融合蛋白AG的原核表达和纯化

发布时间：2018-04-28 16:17

本文选题：免疫吸附 + 蛋白A　；参考：《大连理工大学》2012年硕士论文

【摘要】：蛋白A免疫吸附柱是目前在自身免疫性疾病治疗中用量最大、治疗效果最好的血液净化吸附柱。但是蛋白A免疫吸附剂在30年的临床应用中仍存在一些弊端,譬如其与人IgG3结合力较弱,导致其对于系统性红斑狼疮、扩张性心肌炎等疾病治疗效果不显著。为了克服蛋白A吸附剂的上述缺陷,本论文将一段与人IgG3具有较强结合能力的蛋白G的基因与蛋白A的抗体结合结构域基因相连接,实现AG融合蛋白的原核重组表达,进而制备的融合蛋白AG吸附剂将弥补蛋白A吸附剂对IgG3结合力弱的不足,提高其对系统性红斑狼疮、扩张性心肌炎等疾病的治疗效果。本论文采用基因工程手段,选用大肠杆菌作为表达宿主菌,以含有蛋白A和蛋白G基因的质粒DNA为模板,成功克隆了全长为1470bp的包含编码蛋白A和蛋白G抗体结合结构域的DNA序列,并连接在表达载体pET23a上,构成重组质粒pET23a-CPAG,转化E.coli BL21(DE3)进行表达筛选,获得表达融合蛋白AG最适菌株E.coli BL21(DE3)(pET23a-CPAG)。所表达的融合蛋白AG仅含有抗体结合功能区,分子量约为54kDa。实验证实融合蛋白AG以可溶状态存在,并且与人IgG亲和活性良好。本论文采用加热沉淀和PEI沉淀等初分离技术、DEAE弱阴离子交换层析及IgG-Fc亲和层析等精分离技术对融合蛋白AG进行纯化。通过合理的选择与组合,最终确定相对较优的融合蛋白AG的分离纯化工艺。终产品经SDS-PAGE、高效液相分析,纯度达到98%以上,总回收率为60%。MALDI-TOF-MS测得分子量为54070.256Da,与理论计算值基本相符。本论文合成了以融合蛋白AG为配基的吸附剂,该吸附剂对血清中抗体的吸附效果与蛋白A吸附剂规律基本相同,但对IgG3的亲和力明显增强,且抗体结合量高于蛋白A吸附剂。配基密度为9.93mg/mL的吸附剂,对IgG的动态结合容量为35.9mg/mL gel,固定化融合蛋白AG与IgG的结合比例约为1：1.3,融合蛋白AG对人IgG的亲和常数为3.8×104L/mol,平衡解离常数为3.92mg/mL,最大理论结合容量为92.19mg/mL。融合蛋白AG既可以弥补蛋白A对于IgG3结合力弱的不足,又可以弥补蛋白G不结合其它类型免疫球蛋白的不足。将其固定于固相基质表面制成抗体亲和吸附剂,有潜力用于血液净化治疗包括系统性红斑狼疮、扩张性心肌炎在内的自身免疫性疾病。
[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.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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