葡激酶T細(xì)胞表位區(qū)域18-34關(guān)鍵氨基酸突變對(duì)其生物學(xué)活性的影響
發(fā)布時(shí)間：2018-10-12 19:51
【摘要】：天然葡激酶(staphylokinase, Sak)是一種由金黃色葡萄球菌合成的具有纖溶酶原激活活性的單鏈蛋白質(zhì),由136個(gè)氨基酸所組成,分子中不含二硫鍵,分子量為15.5kD。臨床治療急性心肌梗塞的研究表明,重組葡激酶(r-Sak)具有與重組組織型纖溶酶原激活劑(rt-PA)相當(dāng)?shù)娜芩ɑ钚?且具有更高的纖維蛋白專一性,除此之外,它還能在大腸桿菌中高效表達(dá),生產(chǎn)成本較低,是一種很有應(yīng)用前景的溶栓藥物。但是Sak是一種細(xì)菌來源的蛋白質(zhì),在臨床用藥后的兩到三周會(huì)產(chǎn)生大量的中和性抗體,病人體內(nèi)將產(chǎn)生免疫反應(yīng),甚至發(fā)生過敏反應(yīng),一定程度上影響了葡激酶在臨床上的廣泛應(yīng)用。 用定點(diǎn)突變的方法對(duì)Sak分子進(jìn)行改造,去除其抗原表位,是獲得新型低免疫原性溶栓藥物的重要方法之一。根據(jù)報(bào)道葡激酶的T細(xì)胞表位與HLA-DR結(jié)合的區(qū)域主要有六個(gè),分別是18-34表位區(qū)域、44-57表位區(qū)域、73-87表位區(qū)域、89-99表位區(qū)域、111-120表位區(qū)域和125-135表位區(qū)域。其中以Y24為核心氨基酸的18-34表位區(qū)域?qū)ζ霞っ附Y(jié)合HLA-DR至關(guān)重要,因此本文主要研究葡激酶T細(xì)胞表位區(qū)域18-34關(guān)鍵氨基酸突變對(duì)其生物活性的影響,從而篩選一種纖溶活性高、免疫原性低的突變體。 采用QuickChang定點(diǎn)突變PCR法,對(duì)葡激酶T細(xì)胞表位區(qū)域18-34的錨定氨基酸Y24以及它附近的位點(diǎn)進(jìn)行突變,得到Sak(Y24A)、Sak(Y24V)、Sak(Y24I)、Sak(Y24L)、Sak(V27A)、Sak(N28A)和Sak(V29A)等突變體,并使它們?cè)诖竽c桿菌DH5a中高效表達(dá),各葡激酶突變體的表達(dá)量均占菌體總蛋白量的40%以上,采用陽離子交換層析、分子篩和陰離子交換層析連續(xù)三步層析工藝純化表達(dá)產(chǎn)物,結(jié)果顯示,三步層析純化后,凝膠掃描顯示其純度均大于95%,HPLC分析其純度均超過97%。下一步對(duì)所得的葡激酶突變體的穩(wěn)定性、纖溶活性和免疫原性進(jìn)行了系統(tǒng)研究。 用酪蛋白平板法測(cè)定各葡激酶突變體纖溶活性,結(jié)果顯示Sak(N28A)和Sak(V29A)保持了與Wt-sak相當(dāng)?shù)睦w溶活性(80%以上),Sak(V27A)的纖溶活性降低到了Wt-sak的50%,其余的纖溶活性非常低。將Sak(V27A)、Sak(N28A)和Sak(V29A)免疫小鼠后,用ELISA檢測(cè)各突變體的特異性抗體水平,發(fā)現(xiàn)突變體產(chǎn)生的抗體均顯著下降(p0.05),其抗體的生成量與Wt-sak相比分別下降了近13%、16%和10%。Sak(V27A)、Sak(N28A)和Sak(V29A)刺激Balb/c小鼠T細(xì)胞增殖的能力與Wt-sak相比均明顯減弱�！� 本研究從構(gòu)建的7個(gè)突變體中,篩選到的突變體Sak(N28A)和Sak(V29A)這兩個(gè)葡激酶突變體既降低了免疫原性又保持了與Wt-sak相當(dāng)?shù)幕钚?為進(jìn)一步構(gòu)建新型的低免疫原性突變體奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：河北師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R341
本文編號(hào)：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.
【學(xué)位授予單位】：河北師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R341
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