基因工程菌株里氏木霉合成t-PA发酵条件及r-PA基因在甲醇毕赤酵母中表达的研究
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摘要
论文首先以基因工程菌株里氏木霉(Trichoderma reesei)为研究对象,对其生物合成组织型纤溶酶原激活剂(tissue-type plasminogen activator,t-PA)的发酵条件和发酵动力学进行了研究,并对里氏木霉重组t-PA的分离纯化及其性质进行了探索;然后进行了t-PA突变体的研究,获得了t-PA突变体r-PA基因,并在甲醇毕赤酵母(Pichia methanolica)中进行了表达。
采用稀释平板分离法对基因工程菌株Trichoderma reesei进行了分离纯化,对平板上生长良好的单菌落进行摇瓶液态发酵,以t-PA的酶活力为指标,挑选高产菌株,选得的Trichoderma reesei(8)号菌株t-PA产量最高,且遗传稳定性良好。
对Trichoderma reesei(8)生物合成组织型纤溶酶原激活剂(t-PA)的代谢调节机制进行了研究。在基础发酵条件下,L-山梨糖、D-蔗糖、可溶性纤维素、CMC、麦芽糖、乳糖和棉子糖等单糖及多糖对t-PA生物合成都有诱导作用,其中L-山梨糖的诱导效果最好,加量以1.0%为宜。葡萄糖及其中间代谢产物对t-PA的生物合成产生分解代谢物阻遏作用。纤维二糖在低浓度时可以促进t-PA的生物合成而在高浓度时对t-PA生物合成起反馈阻遏作用。
应用单因素和正交设计试验法,确定了种子培养基的最佳组成和最佳培养条件。应用正交设计试验法和响应面分析试验法优化出摇瓶分批发酵培养基的最佳组成。通过研究不同发酵条件对t-PA生物合成的影响,获得了最佳摇瓶分批发酵培养条件。优化条件与初始条件的摇瓶分批发酵比较试验结果表明,Trichoderma reesei(8)菌株在优化条件下的t-PA产量(3275.26U/mL)比初始条件下(1.14U/mL)显著提高。
对5L发酵罐上的分批发酵过程进行了分析。选择了适宜发酵条件并以5L发酵罐分批发酵试验数据为依据,采用MATLAB工具软件,利用全局收敛的修正的高斯-牛顿法算法,以误差平方和最小为目标,获得发酵动力学方程待估参数,建立了发酵动力学模型。通过拟合分析证明得到的数学模型误差较小,能较好的反映t-PA的分批发酵过程。
对里氏木霉重组t-PA的分离纯化方法进行了研究,建立了里氏木霉重组t-PA的分离提纯工艺。通过超滤、硫酸铵盐析、Butyl Sepharose 4 F F疏水层析、Sephadex G-25凝胶过滤脱盐、Q-Sepharose F F离子交换层析分离等分离步骤,里氏木霉重组t-PA的比活力由347.78U/mg提高到10619.58U/mg,提纯倍数30.54,活力回收率2.63%。通过SDS-PAGE电泳对各步纯化后样品进行分析,经上述各步将里氏木霉重组t-PA分离为电泳纯。
通过SDS-PAGE纤维蛋白自显影证明了Trichoderma reesei(8)的发酵液中
The dissertation focuses on the fermentation conditions, the fermentation kinetics, the purification and the enzyme characteristics of tissue-type plasminogen activator(t-PA) produced by recombinant Trichoderma reesei.The clone of r-PA cDNA and expression in Pichia methanolica was also investigated.Recombinant Trichoderma reesei strain was screened through dilution-plate method. With t-PA activity as target ,the high-producing Trichoderma reesei (8) with highly genetic stability was selected by shaking flask liquid cultivation of the strains from single colony on plate.The metabolic control mechanism of tissue-type plasminogen activator(t-PA) produced by recombinant Trichoderma reesei was studied.Under the minimal fermentation conditions,the saccharides including sucrose,carboxymethyl cellulose, raffinose,L-sorbose ,lactose ,dissolubility cellulose and maltose could induce the t-PA production. L-sorbose is the best inducer when its concentration is 1.0%. Glucose and its metabolites could produce catabolite repression . Cellobiose could promote the biosynthesis of t-PA when its concentration is low and could produce feedback repression when its concentration is high.The seed medium composition and culture conditions were optimized by single factor experiment and orthogonal experiment. The shaking flask batch fermentation medium composition and conditions were also determined through orthogonal experiment and response surface methodology.The maximum t-PA yield was 3275.26 U/mL,and was improved by thousand times as great as 1.14 U/mL in original fermentation conditions.Based on the optimal conditions obtained in shaking flask, the batch fermentation was performed in 5-liter fermentor. The batch fermentation kinetics of t-PA produced by recombinant Trichoderma reesei were studied based on the experimental data from the fermentation in 5-liter fermentor. The kinetic models of fermentation were established by MATLAB software.Purification process of recombinant t-PA from Trichoderma reesei (8) was explored and established.The recombinant t-PA was purified using ultrafiltration, salting-out, Butyl-Sepharose 4FF hydrophobic interaction chromatography, Sephadex G-25 gel filtration chromatography and Q-Sepharose FF ion exchange chromatography.The purified t-PA was homogeneous examined by SDS-PAGE electrophoresis.The recombinant t-PA was analysed by SDS-PAGE fibrin autography technique .The results showed that there were two kinds of hydrolysis fibrin products in the broth of Trichoderma reesei(8)with 6.6×10~4 and 3.3×10~4 molecular weight respectively.The fibrinolytic characteristic of the recombinant t-PA was also analysed by negative and positive plates,which revealed the t-PA only activated plasminogen to plasmin.there wasnot hydrolysis fibrin product in the broth of host. The preservation
