位論文

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【摘要】：豬鏈球菌(Streptococcus suis)屬于蘭氏(Lancefield)分類法的R群,能夠感染人和豬,是重要的新發(fā)傳染病病原體。35個血清型中以2型(Streptococcus suis serotype2, SS2)流行最廣,致病性最強。SS2表面假說蛋白0272(hypothetical protein0272, Hp0272),作為粘附配體分子還具有強免疫原性,可作為候選的疫苗分子,該蛋白具有增強豬鏈球菌逃逸宿主天然免疫及抗吞噬殺傷的功能,但其分子機制還不清楚。 本研究建立了豬鏈球菌-人血清相互作用模型,利用流式細胞術(fluorescence activted cell sorter, FACS)分析確定了豬鏈球菌激活補體的途徑,通過比較SS2強毒力株05ZYH33,突變株Δ0272和互補株CΔ0272表面補體的沉積,確定Hp0272在豬鏈球菌抑制替代補體途徑激活中的作用,驗證了Hp0272與人替代補體途徑的負調(diào)節(jié)子H因子(human Factor H, hFH)的相互作用以及該相互作用對抑制人血清補體激活的影響；重組純化了GST(glutathione S-transferase)標簽Hp0272融合蛋白,通過GST-Pulldown技術初步研究了Hp0272與hFH的結(jié)合方式及其與其他血漿蛋白的結(jié)合。揭示了Hp0272在豬鏈球菌逃避宿主天然免疫反應,抗吞噬殺傷中的功能及分子機制。具體結(jié)果如下： 1.Hp0272對豬鏈球菌激活人血清補體的影響 通過流式細胞術分析比較了SS2強毒力株05ZYH33,突變株Δ0272和互補株CΔ0272表面補體(C3b/iC3b和iC3b)沉積,發(fā)現(xiàn)血清濃度為50%時,Δ0272表面沉積的C3b/iC3b(熒光指數(shù)(fluorescence index, FI)=(10.7±5.0)×107)顯著高于05ZYH33(FI=(3.8±0.2)×107)和CΔ0272(FI=(4.6±2.1)×107),而Δ0272表面結(jié)合的iC3b(FI=(3.5±0.2)×106)顯著低于05ZYH33(FI=(5.7±0.0)×106)和CΔ0272(FI=(4.2±0.0)×106)。提示Hp0272對豬鏈球菌激活人血清補體的過程中有抑制,Δ0272表面結(jié)合的iC3b顯著低于05ZYH33和CΔ0272,提示Hp0272很可能與補體途徑的負調(diào)節(jié)子有相互作用,將C3b降解為iC3b。 2.豬鏈球菌激活人血清補體的途徑分析 構(gòu)建FACS模型,并從抗體濃度方面優(yōu)化了該模型,采用此模型通過經(jīng)典(classical pathway, CP),替代(alternative pathway, AP)和凝集素(lectin pathway, LP)途徑抑制劑進行抑制實驗,發(fā)現(xiàn)經(jīng)典途徑和凝途徑的抑制劑均不能起到抑制作用,而替代途徑的抑制劑能抑制90%以上的C3沉積,說明豬鏈球菌主要通過替代途徑激活補體,進一步推測Hp0272可能與hFH結(jié)合。 3.Hp0272與hFH相互作用的鑒定 采用配體印跡的方法鑒定到Hp0272與人血清中的hFH的特異性結(jié)合但不結(jié)合純的hFH蛋白,提示Hp0272與hFH的結(jié)合可能是間接結(jié)合。通過FACS進行了菌株原位水平的驗證,即結(jié)合到Δ0272表面的hFH(FI=(0.7±0.2)×106)顯著低于05ZYH33(FI=(1.4±0.3)×106)和C△0272(FI=(2.4±0.6)×106);抗hFH抗體阻封實驗發(fā)現(xiàn)05ZYH33和C△0272hFH封閉后補體結(jié)合量提高到2-3倍,而Δ0272無顯著變化,說明Hp0272與hFH的結(jié)合抑制豬鏈球菌表面補體的激活。 4.Hp0272與hFH的結(jié)合方式及Hp0272與其他血漿蛋白配體的鑒定 構(gòu)建立了pGEX-4T-1-0272的重組質(zhì)粒,轉(zhuǎn)化大腸桿菌Escherichia coli BL21,表達并純化GST-Hp0272融合蛋白。采用此蛋白進行特異性強的GST-Pulldown,選取兩條差異點進行質(zhì)譜分析,結(jié)果發(fā)現(xiàn)其中一條為hFH與C3d的復合物,提示C3d介導了Hp0272與hFH的結(jié)合。另一條為人血漿激肽原,同時通過ELISA初步鑒定到Hp0272能與hIgG和hIgA結(jié)合,且結(jié)合位點位于Hp0272N端41-318aa區(qū)域內(nèi)。
[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.

【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

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