表达人血清白蛋白-C肽融合蛋白毕赤酵母工程菌的构建
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摘要
C肽(C-Peptide,CP)是胰岛素A链与B链之间的连接肽,近年来研究发现CP通过与细胞膜上G蛋白耦联的受体结合,使Ca2+通道开放,激活Na+-K+-ATP酶活性和一氧化氮合酶的活性,增加血流量,扩张血管,从而改善糖尿病患者的肾脏、神经、微血管病变。为了加快CP的药物化进展,提高CP在体内的半衰期,我们构建了人血清白蛋白(HSA)-CP融合蛋白,将其在毕赤酵母系统中进行表达,并对表达的融合蛋白进行了鉴定、活性分析和初步的分离纯化。
根据表达系统的密码子偏好性优化CP基因,PCR从T-CP质粒中扩增CP基因,酶切连接pBlue-HSA质粒(HSA1800bp)和CP(100bp)基因,构建pBlue-HSA-CP质粒。测序结果显示得到的融合基因HSA-CP序列与预期一致。用EcoRⅠ和NotⅠ双酶切重组质粒pBlue-HSA-CP,回收HSA-CP片段,插入分泌表达载体pPIC9k中,构建表达载体pPIC9k-HSA-CP。表达载体pPIC9k-HSA-CP经限制性内切酶分析和PCR验证表明,融合基因已经成功的插入到载体pPIC9k中。
表达载体pPIC9k-HSA-CP经SalⅠ线性化后电击转化毕赤酵母GS115,用MM和MD平板筛选Mut+转化子,PCR鉴定后,用甲醇诱导摇瓶分泌表达。将表达量较高的重组子进行表达产物鉴定和活性分析,优化诱导时间和诱导剂浓度后,对融合蛋白进行初步的分离纯化。经G418筛选得到能耐受3 mg/mL G418重组菌8株,进一步摇瓶诱导筛选得到两株表达量120 mg/L的重组菌rHC1, rHC2。优化摇瓶培养条件后,重组菌表达量由120 mg/L提高到140 mg/L。Western blot鉴定显示表达的融合蛋白具有HSA和CP的抗原性,为HSA和CP的杂合分子,分子量约为70 kDa。细胞活性研究表明HSA-CP融合蛋白对人胚肾293细胞的生长具有一定的促进作用。
C-peptide(CP), a linking chain with 31 amino acids in length, locates between A and B chain of insulin. The recent researches indicate that it elicits a number of cellular responses by specific binding to G-protein coupled receptor. Including increasing Ca2+ influx, stimulation of Na+-K+-ATPase and endothelial NO synthase activities resulting in the increase of blood flow and expansion of blood vessel. Therefore, CP can ameliorate sensory nerve dysfunction, nephropathy, neuropathy and microvascular dysfunction. In order to prolong the half-life of CP in vivo, we developed a potential long-acting CP(HSA-CP) by albumin fusion technology. The fused gene HSA-CP was expressed in pichia pastoris. Confirmed by Western blot and cell proliferation analysis, the fusion protein was purified preliminarily.
To improve the expression of HSA-CP, the codon usage of the gene encoding for CP was optimized according to Pichia pastoris codon bias and reduced repetitive AT and GC content. The artificial CP gene was fused to the HSA cDNA in the same reading frame and then sequenced. The recombinant plasmid named pBlue-HSA-CP. The fused gene HSA-CP was recovered from pBlue-HSA-CP digested with DNA restriction enzyme EcoRⅠand NotⅠ, and then inserted into expression vector pPIC9k to construct recombinant plasmid pPIC9k-HSA-CP. The results showed that the cloned fused gene was in correspondence with our anticipation, and expression vector pPIC9k-HSA-CP was constructed correctly.
The recombinant vector pPIC9k-HSA-CP was linearized by SalⅠ, then transformed into Pichia pastoris strain GS115 by electroporation. The Mut+ transformants were selected out from histidine-deficient medium plates and screening for Mut phenotype. Confirmed integration by PCR, The recombinant strains were induced to express fusion protein HSA-CP by methanol in shake. The genetic engineering Pichia pastoris whose expression level was relative high was identified by western bolt and cell proliferation analysis. After optimized the inducing time and concentration of inducer, the secreted fusion protein HSA-CP was purified. Finally, 8 strains were screened out from contained 3 mg/mL G418 MD plate, in which two high expression strain, named as rHC1, rHC2 could secrete 120 mg/L fusion protein was obtained with culturation in shakes. The expressed fusion protein has apparent molecular weight of 70 kDa observed by SDS-PAGE. It was specifically recognized by an anti-human HSA polyclonal antibody and CP polyclonal antibody in Western blot assay. After optimized the culture condition, the yield of the recombinant strain was raised up to 140mg/L determined by Microalbuminuria Immunoassay kit and C-peptide Chemilumin escence Immunoassay kit. Further studies on its cell activites, we found that C-peptide and HSA-CP in low concentration can effect on 293 cell proliferation.