time (4°C) and the freezing-thawing times have much influence on the enzyme activity.The longer preservation time and the more freezing-thawing times ,the more enzyme activity lost. The optimum temperature of the crude and pure recombinant t-PA were 37°C and 45 °C respectively. The optimum pH of the recombinant t-PA was 9.4. The recombinant t-PA was stable in the pH range of 6.4~~8.4 at 37 "C.The isoelectric point of the recombinant t-PA was 3.82 estimated by isoelectrofocusing electrophoresis.The preparation methods of recombinant Trichoderma reesei chromosome DNA were studied.Three kinds of methods of freezing-grinding-CTAB, freezing-grinding-SDS and chloride benzyl-SDS were compared and DNA were detected by agarose gel electrophoresis.The result showed that freezing-grinding -CTAB was a better method.The preparation conditions of the method were optimized.The chromosome DNA prepared by this method meet the requirements of PCR and other molecular biological manipulation.According to the published sequence of r-PA gene, a pair of primers which can amplify the r-PA region was designed. The r-PA gene was amplified by PCR with recombinant Trichoderma reesei chromosome DNA as template, then the r-PA gene was inserted into pMD18-T vector. The recombinant plasmid was used to transform the competent E.coli DH-5a cells. The transformed cells were spread on LB agar plates with ampicillin,X-gal and IPTG to isolation of recombinant strains according to blue-white reaction. A positive clone harboring r-PA gene was identified by restriction enzyme analysis and PCR technique. Collecting recombinant plasmid pMD18-r-PA and sequencing the recombinant plasmid,the sequencing result showed that the cloned 1.1kb fragment of r-PA sequence homology was 99.91% with the r-PA gene published , the amino acid sequence was same as that published.The secreted expression plasmid pMETaA-r-PA of Pichia methanolica was constructed and digested with Pac I and transformed into Pichia methanolica PMAD16 by electroporation.The method of high efficient electrotransformation for pMETotA-r-PA to Pichia methanolica was established.The positive transformants which integrated r-PA gene in their genomes were obtained by screening on MD plates and identified by PCR technique.The Mut phenotypes of these transformants were Muts.Under shake-flask culture and induced using methanol as a carbon source, the extracellular r-PA reached the largest activity of 1675.62U/mL at 72h. The expression products were analyzed by SDS-PAGE.The results indicated that the r-PA from Pichia methanolica PMAD16 was secreted into broth and not glycosylated with 3.96 X 104 molecular weight.
采用稀释平板分离法对基因工程菌株Trichoderma reesei进行了分离纯化,对平板上生长良好的单菌落进行摇瓶液态发酵,以t-PA的酶活力为指标,挑选高产菌株,选得的Trichoderma reesei(8)号菌株t-PA产量最高,且遗传稳定性良好。
对Trichoderma reesei(8)生物合成组织型纤溶酶原激活剂(t-PA)的代谢调节机制进行了研究。在基础发酵条件下,L-山梨糖、D-蔗糖、可溶性纤维素、CMC、麦芽糖、乳糖和棉子糖等单糖及多糖对t-PA生物合成都有诱导作用,其中L-山梨糖的诱导效果最好,加量以1.0%为宜。葡萄糖及其中间代谢产物对t-PA的生物合成产生分解代谢物阻遏作用。纤维二糖在低浓度时可以促进t-PA的生物合成而在高浓度时对t-PA生物合成起反馈阻遏作用。
应用单因素和正交设计试验法,确定了种子培养基的最佳组成和最佳培养条件。应用正交设计试验法和响应面分析试验法优化出摇瓶分批发酵培养基的最佳组成。通过研究不同发酵条件对t-PA生物合成的影响,获得了最佳摇瓶分批发酵培养条件。优化条件与初始条件的摇瓶分批发酵比较试验结果表明,Trichoderma reesei(8)菌株在优化条件下的t-PA产量(3275.26U/mL)比初始条件下(1.14U/mL)显著提高。
对5L发酵罐上的分批发酵过程进行了分析。选择了适宜发酵条件并以5L发酵罐分批发酵试验数据为依据,采用MATLAB工具软件,利用全局收敛的修正的高斯-牛顿法算法,以误差平方和最小为目标,获得发酵动力学方程待估参数,建立了发酵动力学模型。通过拟合分析证明得到的数学模型误差较小,能较好的反映t-PA的分批发酵过程。
对里氏木霉重组t-PA的分离纯化方法进行了研究,建立了里氏木霉重组t-PA的分离提纯工艺。通过超滤、硫酸铵盐析、Butyl Sepharose 4 F F疏水层析、Sephadex G-25凝胶过滤脱盐、Q-Sepharose F F离子交换层析分离等分离步骤,里氏木霉重组t-PA的比活力由347.78U/mg提高到10619.58U/mg,提纯倍数30.54,活力回收率2.63%。通过SDS-PAGE电泳对各步纯化后样品进行分析,经上述各步将里氏木霉重组t-PA分离为电泳纯。
通过SDS-PAGE纤维蛋白自显影证明了Trichoderma reesei(8)的发酵液中
The dissertation focuses on the fermentation conditions, the fermentation kinetics, the purification and the enzyme characteristics of tissue-type plasminogen activator(t-PA) produced by recombinant Trichoderma reesei.The clone of r-PA cDNA and expression in Pichia methanolica was also investigated.Recombinant Trichoderma reesei strain was screened through dilution-plate method. With t-PA activity as target ,the high-producing Trichoderma reesei (8) with highly genetic stability was selected by shaking flask liquid cultivation of the strains from single colony on plate.The metabolic control mechanism of tissue-type plasminogen activator(t-PA) produced by recombinant Trichoderma reesei was studied.Under the minimal fermentation conditions,the saccharides including sucrose,carboxymethyl cellulose, raffinose,L-sorbose ,lactose ,dissolubility cellulose and maltose could induce the t-PA production. L-sorbose is the best inducer when its concentration is 1.0%. Glucose and its metabolites could produce catabolite repression . Cellobiose could promote the biosynthesis of t-PA when its concentration is low and could produce feedback repression when its concentration is high.The seed medium composition and culture conditions were optimized by single factor experiment and orthogonal experiment. The shaking flask batch fermentation medium composition and conditions were also determined through orthogonal experiment and response surface methodology.The maximum