重组人CR1-SCR1-3活性片段的制备及其对补体介导的脓毒症炎症损伤的保护性研究
发布时间：2019-06-27 18:24
【摘要】：脓毒症（sepsis）是由感染引起的全身性炎症反应综合征（systemic inflammatoryresponse syndrome,SIRS）,是严重创（烧）伤、感染性疾病以及大手术后患者常见并发症，如不及时治疗和控制，可导致休克及多器官功能障碍综合症（Multiple organdysfunction syndrome，MODS），是当前医院住院患者最主要的死亡原因之一。目前尚无有效的防治药物用于临床治疗。脓毒症的发病机制复杂，涉及到补体、凝血、纤溶等多个系统及它们之间复杂的相互作用，对脓毒症的病理生理学和流行病学还缺乏全面的认识。近年来国内外学者针对脓毒症的发病机制开展了许多研究，其中越来越多的研究表明补体系统的过度激活是脓毒症发生和发展的关键环节之一，抑制补体的过度活化已成为防治脓毒症炎症损伤的重要策略。 在经典、旁路、甘露糖结合凝集素(MBL)三条补体激活途径中，虽然活化起始点不同，但都在C3处汇合，形成C3转化酶，由此启动下游一系列介导炎症损伤的介质生成。CR1-SCR1-3段能结合C4b，抑制C3/C5转化酶，可抑制过量C5a、C5b-9等片段的生成，在始动部位防止脓毒症的发生和发展。本研究旨在重组人CR1-SCR1-3活性片段的制备及其对补体介导的脓毒症炎症损伤的保护性研究，为补体介导的脓毒症的防治提供新的思路和途径。 本课题在我室已构建的重组工程菌pPIC9k-CR1-SCR1-3/KM71的基础上，复苏工程菌，挑取优势菌落以无水甲醇诱导分泌表达目的蛋白，SDS-PAGE电泳分析，Western-Blot目的蛋白鉴定，Ni-NTA树脂亲和层析纯化目的蛋白，CH50酶联免疫分析（Enzyme-linkedimmuno sorbent assay，ELISA）检测体外补体抑制活性，观察目的蛋白对补体介导的脓毒症小鼠炎症损伤的保护作用。该研究主要获得以下结果： 1、复苏工程菌pPIC9k-CR1-SCR1-3/KM71后，以无水甲醇诱导表达，表达产物经SDS-PAGE电泳分析，可见表达蛋白的大小约为27kD，Western-Blot鉴定，在分子量约27kD处出现单一条带，呈现阳性反应。证实表达出的蛋白是所构建His标签的重组蛋白，且复苏后的工程菌仍可以稳定的表达重组蛋白CR1-SCR1-3。 2、表达产物经镍柱亲和层析纯化后，SDS-PAGE显示目的蛋白纯度较高，用BCA试剂盒测定蛋白浓度并计算纯化前后目的蛋白产率，CR1-SCR1-3约为30%。 3、体外CH50ELISA法检测补体抑制活性，结果显示：重组人CR1-SCR1-3蛋白具有较高的补体抑制活性，其补体抑制率随蛋白浓度的增高而增高，当重组人CR1-SCR1-3蛋白浓度达到50μg/ml时，具有抑制50%补体的生物活性，表明重组人CR1-SCR1-3蛋白具有良好的抑制补体活性的功能。 4、采用D-氨基半乳糖增敏内毒素攻击小鼠成功构建了脓毒症（sepsis）模型。观察重组人CR1-SCR1-3蛋白对补体介导的脓毒症小鼠炎症损伤的保护作用，我们发现：脓毒症组的脓毒症反应最强烈，16h后全部死亡，保护组的脓毒症症状减轻，16h后生存率达40%，显著提高（P＜0.05）。脓毒症组的血清促炎症介质IL-1β和肺组织MPO水平均明显升高，保护组的显著降低（P＜0.001）。脓毒症组的肺组织原位补体C4b沉积明显增多，保护组的明显减少。病理学检查显示，脓毒症组的可见肺间质内有大量中性粒细胞聚集和浸润，肺泡壁内毛细血管扩张淤血，肺泡和间质有不同程度的肺水肿；部分肺泡壁变薄断裂，泡腔膨大，呈现不同程度的肺气肿；部分肺间质细支气管周围肺泡隔纤维增生，，以致萎陷。CR1-SCR1-3保护组的上述病变明显减轻。结果表明重组人CR1-SCR1-3蛋白可以抑制补体过度活化，减轻补体介导的脓毒症炎症损伤。 综上所述，通过甲醇诱导工程菌pPIC9k-CR1-SCR1-3/KM71大剂量的表达目的蛋白,经镍柱亲和层析一步纯化获得纯度较高的目的蛋白；并证实重组人CR1-SCR1-3蛋白在体外具有抑制补体活性；在小鼠体内对补体介导的脓毒症炎症损伤有较好的保护效应。本研究结果为补体介导脓毒症的防治提供了新思路和途径。
[Abstract]:Sepsis is a systemic inflammatory response syndrome (SIRS) caused by infection, which is a common complication of severe (burn), infectious, and post-operative patients, such as non-timely treatment and control, Multiple organ dysfunction syndrome (MODS), which can lead to shock and multiple organ dysfunction syndrome (MODS), is one of the most important causes of death in the hospital in the current hospital. There is no effective prevention and control drug for clinical treatment at present. The pathogenesis of sepsis is complicated, involving multiple systems such as complement, coagulation, and fibrinolysis, and the complex interaction between them, and the pathophysiology and epidemiology of sepsis also lack a comprehensive understanding. In recent years, a number of studies have been carried out on the pathogenesis of sepsis, and more and more studies have shown that the excessive activation of the complement system is one of the key links in the occurrence and development of sepsis, and the inhibition of the excessive activation of the complement has become an important strategy for preventing and treating the inflammatory injury of sepsis. In the three complement activation pathway of classical, by-pass, mannose-binding lectin (MBL), although the activation initiation point is different, all at C3 are combined to form a C3 convertase, thereby starting a series of media to mediate the inflammatory injury downstream. The CR1-SCR1-3 segment can bind to the C4b, inhibit the C3/ C5 convertase, inhibit the generation of the excess C5a, C5b-9, and the like, and prevent the occurrence and the hair of the sepsis at the starting position. The present study was aimed at the preparation of recombinant human CR1-SCR1-3 active fragment and its protective study on complement-mediated sepsis, and to provide a new way for the prevention and treatment of complement-mediated sepsis. On the basis of the construction of the recombinant engineering bacteria pPIC9k-CR1-SCR1-3/ KM71 in our room, the engineering bacteria were recovered, and the dominant colonies were selected to induce the secretion expression target protein by the anhydrous methanol, the SDS-PAGE electrophoresis analysis, the Western-Blot target protein identification, the Ni-NTA resin affinity chromatography purification target protein, the CH50 enzyme-linked immunosorbent assay (Enzyme-linkedimmuno sorbrent as Say (ELISA) for the detection of complement-inhibitory activity in vitro and to observe the protective effects