重组克胰素的基因工程表达及药效学研究
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摘要
第一章重组克胰素的真核表达和鉴定
目的构建克胰素真核表达体系,纯化和鉴定重组克胰素,探索规模化重组克胰素制备技术。
方法根据克胰素的cDNA序列,设计引物,在虎纹蜘蛛毒素的cDNA文库中,用PCR扩增基因,并将目的DNA与载体pVT102U连接构建质粒,将质粒转入酿酒酵母S-78酵母菌,在YPD培养基中培养。培养的菌液经离心、过滤、阳离子交换、反相HPLC分离后冻干保存。通过MALDI—TOF测定分子量、N-末端序列测定以及SDS—PAGE和反相HPLC等分析对表达产物进行鉴定。
结果1、构建目的DNA与载体pVT102U连接的质粒pVT-HW11;
2、获得高效表达重组克胰素的菌种,平均产率约为13 mg/L;
3、建立由离心、过滤、阳离子交换、反相HPLC分离组成的简单可行的纯化方法,产物纯度超过98%;
4、重组克胰素与天然多肽具有相同的分子量和氨基酸序列。
结论成功构建克胰素酿酒酵母S-78酵母菌真核表达体系,表达稳定高效;建立了简单有效的纯化方法;经鉴定重组克胰素与天然克胰素分子量和氨基酸序列相同。重组克胰素具备规模化制备的可能。本研究有开创性和实际应用价值。
第二章重组克胰素的药效学研究
目的研究重组克胰素的生物学活性,探讨其临床应用前景。
方法用酶和特定的底物反应原理,采用分光光度计可以测量产物的生成量。测定重组克胰素和牛胰蛋白酶抑制剂(bovine pancreatictrypsin inhibitor,BPTI)抑制胰蛋白酶、胰凝乳蛋白酶、组织激肽释放酶、凝血酶等丝氨酸蛋白酶的抑制常数,从而衡量其酶抑制活性的强弱。重组克胰素抑制胰蛋白酶的活性极强,无法采用分光光度法准确测定抑制动力学,因此通过等温滴定量热法,从热变化的角度对重组克胰素与胰蛋白酶的结合动力学进行研究,同时也通过这种方法比较了重组克胰素和BPTI的胰蛋白酶抑制能力;构建L—Arg诱导的小鼠急性胰腺炎模型,从生化指标、病理切片方面观察重组克胰素对急性胰腺炎的药效;应用膜片钳技术,研究重组克胰素在全细胞模式下对于大鼠背根神经节(DRG)细胞上的电压门控钾离子通道的作用,包括不同电压下、不同重组克胰素浓度下的抑制效果。
结果1、重组克胰素和BPTI对胰蛋白酶活性均有明显的抑制作用,但重组克胰素对胰凝乳蛋白酶、组织激肽释放酶、凝血酶活性的抑制作用低于BPTI,而对胰蛋白酶活性的抑制作用重组克胰素比BPTI强30倍,是目前发现的较强的胰蛋白酶抑制剂。
2、重组克胰素能明显降低急性胰腺炎小鼠血清淀粉酶和脂肪酶的活性,主要通过对胰蛋白酶的抑制,早期给药可显著降低胰腺高分泌状态及保护自身消化损伤的胰腺细胞,对小鼠急性胰腺炎有明显保护作用,且能明显改善其病理表现。
3、重组克胰素对于电压门控钾离子通道的阻断作用呈现浓度依赖性,10~(-6)M浓度钾电流下降达到饱和(达到最强抑制效果),重组克胰素完全抑制胰蛋白酶活性的浓度为10~(-12)M,提示对电压门控钾离子通道有较弱抑制作用,在发生胰蛋白酶抑制效用时,产生与钾通道药理有关副作用较小。
结论重组克胰素是目前已知胰蛋白酶抑制活性最强的多肽,对胰蛋白酶抑制专一性强,对小鼠急性胰腺炎有保护作用,且能明显改善其病理表现。具有较好的治疗急性胰腺炎等与胰蛋白酶相关疾病的临床应用前景。
PartⅠThe Eukaryotic Expression and Characterization of the Recombinant Trypsin Inhibitor
Objective:To construct the eukaryofic expression system to produce the recombinant trypsin inhibitor;To purify and characterize recombinant peptide and explore its large-scale production techniques.