t-PA yield was 3275.26 U/mL,and was improved by thousand times as great as 1.14 U/mL in original fermentation conditions.Based on the optimal conditions obtained in shaking flask, the batch fermentation was performed in 5-liter fermentor. The batch fermentation kinetics of t-PA produced by recombinant Trichoderma reesei were studied based on the experimental data from the fermentation in 5-liter fermentor. The kinetic models of fermentation were established by MATLAB software.Purification process of recombinant t-PA from Trichoderma reesei (8) was explored and established.The recombinant t-PA was purified using ultrafiltration, salting-out, Butyl-Sepharose 4FF hydrophobic interaction chromatography, Sephadex G-25 gel filtration chromatography and Q-Sepharose FF ion exchange chromatography.The purified t-PA was homogeneous examined by SDS-PAGE electrophoresis.The recombinant t-PA was analysed by SDS-PAGE fibrin autography technique .The results showed that there were two kinds of hydrolysis fibrin products in the broth of Trichoderma reesei(8)with 6.6×10~4 and 3.3×10~4 molecular weight respectively.The fibrinolytic characteristic of the recombinant t-PA was also analysed by negative and positive plates,which revealed the t-PA only activated plasminogen to plasmin.there wasnot hydrolysis fibrin product in the broth of host. The preservation
time (4°C) and the freezing-thawing times have much influence on the enzyme activity.The longer preservation time and the more freezing-thawing times ,the more enzyme activity lost. The optimum temperature of the crude and pure recombinant t-PA were 37°C and 45 °C respectively. The optimum pH of the recombinant t-PA was 9.4. The recombinant t-PA was stable in the pH range of 6.4~~8.4 at 37 "C.The isoelectric point of the recombinant t-PA was 3.82 estimated by isoelectrofocusing electrophoresis.The preparation methods of recombinant Trichoderma reesei chromosome DNA were studied.Three kinds of methods of freezing-grinding-CTAB, freezing-grinding-SDS and chloride benzyl-SDS were compared and DNA were detected by agarose gel electrophoresis.The result showed that freezing-grinding -CTAB was a better method.The preparation conditions of the method were optimized.The chromosome DNA prepared by this method meet the requirements of PCR and other molecular biological manipulation.According to the published sequence of r-PA gene, a pair of primers which can amplify the r-PA region was designed. The r-PA gene was amplified by PCR with recombinant Trichoderma reesei chromosome DNA as template, then the r-PA gene was inserted into pMD18-T vector. The recombinant plasmid was used to transform the competent E.coli DH-5a cells. The transformed cells were spread on LB agar plates with ampicillin,X-gal and IPTG to isolation of recombinant strains according to blue-white reaction. A positive clone harboring r-PA gene was identified by restriction enzyme analysis and PCR technique. Collecting recombinant plasmid pMD18-r-PA and sequencing the recombinant plasmid,the sequencing result showed that the cloned 1.1kb fragment of r-PA sequence homology was 99.91% with the r-PA gene published , the amino acid sequence was same as that published.The secreted expression plasmid pMETaA-r-PA of Pichia methanolica was constructed and digested with Pac I and transformed into Pichia methanolica PMAD16 by electroporation.The method of high efficient electrotransformation for pMETotA-r-PA to Pichia methanolica was established.The positive transformants which integrated r-PA gene in their genomes were obtained by screening on MD plates and identified by PCR technique.The Mut phenotypes of these transformants were Muts.Under shake-flask culture and induced using methanol as a carbon source, the extracellular r-PA reached the largest activity of 1675.62U/mL at 72h. The expression products were analyzed by SDS-PAGE.The results indicated that the r-PA from Pichia methanolica PMAD16 was secreted into broth and not glycosylated with 3.96 X 104 molecular weight.
引文
[1] 马大龙主编.生物技术药物.北京:科学出版社,2001,2:29-41.
[2] Kenneth Ouriel. Level and Current status of Thrombolysis for Peripheral Arterial OcclusiveDisease.Analysis of vascular surgery,2004,799-801
[3] 褚志义主编.生物合成药物学.北京:化学工业出版社,1999,4:787—792
[4] Lewandowski C, Barsan W. Treament of acute ischemic stroke. An Emerg Med,2001,37(2):202-206
[5] Zoppo G J. Antithrombotic treatments in acute ischemic stroke.Curt Opin Hematol,2000,46:261-265
[6] 李敏,扈荣良,王玉炯等.重组纤溶酶原激活剂及其突变体的表达研究.宁夏大学学报2002,23(1):71-75
[7] Pohl G, et al.Biochemistry,1984,23:3701-3701
