葡激酶T细胞表位区域18-34关键氨基酸突变对其生物学活性的影响
发布时间：2018-10-12 19:51
【摘要】：天然葡激酶(staphylokinase, Sak)是一种由金黄色葡萄球菌合成的具有纤溶酶原激活活性的单链蛋白质,由136个氨基酸所组成,分子中不含二硫键,分子量为15.5kD。临床治疗急性心肌梗塞的研究表明,重组葡激酶(r-Sak)具有与重组组织型纤溶酶原激活剂(rt-PA)相当的溶栓活性,且具有更高的纤维蛋白专一性,除此之外,它还能在大肠杆菌中高效表达,生产成本较低,是一种很有应用前景的溶栓药物。但是Sak是一种细菌来源的蛋白质,在临床用药后的两到三周会产生大量的中和性抗体,病人体内将产生免疫反应,甚至发生过敏反应,一定程度上影响了葡激酶在临床上的广泛应用。 用定点突变的方法对Sak分子进行改造,去除其抗原表位,是获得新型低免疫原性溶栓药物的重要方法之一。根据报道葡激酶的T细胞表位与HLA-DR结合的区域主要有六个,分别是18-34表位区域、44-57表位区域、73-87表位区域、89-99表位区域、111-120表位区域和125-135表位区域。其中以Y24为核心氨基酸的18-34表位区域对葡激酶结合HLA-DR至关重要,因此本文主要研究葡激酶T细胞表位区域18-34关键氨基酸突变对其生物活性的影响,从而筛选一种纤溶活性高、免疫原性低的突变体。 采用QuickChang定点突变PCR法,对葡激酶T细胞表位区域18-34的锚定氨基酸Y24以及它附近的位点进行突变,得到Sak(Y24A)、Sak(Y24V)、Sak(Y24I)、Sak(Y24L)、Sak(V27A)、Sak(N28A)和Sak(V29A)等突变体,并使它们在大肠杆菌DH5a中高效表达,各葡激酶突变体的表达量均占菌体总蛋白量的40%以上,采用阳离子交换层析、分子筛和阴离子交换层析连续三步层析工艺纯化表达产物,结果显示,三步层析纯化后,凝胶扫描显示其纯度均大于95%,HPLC分析其纯度均超过97%。下一步对所得的葡激酶突变体的稳定性、纤溶活性和免疫原性进行了系统研究。 用酪蛋白平板法测定各葡激酶突变体纤溶活性,结果显示Sak(N28A)和Sak(V29A)保持了与Wt-sak相当的纤溶活性(80%以上),Sak(V27A)的纤溶活性降低到了Wt-sak的50%,其余的纤溶活性非常低。将Sak(V27A)、Sak(N28A)和Sak(V29A)免疫小鼠后,用ELISA检测各突变体的特异性抗体水平,发现突变体产生的抗体均显著下降(p0.05),其抗体的生成量与Wt-sak相比分别下降了近13%、16%和10%。Sak(V27A)、Sak(N28A)和Sak(V29A)刺激Balb/c小鼠T细胞增殖的能力与Wt-sak相比均明显减弱。■ 本研究从构建的7个突变体中,筛选到的突变体Sak(N28A)和Sak(V29A)这两个葡激酶突变体既降低了免疫原性又保持了与Wt-sak相当的活性,为进一步构建新型的低免疫原性突变体奠定了基础。
[Abstract]:Natural staphylokinase (staphylokinase, Sak) is a plasminogen activated single-stranded protein synthesized from Staphylococcus aureus. It is composed of 136 amino acids and does not contain disulfide bonds with a molecular weight of 15.5kD. Clinical studies on the treatment of acute myocardial infarction have shown that recombinant staphylokinase (r-Sak) has the same thrombolytic activity as recombinant tissue plasminogen activator (rt-PA) and has higher fibrin specificity. It is a promising thrombolytic drug for its high expression in Escherichia coli and low production cost. But Sak is a bacterial-derived protein that produces a large number of neutralizing antibodies two to three weeks after treatment, leading to immune reactions or even allergic reactions in the patient's body. To some extent, it affects the wide application of staphylokinase in clinic. It is one of the important methods to obtain new low-immunogenic thrombolytic drugs by modifying Sak molecule and removing its antigenic epitopes by site-directed mutation. According to the reported T-cell epitope binding to HLA-DR, there are six regions, 18-34 epitope region, 44-57 epitope region, 73-87 epitope region, 89-99 epitope region, 111-120 epitope region and 125-135 epitope region. The 18-34 epitope region with Y24 as the core amino acid is very important for staphylokinase binding to HLA-DR. Therefore, this paper mainly studies the effect of 18-34 key amino acid mutation in staphylokinase T cell epitope on its biological activity, so as to screen a kind of high fibrinolytic activity. A mutants with low immunogenicity. The anchored amino acid Y24 of staphylokinase T cell epitope 18-34 and its adjacent sites were mutated by QuickChang site-directed mutagenesis PCR. Sak (Y24A), Sak (Y24V), Sak (Y24I), Sak (Y24L), Sak (V27A), Sak (N28A) and Sak (V29A) were obtained, and they were highly expressed in DH5a of Escherichia coli. All the staphylokinase mutants accounted for more than 40% of the total protein. The expressed products were purified by cation exchange chromatography, molecular sieve chromatography and anion exchange chromatography. Gel scanning showed that its purity was higher than 95% and its purity was more than 97% by HPLC. The stability, fibrinolytic activity and immunogenicity of the staphylokinase mutants were studied. The fibrinolytic activity of all staphylokinase mutants was determined by casein plate method. The results showed that Sak (N28A) and Sak (V29A) maintained the same fibrinolytic activity as Wt-sak (more than 80%), Sak (V27A) and decreased the fibrinolytic activity to 50% of Wt-sak, while the other plasminogen activity was very low. After immunizing mice with Sak (V27A), Sak (N28A) and Sak (V29A), the specific antibody levels of each mutant were detected by ELISA. It was found that the antibodies produced by the mutants were significantly decreased (p0.05), and the antibody production of the mutants was decreased by nearly 1316% compared with Wt-sak, and the ability of 10%.Sak (V27A), Sak (N28A) and Sak (V29A) to stimulate the proliferation of T cells in Balb/c mice was significantly decreased compared with Wt-sak. From the seven mutants constructed, The two mutants, Sak (N28A) and Sak (V29A), not only reduced the immunogenicity but also kept the same activity as Wt-sak, which laid the foundation for further construction of new low-immunogenicity mutants.