Methods:The primers were designed according on the cDNA sequence of the trypsin inhibitor.The gene encoding the trypsin inhibitor was amplified from the spider venom cDNAs.The expressing vector of the trypsin inhibitor was constructed by linking the gene with the plasmid pVT102U,and then transfected into the yeast Saccharomyces cerevisiae S-78.After cultured YPD,the yeasts were centrifuged and the supernatant was collected.The recombinant peptide was purified with a combined use of ion exchange chromatography and RP-HPLC.The expressed product was characterized by Edmam degradation,SDS-PAGE and RP-HPLC, and MALDI-TOF.
Results:
1.The expression vector pVT-HW11 was constructed successfully.
2.The trypsin inhibitor could be produced with high yield by using the yeasts S-78 expression system,and the expression yield is about 13 mg/L.
3.The recombinant peptide could be purified by a combined use of centrifugation,filtration,ion exchange chromatography and RP-HPLC.The purity of the recombinant peptide was over 98%.
4.The recombinant peptide has the same molecular weight and amino acid sequence as natural one.
Conclusion:The eukaryotic expression system of yeast Saccharomyces cerevisiae S-78 for recombinant trypsin inhibitor was successfully constructed and a simple and effective purification method was established.The recombinant peptide has the same molecule weight and amino acid sequence with its natural one.By using the method established in this study,the trypsin inhibitor would be produced with large-scale.Therefore,the present research may be innovative and has the value of practical application.
PartⅡThe Pharmacodynamic Study of the Recombinant Trypsin Inhibitor
Objective:To inestigate the pharmacodynamic activities of the recombinant trypsin inhibitor and explore its prospect of clinical application
Methods:Firstly,the inhibition constants of the recombinant peptide and BPTI against serine proteinases such as trypsin,chymotrypsin,kallikrein and thrombin,were assayed by analyzing the reaction of the enzyme with the relative substrate with spectrophotometer.As the inhibition of recombinant peptide against trypsin was very potent,spectrophotometric method could hardly be used to evaluate its inhibition dynmics.Therefore Isothermal Titration Calorimetry(ITC) was applied,in which the binding dynamics of recombinant peptide and trypsin was studied by thermodynamic changes.To construct a L-Arg-induced acute necrosis pancreatitis in mice model in which therapeutic effect of recombinant peptide upon acute necrosis pancreatitis could be studied from biochemical indicators and pathological sections.The inhibition of the recombinant peptide against voltage-gated potassium channels was studied by whole cell clamp recordings on rat dorsal root ganglion(DRG) neurons,in which the inhibition effects were examined under variant voltage and different concentration of recombinant peptide.
Results:
1.Both recombinant trypsin inhibitor and BPTI have potent inhibition effect on the activity of trypsin.Though recombinant peptide had lower inhibition effect upon the activities of chymotrypsin,kallikrein and thrombin than BPTI,it has 30 times stronger inhibition effect on the activity of trypsin than BPTI and could be one of the most potent inhibitors against trypsin indentified so far.
2.With the inhibition against trypsin,the recombinant peptide could potently suppress the activity of amylase and lipase in the plasma of the mice with acute necrosis pancreatitis.Earlier medication could markedly reduce high secrete of pancreas and protect the pancreas cell from digestion injuries with the result of good protection for the acute necrosis pancreatitis in mice and great improvement of pathological symptom.
3.The blocking function on the voltage-gated potassium channels of the recombinant peptide was concentration-dependant.With the concentration of 10~(-6)M,potassium current could not be further reduced and were saturated,which means the strongest inhibitation was achieved.The concentration of 10~(-12)M was the one which recombinant peptide completely inhibitated the activity of trypsin, which indicated that it had weaker inhibition effect on voltage-gated potassium channels.And when the inhibitation against trypsin happened,the side effects in relation to the pharmacology of potassium channels were very low.
Conclusion:The recombinant trypsin inhibitor is the most potent peptide inhibitor against trypsin identified so far.It has unique biological function that inhibits trypsin.It has potent protection from acute necrosis pancreatitis in mice and markedly improves its pathological symptoms. The recombinant trypsin inhibitor has promising clinical application for the better treatment of acute necrosis pancreatitis and trypsin-related diseases.