[8] Smith B O, Downing A K, Driscoll P C,et al. The solution structure and backbone dynamics of the fibronectin type I and epidermal growth factor-like pair of modules of tissue-type plasminogen activator. Structure,1995,3(8):823
[9] Shoichi A, Norihide S, Msnabu S, et al. Influence of sugar chain on fibrin affinity of recombinant t-PA. Biol Pharm Bu11,2001,24(3):295
[10] Shoichi A,Norihide S,Msnabu S,et al.lnfluence of sugar chain on fibrin affinity of recombinant t-PA.Bio Pharm Bull,2001,24(3):295
[11] Rijken D C, et al. Measurement of human tissue type plasminogen activator by a two-sites immunoradiometric assay. J. Lab.Clin. Med., 1983, 101:274-284
[12] 王鸿利,王学锋.血栓病临床新技术.北京:人民军医出版社,2003
[13] Ny T, et al.Proc Nati Acad Sci USA,1983,80:349
[14] Browne M J, et al. Gene,1985,33:279
[15] Sandra J,et al. J. Biol Chem, 1986,261:6972
[16] 钱民章,宋后燕.人组织型纤溶酶原激活剂(t-PA)基因.生物工程学报.1998,4(1):1
[17] Pennica D ,EW Holmes, JW Kohr, et al .Cloning and expression of human t-PA cDNA in E. coli. Nature, 1983, 301:214-221
[18] Gamani C,Gavin O,Bertossa C,et al.Studies on the effect of fucosylated and non-fucosylated finger/growth factor constructs on the clearance of tissue-type plasminogen activator mediated by the Iow-density-lipoprotein-receptor-related protein. Eur J Biochem.,1998,251:804
[19] 焦建伟,茹炳根.溶栓剂研究的新进展,生物工程进展,2002,22:30-33
[20] Lamba B, Bauer M, Huber R, et al. The 2.3A Crystal Structute of the Catalytic Domain of Recombinant Two chain Human Tissue-type Plasminogen Activator.J Mol Bio1,1996,258:117-135
[21] Renatus M, Engh R A,Stubbs M T. Lysine 156 Promotes the Anomalous Proenzyme Activity of tPA:Crystal Structure of Single-chain Human tPA. EMBO J, 1997,16:4797-4805
[22] Tachias K, Madison E L.Converting Tissue-type Plasminogen Activator into a Zymogen.J Biol Chem, 1996,271:28749-28752
[23] Tachias K, Madison E L. Vsriants of Tissue-type Plasminogen Activator Which Display Substantially Enhanced Stimulation by Fibrin.J.Boil Chem, 1995,270:18319-18322
[24] Ke S-H,Tachias K,Lamba D,et al.ldentification of a Hydrophobic Exosite on Tissue Type Plasminogen Activator That Modulates Specificity for Plasminogen.J Biol Chem,1997,272:1811-1816
[25] Toschi L,BringmannP, Petri T, et al.Fibrin Selectivity of the Isolated Protease Domains of Tissue-type and Vampire Bat Salivary Gland Plasminogen Activators. Eur J Biochem, 1998,252:108-112
[26] Yamada T, Shimada, Y, Kikuchi M.Integrin-specific Tissue-type Plasminogen Activator Engineered by Introduction of the Arg-Gly-Asp Sepuence. Biochem Biophys Res Commun, 1996,228:306-311
[27] Smith J W, Tachias K, Madison E L. Protein Loop Grafting to Construct a Variant of Tissue-type Plasminogen Activator That Binds Platelet Integrin Alpha II b Beta 3. J. Biol Chern, 1995,270:30486-30490
[28] Noble S,Mctavish D.A review of its pharmacological properties and clinical efficacy in the management of acute myocardial infarction.Drug Evaluation, 1996,52:589
[29] 周红.第三代溶栓剂的作用特点及其研究现状.国外医学心血管分册,2000,5:274-276
[30] Den Heijer R,Vermeer F, Ambrosiono E,et al. Evaluation of a weight-adjusted single-bolus plasminogen activator inpatients with myocardial Infarction. Circulation, 1998,98:2117
[31] Tucker H M, Gerard R D. Sepuence Requirements in the Reactive-center Loop of Plasminogen-activator Inhibitor-1 for Recognition of Plasminogen Activators.Eur J Biochem,1996,237:180-187
[32] Tachias K,Madison E L. Variants of Tissue-type Plasminogen Activator That Display xtraordinary Resistance to Inhibition by the Serpin Plasminogen Activator Inhibitor Type l.J Biol Chem,1997,272:14580-14585
[33] Modi N B, Eppler S,Breed J,et al. Pharmacokinetics of a slower clearing tissue plasminogen activator variant TNK-t-PA in patients with acute myocardial imfarction. Thromb Haemostas, 1998,79:134
[34] 刘士辉等.半衰期延获得PAI-1抗性的突变体构建、表达及特性分析.生物工程学报,1995,11(1):13-19
[35] 赵庆国等.组织型纤溶酶原激活剂(t-PA)突变体在CHO细胞中表达产物的纯化.解放军药学学报,2000,16(3):117-119
[36] Hua Z.C.Renaturation and purification of recombinant tissue -plasminogen activator expressed in E. coli .Biochem. Mol. Biol. International , 1997,41(4):815
[37] Sarmientos P, Duchesns M ,Deneffe P ,et al .Synthesis and purification of active human tissue plasminogen activator from Escherichia coli. Bio/Technology, 1989,7:495
[38] Harris TJR ,Patel T ,Marston FAO, et al .Cloning of cDNA coding for human tissue-type plasminogen activator and its expression in Escherichia coli. Mol Biol Med, 1986,3:279
[39] Geunfeld H . Effector-assisted refolding of recombinant tissue -plasminogen activator producing in Escherichia coli .Applied Biochem. Biotech., 1992,33(2):117
[40] 夏焱,华子春,陈晓春等.人组织型纤溶酶原激活剂在大肠杆菌中的表达、复性及分离纯化.南京大学学报,1994,30(4):627-632
[41] 熊凌霜,黄培堂,董陆佳等.组织型纤溶酶原激活剂cDNA在大肠杆菌中表达的研究.军事医学科学院院刊,1994,18(4):249-252
