重組人CR1-SCR1-3活性片段的制備及其對補體介導的膿毒癥炎癥損傷的保護性研究
發(fā)布時間：2019-06-27 18:24
【摘要】：膿毒癥（sepsis）是由感染引起的全身性炎癥反應綜合征（systemic inflammatoryresponse syndrome,SIRS）,是嚴重創(chuàng)（燒）傷、感染性疾病以及大手術(shù)后患者常見并發(fā)癥，如不及時治療和控制，可導致休克及多器官功能障礙綜合癥（Multiple organdysfunction syndrome，MODS），是當前醫(yī)院住院患者最主要的死亡原因之一。目前尚無有效的防治藥物用于臨床治療。膿毒癥的發(fā)病機制復雜，涉及到補體、凝血、纖溶等多個系統(tǒng)及它們之間復雜的相互作用，對膿毒癥的病理生理學和流行病學還缺乏全面的認識。近年來國內(nèi)外學者針對膿毒癥的發(fā)病機制開展了許多研究，其中越來越多的研究表明補體系統(tǒng)的過度激活是膿毒癥發(fā)生和發(fā)展的關(guān)鍵環(huán)節(jié)之一，抑制補體的過度活化已成為防治膿毒癥炎癥損傷的重要策略。 在經(jīng)典、旁路、甘露糖結(jié)合凝集素(MBL)三條補體激活途徑中，雖然活化起始點不同，但都在C3處匯合，形成C3轉(zhuǎn)化酶，由此啟動下游一系列介導炎癥損傷的介質(zhì)生成。CR1-SCR1-3段能結(jié)合C4b，抑制C3/C5轉(zhuǎn)化酶，可抑制過量C5a、C5b-9等片段的生成，在始動部位防止膿毒癥的發(fā)生和發(fā)展。本研究旨在重組人CR1-SCR1-3活性片段的制備及其對補體介導的膿毒癥炎癥損傷的保護性研究，為補體介導的膿毒癥的防治提供新的思路和途徑。 本課題在我室已構(gòu)建的重組工程菌pPIC9k-CR1-SCR1-3/KM71的基礎上，復蘇工程菌，挑取優(yōu)勢菌落以無水甲醇誘導分泌表達目的蛋白，SDS-PAGE電泳分析，Western-Blot目的蛋白鑒定，Ni-NTA樹脂親和層析純化目的蛋白，CH50酶聯(lián)免疫分析（Enzyme-linkedimmuno sorbent assay，ELISA）檢測體外補體抑制活性，觀察目的蛋白對補體介導的膿毒癥小鼠炎癥損傷的保護作用。該研究主要獲得以下結(jié)果： 1、復蘇工程菌pPIC9k-CR1-SCR1-3/KM71后，以無水甲醇誘導表達，表達產(chǎn)物經(jīng)SDS-PAGE電泳分析，可見表達蛋白的大小約為27kD，Western-Blot鑒定，在分子量約27kD處出現(xiàn)單一條帶，呈現(xiàn)陽性反應。證實表達出的蛋白是所構(gòu)建His標簽的重組蛋白，且復蘇后的工程菌仍可以穩(wěn)定的表達重組蛋白CR1-SCR1-3。 2、表達產(chǎn)物經(jīng)鎳柱親和層析純化后，SDS-PAGE顯示目的蛋白純度較高，用BCA試劑盒測定蛋白濃度并計算純化前后目的蛋白產(chǎn)率，CR1-SCR1-3約為30%。 3、體外CH50ELISA法檢測補體抑制活性，結(jié)果顯示：重組人CR1-SCR1-3蛋白具有較高的補體抑制活性，其補體抑制率隨蛋白濃度的增高而增高，當重組人CR1-SCR1-3蛋白濃度達到50μg/ml時，具有抑制50%補體的生物活性，表明重組人CR1-SCR1-3蛋白具有良好的抑制補體活性的功能。 4、采用D-氨基半乳糖增敏內(nèi)毒素攻擊小鼠成功構(gòu)建了膿毒癥（sepsis）模型。觀察重組人CR1-SCR1-3蛋白對補體介導的膿毒癥小鼠炎癥損傷的保護作用，我們發(fā)現(xiàn)：膿毒癥組的膿毒癥反應最強烈，16h后全部死亡，保護組的膿毒癥癥狀減輕，16h后生存率達40%，顯著提高（P＜0.05）。膿毒癥組的血清促炎癥介質(zhì)IL-1β和肺組織MPO水平均明顯升高，保護組的顯著降低（P＜0.001）。膿毒癥組的肺組織原位補體C4b沉積明顯增多，保護組的明顯減少。病理學檢查顯示，膿毒癥組的可見肺間質(zhì)內(nèi)有大量中性粒細胞聚集和浸潤，肺泡壁內(nèi)毛細血管擴張淤血，肺泡和間質(zhì)有不同程度的肺水腫；部分肺泡壁變薄斷裂，泡腔膨大，呈現(xiàn)不同程度的肺氣腫；部分肺間質(zhì)細支氣管周圍肺泡隔纖維增生，，以致萎陷。CR1-SCR1-3保護組的上述病變明顯減輕。結(jié)果表明重組人CR1-SCR1-3蛋白可以抑制補體過度活化，減輕補體介導的膿毒癥炎癥損傷。 綜上所述，通過甲醇誘導工程菌pPIC9k-CR1-SCR1-3/KM71大劑量的表達目的蛋白,經(jīng)鎳柱親和層析一步純化獲得純度較高的目的蛋白；并證實重組人CR1-SCR1-3蛋白在體外具有抑制補體活性；在小鼠體內(nèi)對補體介導的膿毒癥炎癥損傷有較好的保護效應。本研究結(jié)果為補體介導膿毒癥的防治提供了新思路和途徑。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R459.7
本文編號：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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R459.7
【參考文獻】
相關(guān)期刊論文 前7條
1 郝進,劉薇,汪正清;旁路活化補體激活內(nèi)皮細胞表達趨化因子及其NF-kB調(diào)控作用的實驗研究[J];創(chuàng)傷外科雜志;2002年01期
2 王瑞蘭;魏喬;康福新;曹戍;李國平;茹松偉;李彩花;;眼鏡蛇毒因子抑制膿毒癥大鼠的炎癥反應[J];基礎醫(yī)學與臨床;2008年11期
3 盛志勇,姚詠明;膿毒癥與多器官功能障礙綜合征[J];中華急診醫(yī)學雜志;2003年10期
4 陳志瑾;叢延廣;周瑩冰;侯瑞;胡福泉;饒賢才;;畢赤酵母表達肽抗生素hPAB-β的純化[J];免疫學雜志;2008年03期
5 楊紹俊;張璇;汪正清;;人補體受體1型SCR1-3功能域基因的克隆表達及生物活性鑒定[J];免疫學雜志;2012年01期
6 鄭江,周紅,魯永玲,肖光夏;殺菌性/通透性增加蛋白模擬肽中和內(nèi)毒素作用的研究[J];中華燒傷雜志;2002年02期
7 郭毅斌,鄭江,呂根法,衛(wèi)國,王良喜,肖光夏;陽離子多肽MP-1拮抗內(nèi)毒素/脂多糖活性的實驗研究[J];中華燒傷雜志;2005年03期
本文編號：2507031
本文鏈接：http://sikaile.net/yixuelunwen/jjyx/2507031.html
最近更新
教材專著