根据表达系统的密码子偏好性优化CP基因,PCR从T-CP质粒中扩增CP基因,酶切连接pBlue-HSA质粒(HSA1800bp)和CP(100bp)基因,构建pBlue-HSA-CP质粒。测序结果显示得到的融合基因HSA-CP序列与预期一致。用EcoRⅠ和NotⅠ双酶切重组质粒pBlue-HSA-CP,回收HSA-CP片段,插入分泌表达载体pPIC9k中,构建表达载体pPIC9k-HSA-CP。表达载体pPIC9k-HSA-CP经限制性内切酶分析和PCR验证表明,融合基因已经成功的插入到载体pPIC9k中。
表达载体pPIC9k-HSA-CP经SalⅠ线性化后电击转化毕赤酵母GS115,用MM和MD平板筛选Mut+转化子,PCR鉴定后,用甲醇诱导摇瓶分泌表达。将表达量较高的重组子进行表达产物鉴定和活性分析,优化诱导时间和诱导剂浓度后,对融合蛋白进行初步的分离纯化。经G418筛选得到能耐受3 mg/mL G418重组菌8株,进一步摇瓶诱导筛选得到两株表达量120 mg/L的重组菌rHC1, rHC2。优化摇瓶培养条件后,重组菌表达量由120 mg/L提高到140 mg/L。Western blot鉴定显示表达的融合蛋白具有HSA和CP的抗原性,为HSA和CP的杂合分子,分子量约为70 kDa。细胞活性研究表明HSA-CP融合蛋白对人胚肾293细胞的生长具有一定的促进作用。
C-peptide(CP), a linking chain with 31 amino acids in length, locates between A and B chain of insulin. The recent researches indicate that it elicits a number of cellular responses by specific binding to G-protein coupled receptor. Including increasing Ca2+ influx, stimulation of Na+-K+-ATPase and endothelial NO synthase activities resulting in the increase of blood flow and expansion of blood vessel. Therefore, CP can ameliorate sensory nerve dysfunction, nephropathy, neuropathy and microvascular dysfunction. In order to prolong the half-life of CP in vivo, we developed a potential long-acting CP(HSA-CP) by albumin fusion technology. The fused gene HSA-CP was expressed in pichia pastoris. Confirmed by Western blot and cell proliferation analysis, the fusion protein was purified preliminarily.
To improve the expression of HSA-CP, the codon usage of the gene encoding for CP was optimized according to Pichia pastoris codon bias and reduced repetitive AT and GC content. The artificial CP gene was fused to the HSA cDNA in the same reading frame and then sequenced. The recombinant plasmid named pBlue-HSA-CP. The fused gene HSA-CP was recovered from pBlue-HSA-CP digested with DNA restriction enzyme EcoRⅠand NotⅠ, and then inserted into expression vector pPIC9k to construct recombinant plasmid pPIC9k-HSA-CP. The results showed that the cloned fused gene was in correspondence with our anticipation, and expression vector pPIC9k-HSA-CP was constructed correctly.
The recombinant vector pPIC9k-HSA-CP was linearized by SalⅠ, then transformed into Pichia pastoris strain GS115 by electroporation. The Mut+ transformants were selected out from histidine-deficient medium plates and screening for Mut phenotype. Confirmed integration by PCR, The recombinant strains were induced to express fusion protein HSA-CP by methanol in shake. The genetic engineering Pichia pastoris whose expression level was relative high was identified by western bolt and cell proliferation analysis. After optimized the inducing time and concentration of inducer, the secreted fusion protein HSA-CP was purified. Finally, 8 strains were screened out from contained 3 mg/mL G418 MD plate, in which two high expression strain, named as rHC1, rHC2 could secrete 120 mg/L fusion protein was obtained with culturation in shakes. The expressed fusion protein has apparent molecular weight of 70 kDa observed by SDS-PAGE. It was specifically recognized by an anti-human HSA polyclonal antibody and CP polyclonal antibody in Western blot assay. After optimized the culture condition, the yield of the recombinant strain was raised up to 140mg/L determined by Microalbuminuria Immunoassay kit and C-peptide Chemilumin escence Immunoassay kit. Further studies on its cell activites, we found that C-peptide and HSA-CP in low concentration can effect on 293 cell proliferation.
引文
1 Y Ido, A Vindigni, K Chang, et al. Prevention of vascular and neural dysfunction in diabetic rats by C-peptide. Science, 1997, 77:563-566.