[42] http://www. Centocor. Com/or http/www. Boehringer-ingelheim.Com/
[43] Martin U,Mollendorff E,Akpan W, et al.Dose-ranging study of the novel recombinant plasminogen activator BM 06.022 in healthy volunteers.J.Clin Pharmacol Ther, 1991,50(4):429
[44] 苏勇,易进华,李军等.人组织型纤溶酶原激活物衍生物在大肠杆菌硫氧化还原蛋白融合表达系统中的表达.生物工程进展,2000,6:63-67
[45] 沉毅珺,李军,易进华等.重组人细织型纤溶酶原激活物衍生物住大肠杆菌中表达的研究.药物生物技术,2001,8(6):306—309
[46] 廖建民,张瑾,沈子龙.组织型纤溶酶原激活剂突变体Reteplase(r-PA)基因的克隆及在大肠杆菌中的表达.药物生物技术,2002,9(2):95—98
[47] Lucas B K ,Giere L M ,DeMarco R A, et al .High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector .Nucleic Acids Res., 1996,24(9):1774-1779
[48] 欧阳应斌,黄培堂,徐秀英等.t-PA cDNA在CHO细胞中的高效表达.生物技术通讯,1995,6:11-15
[49] Fussengger M ,Bailey J E ,Hauser H, et al .Genetic optimization of recombinanl glycoprotein production by mammalian cells .Trends Biotechnol , 1999,17(1):35-42
[50] Khan M W ,Musgrave S C ,Jenkins N, et al .N-linked glycosylation of tissue plasminogen activator in Namalwa cells .Biochem. Soc. Trans, 1995,23(1):99
[51] Lo K M,Gillies S D.High level expression of human proteins in murine hybridome cells:induction by methotrexate in the absence of gene amplification.Biochem.Biophys.Acta, 1991,1088:217-224
[52] Traunecker A ,Oliveri F ,Karjalainen K .Myeloma based expression system for production of large mammalian proteins .Trends Biotechnol , 1991,9(4):109-113
[53] Werner R G, Noe W, Kopp K, et al. Appropriate mammalian expression system .Arzeim-Forsch/Drug Res., 1998,48(8):870-880
[54] Romanos M A ,Scorer C A ,Clare J, et al.J. Yeast ,1992,8:423-488
[55] Cregg J M ,Vedvick T S ,Raschke W C .Recent advances in expression of foreign genes in Pichia pastoris .Biotechnology, 1993,11 (8):905-910
[56] Ellis S B,Brust P F, Koutz P J,et al.lsolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris.Mol Cell Biol, 1985,5:1111
[57] Cregg J M,Barringer K J,Hessler A Y, et al. Pichia pastoris as a host system for transformations. Mol .Cell Biol,1985,5:3376
[58] Cereghino J L,Cregg J M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiol Rev,2000,24:45
[59] Sreekrishna K,Brankamp R G,Kropp K E,et al.Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.Gene, 1997,190:55
[60] 涂宣林,朱运松,宋后燕等.t-PA cDNA的克隆及其在毕赤酵母中的表达.生物工程学报,1998,14(4):439-444
[61] 黄坚,龙青,任启生等.重组rPA基因在毕赤酵母细胞中的胞内表达研究.中国生化药物杂志,2003,24(2):58-61
[62] Luckow VA ,Summers M D .Trends in the development of baculovirus expression vectors .Biotechnology, 1988,6:47-55
[63] Marshall A .The insects are coming .Nature Biotechnol. 1998,16:530-533
[64] 刘军波,杨凯,薛爱群等.t-PA昆虫细胞表达及特性分析.实验生物学报,2000,33(4):293-300
[65] Rohrmann G F.Baculovirus structural proteins.Journal of General Virology, 1992,73:749-761
[66] 赵春梅,张洪涛,胡美浩.组织型纤溶酶原激活剂A链与尿激酶原B链嵌合分子的构建与表达.中国生物化学与分子生物学报,1999,15(4):528-531
[67] Gordon K,Lee E ,Vitale J, et al.Production of human tissue plasminogen activator in transgenic mause milk.Biotechnology, 1987,5:1183-1187
[68] Pittus C W, Hennighausen L, Lee E, et al.A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice.Proc Natl Acad Sci USA, 1988,85:5874-5878
[69] 卢一凡,邓继先,程萱等.组织型纤溶酶原激活剂突变体乳腺表达载体构建及在转基 因鼠中表达的研究.生物工程学报,1999,4:166—170
[70] 陈红星,程萱,杨晓等.牛β-乳球蛋白基因调控序列指导组织型纤溶酶原激活剂在小鼠乳腺中的表达.生物工程学报,2001,2:135-139
[71] 徐寒梅,成国祥,沈子龙.利用转基因动物乳腺生物反应器生产溶栓药物的研究进展.中国药科大学学报,2001,32(6):474-477
[72] 李育阳主编.基因表达技术.北京:科学出版社,2001,2:96—119
[73] Upshall A ,Kuonar A ,Baileys M , et al .Secretion of active human tissue plasminogen activator from the filamentous fungus Aspergillus nidulans. Biotechnology, 1987,5:1301-1304
[74] Wiebe M G,Karandikar A ,Robson G D, et al.Production of tissue plasminogen activator in Aspergillus niger.Biotechnology and Bioengineering ,2001,76(2):164-174
[75] 汪天虹,吴静,邹玉霞等.瑞氏木霉分子生物学研究进展.菌物系统,2000,19(1):147-152
[76] Marder V J, Sherry S. Thrombolytic therapy:current status. N Engl J Med., 1998,318:1512-1520
[77] 黄柄辉,陈春麟.临床溶栓药UK与SK生化、药理和临床应用比较.生化药物杂志,1990,52(2):19-20
[78] Kohler M, Sen S, Hermes R, et al. Pharmacokinetics of single-chain urokinase-type plasminogen activator (scu-PA) and two-chain urokinase-type plasminogen activator(tcu-PA) in patients with acute myocardial infarction. Ann H aematol, 62 suppl. 1991,A: A75
[79] Ross A M.New plaminogen activators:a clinical review.Clin Cardial, 1999,22 : 165-171
[80] Verstraete M,Lijnen H R,Collen D.Thrombolytic agents in development .Drugs, 1995,50 : 29-42
[81] Sumi H, Hamada H, Tsushima H, et al. A novel fibrinolytic enzyme (Nattokinase) in the vegetable cheese Natto, a typical and popular soybean food in the Japanese diet. Experiment, 1987, 43:1110-1111
[82] Fujita M, Nomura K, Hong K, et al. Purification and characterization of a strong fermented food in Japan. Biochem Biophy Res Commun 1993, 197(3): 1340-1346.