of the target protein on the inflammation of the complement-mediated sepsis mice The protective effect. The study was mainly obtained with The expression of pPIC9k-CR1-SCR1-3/ KM71 was induced by SDS-PAGE. It is confirmed that the expressed protein is the recombinant protein of the constructed His tag, and the recovered engineering bacteria can still stably express the recombinant protein CR1-S. CR1-3.2. After the expression product was purified by the nickel column affinity chromatography, the purity of the target protein was higher than that of the SDS-PAGE, the protein concentration was determined by the BCA kit and the yield of the target protein before and after purification was calculated, and CR1-SCR1 The results showed that the complement inhibition activity of the recombinant human CR1-SCR1-3 protein increased with the increase of the protein concentration. When the concentration of the recombinant human CR1-SCR1-3 protein reached 50. m The biological activity of 0% complement indicates that the recombinant human CR1-SCR1-3 protein is good Inhibition of the function of complement activity.4. The use of D-amino-galactose-sensitized endotoxin attack mice successfully constructed the sepsis The protective effect of the recombinant human CR1-SCR1-3 protein on the inflammatory injury of the complement-mediated sepsis mice was observed, and we found that the sepsis reaction in the sepsis group was the most intense and all died after 16 h, the sepsis symptom of the protection group was relieved, and the survival rate after 16 h was up to 40%. The levels of MPO in the serum pro-inflammatory mediators, IL-1 and MPO in the sepsis group were significantly increased, and the protection group was significant. The in situ complement of the lung tissue of the sepsis group was decreased (P <0.001). There was a significant decrease in the protection group. The pathological examination showed that there was a large number of neutrophils in the lungs of the sepsis group and the infiltration of the pulmonary capillary in the wall of the alveoli. There was a different degree of pulmonary edema in the alveoli and the stroma of the alveoli. Some of the alveolar walls were thinned and the cavity was expanded. A different degree of emphysema; a partial lung interstitial lung surrounding the lung. Blister fiber hyperplasia, so as to collapse. CR1-SCR1-3 protection The results showed that the recombinant human CR1-SCR1-3 protein could inhibit the complement activation and reduce the complement. In the above, the recombinant human CR1-SCR1-3 is confirmed by one-step purification of a nickel-column affinity chromatography to obtain a target protein with high purity through the purification of a high-dose expression target protein of the engineering bacteria pPIC9k-CR1-SCR1-3/ KM71 by the methanol, in vitro, the protein has the effect of inhibiting the complement activity; in vivo, the complement-mediated sepsis The results of this study are complement-mediated sepsis.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7
本文编号：2507031
[Abstract]:Sepsis is a systemic inflammatory response syndrome (SIRS) caused by infection, which is a common complication of severe (burn), infectious, and post-operative patients, such as non-timely treatment and control, Multiple organ dysfunction syndrome (MODS), which can lead to shock and multiple organ dysfunction syndrome (MODS), is one of the most important causes of death in the hospital in the current hospital. There is no effective prevention and control drug for clinical treatment at present. The pathogenesis of sepsis is complicated, involving multiple systems such as complement, coagulation, and fibrinolysis, and the complex interaction between them, and the pathophysiology and epidemiology of sepsis also lack a comprehensive understanding. In recent years, a number of studies have been carried out on the pathogenesis of sepsis, and more and more studies have shown that the excessive activation of the complement system is one of the key links in the occurrence and development of sepsis, and the inhibition of the excessive activation of the complement has become an important strategy for preventing and treating the inflammatory injury of sepsis. In the three complement activation pathway of classical, by-pass, mannose-binding lectin (MBL), although