本文選題：免疫吸附 + 蛋白A��； 參考：《大連理工大學(xué)》2012年碩士論文

【摘要】：蛋白A免疫吸附柱是目前在自身免疫性疾病治療中用量最大、治療效果最好的血液凈化吸附柱。但是蛋白A免疫吸附劑在30年的臨床應(yīng)用中仍存在一些弊端,譬如其與人IgG3結(jié)合力較弱,導(dǎo)致其對于系統(tǒng)性紅斑狼瘡、擴張性心肌炎等疾病治療效果不顯著。 為了克服蛋白A吸附劑的上述缺陷,本論文將一段與人IgG3具有較強結(jié)合能力的蛋白G的基因與蛋白A的抗體結(jié)合結(jié)構(gòu)域基因相連接,實現(xiàn)AG融合蛋白的原核重組表達,進而制備的融合蛋白AG吸附劑將彌補蛋白A吸附劑對IgG3結(jié)合力弱的不足,提高其對系統(tǒng)性紅斑狼瘡、擴張性心肌炎等疾病的治療效果。 本論文采用基因工程手段,選用大腸桿菌作為表達宿主菌,以含有蛋白A和蛋白G基因的質(zhì)粒DNA為模板,成功克隆了全長為1470bp的包含編碼蛋白A和蛋白G抗體結(jié)合結(jié)構(gòu)域的DNA序列,并連接在表達載體pET23a上,構(gòu)成重組質(zhì)粒pET23a-CPAG,轉(zhuǎn)化E.coli BL21(DE3)進行表達篩選,獲得表達融合蛋白AG最適菌株E.coli BL21(DE3)(pET23a-CPAG)。所表達的融合蛋白AG僅含有抗體結(jié)合功能區(qū),分子量約為54kDa。實驗證實融合蛋白AG以可溶狀態(tài)存在,并且與人IgG親和活性良好。 本論文采用加熱沉淀和PEI沉淀等初分離技術(shù)、DEAE弱陰離子交換層析及IgG-Fc親和層析等精分離技術(shù)對融合蛋白AG進行純化。通過合理的選擇與組合,最終確定相對較優(yōu)的融合蛋白AG的分離純化工藝。終產(chǎn)品經(jīng)SDS-PAGE、高效液相分析,純度達到98%以上,總回收率為60%。MALDI-TOF-MS測得分子量為54070.256Da,與理論計算值基本相符。 本論文合成了以融合蛋白AG為配基的吸附劑,該吸附劑對血清中抗體的吸附效果與蛋白A吸附劑規(guī)律基本相同,但對IgG3的親和力明顯增強,且抗體結(jié)合量高于蛋白A吸附劑。配基密度為9.93mg/mL的吸附劑,對IgG的動態(tài)結(jié)合容量為35.9mg/mL gel,固定化融合蛋白AG與IgG的結(jié)合比例約為1：1.3,融合蛋白AG對人IgG的親和常數(shù)為3.8×104L/mol,平衡解離常數(shù)為3.92mg/mL,最大理論結(jié)合容量為92.19mg/mL。 融合蛋白AG既可以彌補蛋白A對于IgG3結(jié)合力弱的不足,又可以彌補蛋白G不結(jié)合其它類型免疫球蛋白的不足。將其固定于固相基質(zhì)表面制成抗體親和吸附劑,有潛力用于血液凈化治療包括系統(tǒng)性紅斑狼瘡、擴張性心肌炎在內(nèi)的自身免疫性疾病。
[Abstract]:Protein A immunosorbent column is the most effective blood purification column in the treatment of autoimmune diseases. However, there are still some drawbacks in the clinical application of protein A immunoadsorbent in 30 years, such as the weak binding ability of protein A to human IgG3, which leads to the lack of effect in the treatment of systemic lupus erythematosus, dilated myocarditis and other diseases, such as systemic lupus erythematosus (SLE) and dilated myocarditis. In order to overcome the above defects of protein A adsorbent, a protein G gene with strong binding ability to human IgG3 was linked to the antibody binding domain gene of protein A to realize the prokaryotic expression of AG fusion protein. Furthermore, the fusion protein AG adsorbent will make up for the weak binding ability of protein A adsorbent to IgG3 and improve its therapeutic effect on systemic lupus erythematosus, dilated myocarditis and other diseases. In this paper, the plasmid DNA containing protein A and protein G genes was used as template, and Escherichia coli was selected as the host strain by genetic engineering. The DNA sequence containing the antibody binding domain of encoding protein A and protein G was cloned successfully and ligated into the expression vector pET23a. The recombinant plasmid pET23a-CPAGG was transformed into E.coli BL21DE3 for expression and screening. The best strain for expressing fusion protein AG, E.coli BL21, DE3, pET23a-CPAGG, was obtained. The expressed fusion protein AG contained only antibody binding domain and its molecular weight was about 54 kDa. The results showed that the fusion protein AG existed in soluble state and had good affinity to human IgG. In this paper, the fusion protein AG was purified by using such primary separation techniques as heating precipitation and PEI precipitation, such as DEAE weak anion exchange chromatography and IgG-Fc affinity chromatography. Through reasonable selection and combination, the better separation and purification process of fusion protein AG was finally determined. The final product was analyzed by SDS-PAGE.The purity of the product was over 98%, and the total recovery rate was 54070.256Da. the total recovery rate was 54070.256Da. it was in good agreement with the theoretical calculation. In this paper, the fusion protein AG as ligand was synthesized. The adsorption effect of this adsorbent on serum antibody was basically the same as that of protein A adsorbent, but the affinity to IgG3 was obviously enhanced, and the binding capacity of antibody was higher than that of protein A adsorbent. When the ligand density is 9.93mg/mL, the dynamic binding capacity to IgG is 35.9mg/mL, the ratio of immobilized fusion protein AG to IgG is about 1: 1.3, the affinity constant of the fusion protein AG to human IgG is 3.8 脳 10 4 L / mol, the equilibrium dissociation constant is 3.92 mg / mL, the maximum theoretical binding capacity is 92.19 mg / mL. The fusion protein AG can not only make up for the weak binding ability of protein A to IgG3, but also make up for the deficiency of protein G not binding to other types of immunoglobulin. It is immobilized on the solid matrix surface to make antibody affinity adsorbent, which has the potential to be used in the treatment of autoimmune diseases including systemic lupus erythematosus and dilated myocarditis.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1