[83] Sumi H, Hamada H, Nakanishi K, et al. Enhancement of the fibrinolytic activity in plasma by oral administration of NK. Acta Haematol 1990, 84:139-143.
[84] Chart C T, Fan M H, Kuo F C, et al. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NKI. J. Agric Food Chem. 2000, 48:3210-3216
[85] Kim W, Choi K, Kim Y, et al. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. Strain CK 11-4 Screened from Chungkook-Jang. Appl Environ Microbiol, 1996, 62(7):2482-2488
[86] Kim S, Choi N. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain D J-4 screened from Doen-Jang. Biosci Biotechnol biochem, 2000, 64(8):1722-1725
[87] 阎家麒,童岩,臧莹安.豆豉纤溶酶的纯化及其性质研究.药物生物技术,2000,7(3):149-152
[88] 韩润林,张小勇,张建安.枯草杆菌溶栓酶的分离与纯化研究.中国生化药物杂志,2000,21(5):219-222
[89] 杨志兴,张淑梅,张云湖等.一株具有纤溶活性的枯草杆菌蛋白激酶的初步研究.药物生物技术,1996,3(3):133-136
[90] 傅俐,李荣萍,李晶等.一株具有纤溶活性的枯草杆菌(Bacillus subtilis)的研究—液体发酵条什的选择.生物工程进展,1997,17(3):31-33
[91] Keranen S, Penttila M .Production of recombinant proteins in the filamentous fungus Trichoderma reesei.Current Opinion in Biotechnology, 1995,6:534-537
[92] Marleen Maras, Andre De Bruyn ,Wouter Vervecken, et al. In vivo synthesis of complex N-glycans by expression of human N-acetylglu-cosaminyltransferase I in the filamentous fungus Trichoderma reesei.FEBS Letters,1999,452:365-370
[93] 王海宽,孙亚范,杜连祥等.木质素过氧化物酶基因的克隆及在甲醇毕赤酵母中的表达.微生物学报,2004,44(2):258-260
[2] Kenneth Ouriel. Level and Current status of Thrombolysis for Peripheral Arterial OcclusiveDisease.Analysis of vascular surgery,2004,799-801
[3] 褚志义主编.生物合成药物学.北京:化学工业出版社,1999,4:787—792
[4] Lewandowski C, Barsan W. Treament of acute ischemic stroke. An Emerg Med,2001,37(2):202-206
[5] Zoppo G J. Antithrombotic treatments in acute ischemic stroke.Curt Opin Hematol,2000,46:261-265
[6] 李敏,扈荣良,王玉炯等.重组纤溶酶原激活剂及其突变体的表达研究.宁夏大学学报2002,23(1):71-75
[7] Pohl G, et al.Biochemistry,1984,23:3701-3701
[8] Smith B O, Downing A K, Driscoll P C,et al. The solution structure and backbone dynamics of the fibronectin type I and epidermal growth factor-like pair of modules of tissue-type plasminogen activator. Structure,1995,3(8):823
[9] Shoichi A, Norihide S, Msnabu S, et al. Influence of sugar chain on fibrin affinity of recombinant t-PA. Biol Pharm Bu11,2001,24(3):295
[10] Shoichi A,Norihide S,Msnabu S,et al.lnfluence of sugar chain on fibrin affinity of recombinant t-PA.Bio Pharm Bull,2001,24(3):295
[11] Rijken D C, et al. Measurement of human tissue type plasminogen activator by a two-sites immunoradiometric assay. J. Lab.Clin. Med., 1983, 101:274-284
[12] 王鸿利,王学锋.血栓病临床新技术.北京:人民军医出版社,2003
[13] Ny T, et al.Proc Nati Acad Sci USA,1983,80:349
[14] Browne M J, et al. Gene,1985,33:279
[15] Sandra J,et al. J. Biol Chem, 1986,261:6972
[16] 钱民章,宋后燕.人组织型纤溶酶原激活剂(t-PA)基因.生物工程学报.1998,4(1):1
[17] Pennica D ,EW Holmes, JW Kohr, et al .Cloning and expression of human t-PA cDNA in E. coli. Nature, 1983, 301:214-221
[18] Gamani C,Gavin O,Bertossa C,et al.Studies on the effect of fucosylated and non-fucosylated finger/growth factor constructs on the clearance of tissue-type plasminogen activator mediated by the Iow-density-lipoprotein-receptor-related protein. Eur J Biochem.,1998,251:804
[19] 焦建伟,茹炳根.溶栓剂研究的新进展,生物工程进展,2002,22:30-33
[20] Lamba B, Bauer M, Huber R, et al. The 2.3A Crystal Structute of the Catalytic Domain of Recombinant Two chain Human Tissue-type Plasminogen Activator.J Mol Bio1,1996,258:117-135
[21] Renatus M, Engh R A,Stubbs M T. Lysine 156 Promotes the Anomalous Proenzyme Activity of tPA:Crystal Structure of Single-chain Human tPA. EMBO J, 1997,16:4797-4805
[22] Tachias K, Madison E L.Converting Tissue-type Plasminogen Activator into a Zymogen.J Biol Chem, 1996,271:28749-28752
[23] Tachias K, Madison E L. Vsriants of Tissue-type Plasminogen Activator Which Display Substantially Enhanced Stimulation by Fibrin.J.Boil Chem, 1995,270:18319-18322
[24] Ke S-H,Tachias K,Lamba D,et al.ldentification of a Hydrophobic Exosite on Tissue Type Plasminogen Activator That Modulates Specificity for Plasminogen.J Biol Chem,1997,272:1811-1816
[25] Toschi L,BringmannP, Petri T, et al.Fibrin Selectivity of the Isolated Protease Domains of Tissue-type and Vampire Bat Salivary Gland Plasminogen Activators. Eur J Biochem, 1998,252:108-112