the activation initiation point is different, all at C3 are combined to form a C3 convertase, thereby starting a series of media to mediate the inflammatory injury downstream. The CR1-SCR1-3 segment can bind to the C4b, inhibit the C3/ C5 convertase, inhibit the generation of the excess C5a, C5b-9, and the like, and prevent the occurrence and the hair of the sepsis at the starting position. The present study was aimed at the preparation of recombinant human CR1-SCR1-3 active fragment and its protective study on complement-mediated sepsis, and to provide a new way for the prevention and treatment of complement-mediated sepsis. On the basis of the construction of the recombinant engineering bacteria pPIC9k-CR1-SCR1-3/ KM71 in our room, the engineering bacteria were recovered, and the dominant colonies were selected to induce the secretion expression target protein by the anhydrous methanol, the SDS-PAGE electrophoresis analysis, the Western-Blot target protein identification, the Ni-NTA resin affinity chromatography purification target protein, the CH50 enzyme-linked immunosorbent assay (Enzyme-linkedimmuno sorbrent as Say (ELISA) for the detection of complement-inhibitory activity in vitro and to observe the protective effects of the target protein on the inflammation of the complement-mediated sepsis mice The protective effect. The study was mainly obtained with The expression of pPIC9k-CR1-SCR1-3/ KM71 was induced by SDS-PAGE. It is confirmed that the expressed protein is the recombinant protein of the constructed His tag, and the recovered engineering bacteria can still stably express the recombinant protein CR1-S. CR1-3.2. After the expression product was purified by the nickel column affinity chromatography, the purity of the target protein was higher than that of the SDS-PAGE, the protein concentration was determined by the BCA kit and the yield of the target protein before and after purification was calculated, and CR1-SCR1 The results showed that the complement inhibition activity of the recombinant human CR1-SCR1-3 protein increased with the increase of the protein concentration. When the concentration of the recombinant human CR1-SCR1-3 protein reached 50. m The biological activity of 0% complement indicates that the recombinant human CR1-SCR1-3 protein is good Inhibition of the function of complement activity.4. The use of D-amino-galactose-sensitized endotoxin attack mice successfully constructed the sepsis The protective effect of the recombinant human CR1-SCR1-3 protein on the inflammatory injury of the complement-mediated sepsis mice was observed, and we found that the sepsis reaction in the sepsis group was the most intense and all died after 16 h, the sepsis symptom of the protection group was relieved, and the survival rate after 16 h was up to 40%. The levels of MPO in the serum pro-inflammatory mediators, IL-1 and MPO in the sepsis group were significantly increased, and the protection group was significant. The in situ complement of the lung tissue of the sepsis group was decreased (P <0.001). There was a significant decrease in the protection group. The pathological examination showed that there was a large number of neutrophils in the lungs of the sepsis group and the infiltration of the pulmonary capillary in the wall of the alveoli. There was a different degree of pulmonary edema in the alveoli and the stroma of the alveoli. Some of the alveolar walls were thinned and the cavity was expanded. A different degree of emphysema; a partial lung interstitial lung surrounding the lung. Blister fiber hyperplasia, so as to collapse. CR1-SCR1-3 protection The results showed that the recombinant human CR1-SCR1-3 protein could inhibit the complement activation and reduce the complement. In the above, the recombinant human CR1-SCR1-3 is confirmed by one-step purification of a nickel-column affinity chromatography to obtain a target protein with high purity through the purification of a high-dose expression target protein of the engineering bacteria pPIC9k-CR1-SCR1-3/ KM71 by the methanol, in vitro, the protein has the effect of inhibiting the complement activity; in vivo, the complement-mediated sepsis The results of this study are complement-mediated sepsis.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7
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