【学位授予单位】：河北师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
本文编号：2267392
[Abstract]:Natural staphylokinase (staphylokinase, Sak) is a plasminogen activated single-stranded protein synthesized from Staphylococcus aureus. It is composed of 136 amino acids and does not contain disulfide bonds with a molecular weight of 15.5kD. Clinical studies on the treatment of acute myocardial infarction have shown that recombinant staphylokinase (r-Sak) has the same thrombolytic activity as recombinant tissue plasminogen activator (rt-PA) and has higher fibrin specificity. It is a promising thrombolytic drug for its high expression in Escherichia coli and low production cost. But Sak is a bacterial-derived protein that produces a large number of neutralizing antibodies two to three weeks after treatment, leading to immune reactions or even allergic reactions in the patient's body. To some extent, it affects the wide application of staphylokinase in clinic. It is one of the important methods to obtain new low-immunogenic thrombolytic drugs by modifying Sak molecule and removing its antigenic epitopes by site-directed mutation. According to the reported T-cell epitope binding to HLA-DR, there are six regions, 18-34 epitope region, 44-57 epitope region, 73-87 epitope region, 89-99 epitope region, 111-120 epitope region and 125-135 epitope region. The 18-34 epitope region with Y24 as the core amino acid is very important for staphylokinase binding to HLA-DR. Therefore, this paper mainly studies the effect of 18-34 key amino acid mutation in staphylokinase T cell epitope on its biological activity, so as to screen a kind of high fibrinolytic activity. A mutants with low immunogenicity. The anchored amino acid Y24 of staphylokinase T cell epitope 18-34 and its adjacent sites were mutated by QuickChang site-directed mutagenesis PCR. Sak (Y24A), Sak (Y24V), Sak (Y24I), Sak (Y24L), Sak (V27A), Sak (N28A) and Sak (V29A) were obtained, and they were highly expressed in DH5a of Escherichia coli. All the staphylokinase mutants accounted for more than 40% of the total protein. The expressed products were purified by cation exchange chromatography, molecular sieve chromatography and anion exchange chromatography. Gel scanning showed that its purity was higher than 95% and its purity was more than 97% by HPLC. The stability, fibrinolytic activity and immunogenicity of the staphylokinase mutants were studied. The fibrinolytic activity of all staphylokinase mutants was determined by casein plate method. The results showed that Sak (N28A) and Sak (V29A) maintained the same fibrinolytic activity as Wt-sak (more than 80%), Sak (V27A) and decreased the fibrinolytic activity to 50% of Wt-sak, while the other plasminogen activity was very low. After immunizing mice with Sak (V27A), Sak (N28A) and Sak (V29A), the specific antibody levels of each mutant were detected by ELISA. It was found that the antibodies produced by the mutants were significantly decreased (p0.05), and the antibody production of the mutants was decreased by nearly 1316% compared with Wt-sak, and the ability of 10%.Sak (V27A), Sak (N28A) and Sak (V29A) to stimulate the proliferation of T cells in Balb/c mice was significantly decreased compared with Wt-sak. From the seven mutants constructed, The two mutants, Sak (N28A) and Sak (V29A), not only reduced the immunogenicity but also kept the same activity as Wt-sak, which laid the foundation for further construction of new low-immunogenicity mutants.
【学位授予单位】：河北师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
【参考文献】
相关期刊论文 前3条
1 何涛,干荣富;我国医院抗血栓药物使用现状与趋势分析[J];世界临床药物;2005年08期
2 钱志贤,葛雷,钱菊英,樊冰,王灏,周京敏,冯琪,葛均波;急诊PTCA与溶栓治疗急性心肌梗死近期疗效对比研究[J];心血管康复医学杂志;2005年02期
3 叶立文,汤其群,马端,宋后燕;葡激酶突变体基因的克隆与表达[J];药物生物技术;1999年01期
，本文编号：2267392
本文链接：https://www.wllwen.com/xiyixuelunwen/2267392.html
最近更新
教材专著