[26] Yamada T, Shimada, Y, Kikuchi M.Integrin-specific Tissue-type Plasminogen Activator Engineered by Introduction of the Arg-Gly-Asp Sepuence. Biochem Biophys Res Commun, 1996,228:306-311
[27] Smith J W, Tachias K, Madison E L. Protein Loop Grafting to Construct a Variant of Tissue-type Plasminogen Activator That Binds Platelet Integrin Alpha II b Beta 3. J. Biol Chern, 1995,270:30486-30490
[28] Noble S,Mctavish D.A review of its pharmacological properties and clinical efficacy in the management of acute myocardial infarction.Drug Evaluation, 1996,52:589
[29] 周红.第三代溶栓剂的作用特点及其研究现状.国外医学心血管分册,2000,5:274-276
[30] Den Heijer R,Vermeer F, Ambrosiono E,et al. Evaluation of a weight-adjusted single-bolus plasminogen activator inpatients with myocardial Infarction. Circulation, 1998,98:2117
[31] Tucker H M, Gerard R D. Sepuence Requirements in the Reactive-center Loop of Plasminogen-activator Inhibitor-1 for Recognition of Plasminogen Activators.Eur J Biochem,1996,237:180-187
[32] Tachias K,Madison E L. Variants of Tissue-type Plasminogen Activator That Display xtraordinary Resistance to Inhibition by the Serpin Plasminogen Activator Inhibitor Type l.J Biol Chem,1997,272:14580-14585
[33] Modi N B, Eppler S,Breed J,et al. Pharmacokinetics of a slower clearing tissue plasminogen activator variant TNK-t-PA in patients with acute myocardial imfarction. Thromb Haemostas, 1998,79:134
[34] 刘士辉等.半衰期延获得PAI-1抗性的突变体构建、表达及特性分析.生物工程学报,1995,11(1):13-19
[35] 赵庆国等.组织型纤溶酶原激活剂(t-PA)突变体在CHO细胞中表达产物的纯化.解放军药学学报,2000,16(3):117-119
[36] Hua Z.C.Renaturation and purification of recombinant tissue -plasminogen activator expressed in E. coli .Biochem. Mol. Biol. International , 1997,41(4):815
[37] Sarmientos P, Duchesns M ,Deneffe P ,et al .Synthesis and purification of active human tissue plasminogen activator from Escherichia coli. Bio/Technology, 1989,7:495
[38] Harris TJR ,Patel T ,Marston FAO, et al .Cloning of cDNA coding for human tissue-type plasminogen activator and its expression in Escherichia coli. Mol Biol Med, 1986,3:279
[39] Geunfeld H . Effector-assisted refolding of recombinant tissue -plasminogen activator producing in Escherichia coli .Applied Biochem. Biotech., 1992,33(2):117
[40] 夏焱,华子春,陈晓春等.人组织型纤溶酶原激活剂在大肠杆菌中的表达、复性及分离纯化.南京大学学报,1994,30(4):627-632
[41] 熊凌霜,黄培堂,董陆佳等.组织型纤溶酶原激活剂cDNA在大肠杆菌中表达的研究.军事医学科学院院刊,1994,18(4):249-252
[42] http://www. Centocor. Com/or http/www. Boehringer-ingelheim.Com/
[43] Martin U,Mollendorff E,Akpan W, et al.Dose-ranging study of the novel recombinant plasminogen activator BM 06.022 in healthy volunteers.J.Clin Pharmacol Ther, 1991,50(4):429
[44] 苏勇,易进华,李军等.人组织型纤溶酶原激活物衍生物在大肠杆菌硫氧化还原蛋白融合表达系统中的表达.生物工程进展,2000,6:63-67
[45] 沉毅珺,李军,易进华等.重组人细织型纤溶酶原激活物衍生物住大肠杆菌中表达的研究.药物生物技术,2001,8(6):306—309
[46] 廖建民,张瑾,沈子龙.组织型纤溶酶原激活剂突变体Reteplase(r-PA)基因的克隆及在大肠杆菌中的表达.药物生物技术,2002,9(2):95—98
[47] Lucas B K ,Giere L M ,DeMarco R A, et al .High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector .Nucleic Acids Res., 1996,24(9):1774-1779
[48] 欧阳应斌,黄培堂,徐秀英等.t-PA cDNA在CHO细胞中的高效表达.生物技术通讯,1995,6:11-15
[49] Fussengger M ,Bailey J E ,Hauser H, et al .Genetic optimization of recombinanl glycoprotein production by mammalian cells .Trends Biotechnol , 1999,17(1):35-42
[50] Khan M W ,Musgrave S C ,Jenkins N, et al .N-linked glycosylation of tissue plasminogen activator in Namalwa cells .Biochem. Soc. Trans, 1995,23(1):99
[51] Lo K M,Gillies S D.High level expression of human proteins in murine hybridome cells:induction by methotrexate in the absence of gene amplification.Biochem.Biophys.Acta, 1991,1088:217-224
[52] Traunecker A ,Oliveri F ,Karjalainen K .Myeloma based expression system for production of large mammalian proteins .Trends Biotechnol , 1991,9(4):109-113
[53] Werner R G, Noe W, Kopp K, et al. Appropriate mammalian expression system .Arzeim-Forsch/Drug Res., 1998,48(8):870-880
[54] Romanos M A ,Scorer C A ,Clare J, et al.J. Yeast ,1992,8:423-488
[55] Cregg J M ,Vedvick T S ,Raschke W C .Recent advances in expression of foreign genes in Pichia pastoris .Biotechnology, 1993,11 (8):905-910
[56] Ellis S B,Brust P F, Koutz P J,et al.lsolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris.Mol Cell Biol, 1985,5:1111