目的构建克胰素真核表达体系,纯化和鉴定重组克胰素,探索规模化重组克胰素制备技术。
方法根据克胰素的cDNA序列,设计引物,在虎纹蜘蛛毒素的cDNA文库中,用PCR扩增基因,并将目的DNA与载体pVT102U连接构建质粒,将质粒转入酿酒酵母S-78酵母菌,在YPD培养基中培养。培养的菌液经离心、过滤、阳离子交换、反相HPLC分离后冻干保存。通过MALDI—TOF测定分子量、N-末端序列测定以及SDS—PAGE和反相HPLC等分析对表达产物进行鉴定。
结果1、构建目的DNA与载体pVT102U连接的质粒pVT-HW11;
2、获得高效表达重组克胰素的菌种,平均产率约为13 mg/L;
3、建立由离心、过滤、阳离子交换、反相HPLC分离组成的简单可行的纯化方法,产物纯度超过98%;
4、重组克胰素与天然多肽具有相同的分子量和氨基酸序列。
结论成功构建克胰素酿酒酵母S-78酵母菌真核表达体系,表达稳定高效;建立了简单有效的纯化方法;经鉴定重组克胰素与天然克胰素分子量和氨基酸序列相同。重组克胰素具备规模化制备的可能。本研究有开创性和实际应用价值。
第二章重组克胰素的药效学研究
目的研究重组克胰素的生物学活性,探讨其临床应用前景。
方法用酶和特定的底物反应原理,采用分光光度计可以测量产物的生成量。测定重组克胰素和牛胰蛋白酶抑制剂(bovine pancreatictrypsin inhibitor,BPTI)抑制胰蛋白酶、胰凝乳蛋白酶、组织激肽释放酶、凝血酶等丝氨酸蛋白酶的抑制常数,从而衡量其酶抑制活性的强弱。重组克胰素抑制胰蛋白酶的活性极强,无法采用分光光度法准确测定抑制动力学,因此通过等温滴定量热法,从热变化的角度对重组克胰素与胰蛋白酶的结合动力学进行研究,同时也通过这种方法比较了重组克胰素和BPTI的胰蛋白酶抑制能力;构建L—Arg诱导的小鼠急性胰腺炎模型,从生化指标、病理切片方面观察重组克胰素对急性胰腺炎的药效;应用膜片钳技术,研究重组克胰素在全细胞模式下对于大鼠背根神经节(DRG)细胞上的电压门控钾离子通道的作用,包括不同电压下、不同重组克胰素浓度下的抑制效果。
结果1、重组克胰素和BPTI对胰蛋白酶活性均有明显的抑制作用,但重组克胰素对胰凝乳蛋白酶、组织激肽释放酶、凝血酶活性的抑制作用低于BPTI,而对胰蛋白酶活性的抑制作用重组克胰素比BPTI强30倍,是目前发现的较强的胰蛋白酶抑制剂。
2、重组克胰素能明显降低急性胰腺炎小鼠血清淀粉酶和脂肪酶的活性,主要通过对胰蛋白酶的抑制,早期给药可显著降低胰腺高分泌状态及保护自身消化损伤的胰腺细胞,对小鼠急性胰腺炎有明显保护作用,且能明显改善其病理表现。
3、重组克胰素对于电压门控钾离子通道的阻断作用呈现浓度依赖性,10~(-6)M浓度钾电流下降达到饱和(达到最强抑制效果),重组克胰素完全抑制胰蛋白酶活性的浓度为10~(-12)M,提示对电压门控钾离子通道有较弱抑制作用,在发生胰蛋白酶抑制效用时,产生与钾通道药理有关副作用较小。
结论重组克胰素是目前已知胰蛋白酶抑制活性最强的多肽,对胰蛋白酶抑制专一性强,对小鼠急性胰腺炎有保护作用,且能明显改善其病理表现。具有较好的治疗急性胰腺炎等与胰蛋白酶相关疾病的临床应用前景。
PartⅠThe Eukaryotic Expression and Characterization of the Recombinant Trypsin Inhibitor
Objective:To construct the eukaryofic expression system to produce the recombinant trypsin inhibitor;To purify and characterize recombinant peptide and explore its large-scale production techniques.