2 Kitabchi AE. The biological and immunological properties of pork and beef insulin, proinsulin and connecting peptides. J Clin Invest , 1970, 49:979-987.
3王志珍,梁栋材.胰岛素分子结构与功能关系的复杂性.生物化学杂志(J Biochem), 1985, 1(1):9-21.
4岳国华,朱尚权(Yue G H, Zhu S Q).胰岛素受体结构与功能研究概况.生物化学与生物物理进展(Prog Biochem Biophys), 1992, 19(1): 1-5.
5 Munte CE, Vilela L, Kalbitzer HR. Garratt RC Solution structure of human proinsulin C-peptide. FEBS J. 2005 (272) 4284-4293.
6 Aladdin Pramanik, Karin Ekberg. C-Peptide Binding to Human Cell Membranes: Importance of Glu27. Biochemical and Biophysical Research Communications . 2001, 284:94-98.
7王先令,陆菊明. C肽作用的研究进展.国外医学内分泌学分册, 2007, 24:4276-278.
8 Johansson BL, Kernell A, Sjoberg S, et al. Influence of combined C-peptide and insulin administration on renal-function and metabolic control in diabetes type-1. Journal of Clinical Endocrinology and Metabolism, 1993, 77(4):976-981.
9 Ido Y, Vindigni A,Chang K , et al. Prevention of vascular and neural dysfunction in diabetic rats by C-peptide. Science, 1997, 277(5325):563-566.
10赵晓龙,高鑫,刘智慧,等.C肽联合胰岛素干预治疗对大鼠早期糖尿病肾病的干预[J].中华内分泌代谢杂志, 2002, 18(4):272-273.
11 Sjoquist M, Huang W, Johansson BL. Effects of C-pep tide on renal function at early stage of experimental diabetes. Kidney Int, 1998, 54 (3) : 758.
12 Rizk N, Dunbar JC. Insulin-mediated increase in sympathetic nerve activity is attenuated by C-peptide in diabetic rats. Exp BiolMed (Maywood) , 2004, 229 (1) : 80.
13孙蔚,高鑫,刘智慧. C肽对链脲佐菌糖尿病大鼠的糖尿病肾病的干预作用[J].中华内分泌代谢杂志, 2003, 4(2):143-144.
15 Samnegard B, Jacobson SH, Jaremko G, et al. Effects of C-peptide on glomerular and renal size and renal function in diabetic rats[J]. Kindey Int, 2001, 60(4):1258-1265.
16 Johansson B L, Brog K, Fernqvist-Forbes E, et al. Beneficial effects of C-peptide on incipient nephropathy and nephropathy in patients with typeⅠdiabetes mellitus[J]. Diabete Medcine, 2000, 17:181-189.
17 Karin Ekberg, Bjorn Jonsson, John Wahren. Amelioration of Sensory Nerve Dysfunction by C-Peptide in Patients With Type 1 Diabetes[J]. DIABETES, 2003 52:536-541.
18田丽萍,张育坚,龚毅,等.人胰岛素原C肽的表达、纯化及其细胞活性研究.生物工程学报, 22(6):928-931.
19 Li ZG, Zhang WX, Sima AAF. C-peptide enhaences insulin mediated cell growth and protection against high glucose induced apoptosis in SH-Sy(5) cells. Diatetes Metabolism Research and Reviews, 2003, 19(5):375-385.
20 Li L, Oshida Y, Kusunnoki M, et al. Rats C-peptideⅠandⅡstimulate glucose utilization in STZ-induced diabetic rat [J]. Diabetologia, 1999, 42:958-964.
21 Wu W, Oshida Y, Wang W P, et al. Effect of C-peptide admistration on whole body glucose utilization in STZ-induced diabetic rats[J]. Acta Physiol Scand, 1996, 157:253-265 .
22 Forst T,Rave K, P fuetzer A, et al, Effect of C-peptide on glucose metabolism in patients with typeⅠdiabetes[J]. Diabetes Care, 2002, 25(6):1096-1097.
23 ZG L i, Qiang X, Sima AA, et al. C-peptide attenuates protein tyrosine phosphatase activity and enhances glycogen synthesis in L6 myoblasts. Biochem Biophys Res Communi, 2001, 28 (3) : 615.
24 Rigler R,Pramani kA, Jonasson P, et al. Specific binding of proinsulin C-peptide to human cell membranes[J]. Proc Natl Acad Sci USA, 1999, 96:13318-23.
25 Wahren J, Ekberg K, Johansson J, et al. Role of C-peptide in human physiology[J]. Am J Physiol Endocrinol Metab, 2000, 78:759-68.
26孟东,于德民. C肽对高糖状态下人脐静脉内皮细胞体外表达eNOS的影响.天津医药, 2004, 32(7):389-391.