[57] Cregg J M,Barringer K J,Hessler A Y, et al. Pichia pastoris as a host system for transformations. Mol .Cell Biol,1985,5:3376
[58] Cereghino J L,Cregg J M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiol Rev,2000,24:45
[59] Sreekrishna K,Brankamp R G,Kropp K E,et al.Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.Gene, 1997,190:55
[60] 涂宣林,朱运松,宋后燕等.t-PA cDNA的克隆及其在毕赤酵母中的表达.生物工程学报,1998,14(4):439-444
[61] 黄坚,龙青,任启生等.重组rPA基因在毕赤酵母细胞中的胞内表达研究.中国生化药物杂志,2003,24(2):58-61
[62] Luckow VA ,Summers M D .Trends in the development of baculovirus expression vectors .Biotechnology, 1988,6:47-55
[63] Marshall A .The insects are coming .Nature Biotechnol. 1998,16:530-533
[64] 刘军波,杨凯,薛爱群等.t-PA昆虫细胞表达及特性分析.实验生物学报,2000,33(4):293-300
[65] Rohrmann G F.Baculovirus structural proteins.Journal of General Virology, 1992,73:749-761
[66] 赵春梅,张洪涛,胡美浩.组织型纤溶酶原激活剂A链与尿激酶原B链嵌合分子的构建与表达.中国生物化学与分子生物学报,1999,15(4):528-531
[67] Gordon K,Lee E ,Vitale J, et al.Production of human tissue plasminogen activator in transgenic mause milk.Biotechnology, 1987,5:1183-1187
[68] Pittus C W, Hennighausen L, Lee E, et al.A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice.Proc Natl Acad Sci USA, 1988,85:5874-5878
[69] 卢一凡,邓继先,程萱等.组织型纤溶酶原激活剂突变体乳腺表达载体构建及在转基 因鼠中表达的研究.生物工程学报,1999,4:166—170
[70] 陈红星,程萱,杨晓等.牛β-乳球蛋白基因调控序列指导组织型纤溶酶原激活剂在小鼠乳腺中的表达.生物工程学报,2001,2:135-139
[71] 徐寒梅,成国祥,沈子龙.利用转基因动物乳腺生物反应器生产溶栓药物的研究进展.中国药科大学学报,2001,32(6):474-477
[72] 李育阳主编.基因表达技术.北京:科学出版社,2001,2:96—119
[73] Upshall A ,Kuonar A ,Baileys M , et al .Secretion of active human tissue plasminogen activator from the filamentous fungus Aspergillus nidulans. Biotechnology, 1987,5:1301-1304
[74] Wiebe M G,Karandikar A ,Robson G D, et al.Production of tissue plasminogen activator in Aspergillus niger.Biotechnology and Bioengineering ,2001,76(2):164-174
[75] 汪天虹,吴静,邹玉霞等.瑞氏木霉分子生物学研究进展.菌物系统,2000,19(1):147-152
[76] Marder V J, Sherry S. Thrombolytic therapy:current status. N Engl J Med., 1998,318:1512-1520
[77] 黄柄辉,陈春麟.临床溶栓药UK与SK生化、药理和临床应用比较.生化药物杂志,1990,52(2):19-20
[78] Kohler M, Sen S, Hermes R, et al. Pharmacokinetics of single-chain urokinase-type plasminogen activator (scu-PA) and two-chain urokinase-type plasminogen activator(tcu-PA) in patients with acute myocardial infarction. Ann H aematol, 62 suppl. 1991,A: A75
[79] Ross A M.New plaminogen activators:a clinical review.Clin Cardial, 1999,22 : 165-171
[80] Verstraete M,Lijnen H R,Collen D.Thrombolytic agents in development .Drugs, 1995,50 : 29-42
[81] Sumi H, Hamada H, Tsushima H, et al. A novel fibrinolytic enzyme (Nattokinase) in the vegetable cheese Natto, a typical and popular soybean food in the Japanese diet. Experiment, 1987, 43:1110-1111
[82] Fujita M, Nomura K, Hong K, et al. Purification and characterization of a strong fermented food in Japan. Biochem Biophy Res Commun 1993, 197(3): 1340-1346.
[83] Sumi H, Hamada H, Nakanishi K, et al. Enhancement of the fibrinolytic activity in plasma by oral administration of NK. Acta Haematol 1990, 84:139-143.
[84] Chart C T, Fan M H, Kuo F C, et al. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NKI. J. Agric Food Chem. 2000, 48:3210-3216
[85] Kim W, Choi K, Kim Y, et al. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. Strain CK 11-4 Screened from Chungkook-Jang. Appl Environ Microbiol, 1996, 62(7):2482-2488
[86] Kim S, Choi N. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain D J-4 screened from Doen-Jang. Biosci Biotechnol biochem, 2000, 64(8):1722-1725
[87] 阎家麒,童岩,臧莹安.豆豉纤溶酶的纯化及其性质研究.药物生物技术,2000,7(3):149-152
[88] 韩润林,张小勇,张建安.枯草杆菌溶栓酶的分离与纯化研究.中国生化药物杂志,2000,21(5):219-222
[89] 杨志兴,张淑梅,张云湖等.一株具有纤溶活性的枯草杆菌蛋白激酶的初步研究.药物生物技术,1996,3(3):133-136
[90] 傅俐,李荣萍,李晶等.一株具有纤溶活性的枯草杆菌(Bacillus subtilis)的研究—液体发酵条什的选择.生物工程进展,1997,17(3):31-33
[91] Keranen S, Penttila M .Production of recombinant proteins in the filamentous fungus Trichoderma reesei.Current Opinion in Biotechnology, 1995,6:534-537
[92] Marleen Maras, Andre De Bruyn ,Wouter Vervecken, et al. In vivo synthesis of complex N-glycans by expression of human N-acetylglu-cosaminyltransferase I in the filamentous fungus Trichoderma reesei.FEBS Letters,1999,452:365-370
[93] 王海宽,孙亚范,杜连祥等.木质素过氧化物酶基因的克隆及在甲醇毕赤酵母中的表达.微生物学报,2004,44(2):258-260