Methods:The primers were designed according on the cDNA sequence of the trypsin inhibitor.The gene encoding the trypsin inhibitor was amplified from the spider venom cDNAs.The expressing vector of the trypsin inhibitor was constructed by linking the gene with the plasmid pVT102U,and then transfected into the yeast Saccharomyces cerevisiae S-78.After cultured YPD,the yeasts were centrifuged and the supernatant was collected.The recombinant peptide was purified with a combined use of ion exchange chromatography and RP-HPLC.The expressed product was characterized by Edmam degradation,SDS-PAGE and RP-HPLC, and MALDI-TOF.
Results:
1.The expression vector pVT-HW11 was constructed successfully.
2.The trypsin inhibitor could be produced with high yield by using the yeasts S-78 expression system,and the expression yield is about 13 mg/L.
3.The recombinant peptide could be purified by a combined use of centrifugation,filtration,ion exchange chromatography and RP-HPLC.The purity of the recombinant peptide was over 98%.
4.The recombinant peptide has the same molecular weight and amino acid sequence as natural one.
Conclusion:The eukaryotic expression system of yeast Saccharomyces cerevisiae S-78 for recombinant trypsin inhibitor was successfully constructed and a simple and effective purification method was established.The recombinant peptide has the same molecule weight and amino acid sequence with its natural one.By using the method established in this study,the trypsin inhibitor would be produced with large-scale.Therefore,the present research may be innovative and has the value of practical application.
PartⅡThe Pharmacodynamic Study of the Recombinant Trypsin Inhibitor
Objective:To inestigate the pharmacodynamic activities of the recombinant trypsin inhibitor and explore its prospect of clinical application
Methods:Firstly,the inhibition constants of the recombinant peptide and BPTI against serine proteinases such as trypsin,chymotrypsin,kallikrein and thrombin,were assayed by analyzing the reaction of the enzyme with the relative substrate with spectrophotometer.As the inhibition of recombinant peptide against trypsin was very potent,spectrophotometric method could hardly be used to evaluate its inhibition dynmics.Therefore Isothermal Titration Calorimetry(ITC) was applied,in which the binding dynamics of recombinant peptide and trypsin was studied by thermodynamic changes.To construct a L-Arg-induced acute necrosis pancreatitis in mice model in which therapeutic effect of recombinant peptide upon acute necrosis pancreatitis could be studied from biochemical indicators and pathological sections.The inhibition of the recombinant peptide against voltage-gated potassium channels was studied by whole cell clamp recordings on rat dorsal root ganglion(DRG) neurons,in which the inhibition effects were examined under variant voltage and different concentration of recombinant peptide.
Results:
1.Both recombinant trypsin inhibitor and BPTI have potent inhibition effect on the activity of trypsin.Though recombinant peptide had lower inhibition effect upon the activities of chymotrypsin,kallikrein and thrombin than BPTI,it has 30 times stronger inhibition effect on the activity of trypsin than BPTI and could be one of the most potent inhibitors against trypsin indentified so far.
2.With the inhibition against trypsin,the recombinant peptide could potently suppress the activity of amylase and lipase in the plasma of the mice with acute necrosis pancreatitis.Earlier medication could markedly reduce high secrete of pancreas and protect the pancreas cell from digestion injuries with the result of good protection for the acute necrosis pancreatitis in mice and great improvement of pathological symptom.
3.The blocking function on the voltage-gated potassium channels of the recombinant peptide was concentration-dependant.With the concentration of 10~(-6)M,potassium current could not be further reduced and were saturated,which means the strongest inhibitation was achieved.The concentration of 10~(-12)M was the one which recombinant peptide completely inhibitated the activity of trypsin, which indicated that it had weaker inhibition effect on voltage-gated potassium channels.And when the inhibitation against trypsin happened,the side effects in relation to the pharmacology of potassium channels were very low.
Conclusion:The recombinant trypsin inhibitor is the most potent peptide inhibitor against trypsin identified so far.It has unique biological function that inhibits trypsin.It has potent protection from acute necrosis pancreatitis in mice and markedly improves its pathological symptoms. The recombinant trypsin inhibitor has promising clinical application for the better treatment of acute necrosis pancreatitis and trypsin-related diseases.
引文
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[1].Corzo G,Escoubas P.Pharmacologically active spider peptide toxins.Cell Mol Life Sci,2003,60:2409-2426
[2].Sollod BL,Wilson D,Zhaxybayeva O,Gogarten JP,Drinkwater R,King GF,Were arachnids the first to use combinatorial peptide libraries? Peptides,2005,26:131-139
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