【參考文獻】

相關(guān)期刊論文 前10條

1 楊曉蘭,馬育,黃維,羅聰;瓊脂包嵌凹凸棒微囊體外人全血凈化作用研究[J];第三軍醫(yī)大學(xué)學(xué)報;2005年11期

2 陳一新;;葡萄球菌A蛋白在免疫學(xué)上的應(yīng)用[J];江西醫(yī)藥;1981年01期

3 張修彥;;金黃色葡萄球菌蛋白A及其應(yīng)用[J];日本醫(yī)學(xué)介紹;1983年04期

4 靳曉紅,初曉翠;鏈球菌G蛋白的特性及應(yīng)用前景[J];上海免疫學(xué)雜志;1997年02期

5 傅辰生,丁小強;葡萄球菌蛋白A免疫吸附治療難治性類風(fēng)濕關(guān)節(jié)炎進展[J];復(fù)旦學(xué)報(醫(yī)學(xué)版);2005年05期

6 馬育,楊曉蘭,湯先覺,景淑華;幾種吸附劑對血漿中亞甲藍的去除研究[J];生物醫(yī)學(xué)工程學(xué)雜志;2003年01期

7 黃維,馬育,楊曉蘭,湯先覺,舒昌達;交聯(lián)瓊脂包嵌凹凸棒微囊重復(fù)血液灌流中白細胞吞噬功能的變化[J];生物醫(yī)學(xué)工程學(xué)雜志;2003年02期

8 馬育,夏云,楊曉蘭,于明安;血液凈化吸附劑CAA去除血漿亞甲藍對人血漿正常成分的影響[J];生物醫(yī)學(xué)工程學(xué)雜志;2003年02期

9 李節(jié)，邱平;金黃色葡萄球菌蛋白A（Staphylococcal　Protein　A，　SPA）分泌型融合表達系統(tǒng)[J];生物工程進展;1994年05期

10 蔡仕英,姚志建,劉亞霞,強伯勤;金黃色葡萄球菌A蛋白基因的克隆及序列分析[J];生物化學(xué)雜志;1992年03期

相關(guān)博士學(xué)位論文 前1條

1 任軍;致病抗體血液凈化吸附劑的合成及性能研究[D];大連理工大學(xué);2009年

相關(guān)碩士學(xué)位論文 前4條

1 林雪;蛋白A的重組表達及重組菌的發(fā)酵過程優(yōu)化[D];大連理工大學(xué);2010年

2 邳志前;抗體類吸附劑的合成過程優(yōu)化及性能評價[D];大連理工大學(xué);2011年

3 于翔;基于生物免疫原理的入侵檢測研究[D];南京理工大學(xué);2007年

4 張丹妮;抗體選擇性吸附材料的合成及性能評價[D];大連理工大學(xué);2008年

，

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