27 Jan Johasson, Jawed Shafqat. Molecular effects of proinsulin C-peptide. Biochemical and Biophysical Research Communications, 2002, 295:1035-1040.
28戚楠,马清钧.长效重组蛋白药物的研究进展[J].中国生物工程杂志, 2006, 26(2): 79-82.
29王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志, 2003, 23(10): 23-27.
30 Paulina Balbás, Argelia Lorence. Recombinant Gene Expression: Reviews and Protocols [M]. 2nd ed. Totowa: Humana Press Inc.
31李育阳主编.基因表达技术[M].北京:科学出版社, 2001.77-91.
32张惠展主编.基因工程概论[M].上海:华东理工大学出版社, 1999. 197-485.
33 Romanos M. Advances in the use of Pichia pastoris for high-level gene expression [J].Current Opinion in Biotechnology, 1995, 6(5): 527-533.
34 Peter E Sudbery. The expression of recombinant proteins in yeasts [J]. Current Opinion in Biotechnology, 1996, 7: 517-524.
35 Joan LC, James MC. Heterologous protein expression in the methylotrophic yeast Pichia pastoris [J]. FEMS Microbiology Reviews, 2000, 24:45-66.
36张伍魁,范清林,宋礼华.毕赤酵母表达系统在外源基因表达中的研究进展及应用[J].中国生物工程杂志, 2006, 26(1): 87-91.
37于平.巴斯德毕赤酵母表达系统研究进展[J].工业微生物, 2005, 35(3): 50-54.
38黄石,邹民吉,徐东刚.毕赤酵母分泌表达系统的研究进展[J].医学研究通讯, 2004, 33(7): 34-36.
39李健仔.巴斯德毕赤酵母外源基因表达系统[J].生物学通报, 2005, 40(3): 21-23.
40 Joan LC, James MC. Heterologous protein expression in the methylotrophic yeast Pichia pastoris [J]. FEMS Microbiology Reviews, 2000, 24:45-66.
41赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析.生物工程学报. 2003, 16(3):308-311.
42 Invitrogen. Pichia Expression Kit: A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris.
43 Koti Sreekrinshna, Robert G, Brankamp, et al. Strategies for optimal protein expression and secretion in the methylotrophic yeast Pichia [J]. gene, 1997, 190: 55-62.
44聂东宋,梁宋平.外源蛋白在巴氏毕赤酵母中高效表达的策略[J].吉林大学学报, 2001, 22(3): 40-44.
45李洪钊,李亮助.巴斯德毕赤酵母表达系统优化策略[J].微生物学报, 2003, 43(2): 288-292.
46李欣,郭树华.影响外源基因在巴氏毕赤酵母中表达的因素[J].生物技术通讯, 2000, 11(2): 132-134,140.
47邬小兵,乐国伟.影响毕赤酵母高效表达外源蛋白的因素[J].生物技术, 2002, 12(4): 44-46.
48 Romanos M. Advances in the use of Pichia pastoris for high-level gene expression [J]. Current Opinion in Biotechnology, 1995, 6(5): 527-533.
49 Vassileva A, Chugh DA, Swaminathan S, etc. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris [J]. Protein expression and purification, 2001, 21(1): 71-80.
50马兴元,谭建华,朱平,等.巴斯德毕赤酵母表达系统及其在外源蛋白生产中的优势与应用前景[J].中国兽医学报. 2003, 23(1):98-101.
51雷楗勇,张莲芬,金坚,等.人β干扰素-血清白蛋白融合蛋白在毕赤酵母中的分泌表达.中国生物工程杂志. 2007, 26(7):13-18.
52 Koti Sreekrinshna, Robert G, Brankamp, et al. Strategies for optimal protein expression and secretion in the methylotrophic yeast Pichia [J]. gene, 1997, 190: 55-62.
53孙卫民,王惠琴主编.细胞因子研究方法学[M].北京:人民卫生出版社, 1999. 76-78.
54彭彦,王勇,宋方洲,等. IL-18在毕赤酵母中的表达和纯化[J].第四军医大学学报, 2005, 26(22):2057-2061.
55张耀东,杨伯伦.强阴离子色谱法从毕赤酵母培养液中分离纯化重组巴西日圆线虫乙酰胆碱酯酶.色谱, 2006, 24(1): 39-41.
56刘战民,陆兆新,吕凤霞,等.毕赤酵母工程菌原果胶酶的分离纯化[J].西北农林科技大学学报, 2006, 34(1): 79-83,92.
57张伟,陈劲春.蛇神经毒素在毕赤酵母中的分泌表达及其分离纯化[J].北京化工大学学报:自然科学版, 2005, 32(6): 20-22.
58刘永东,王云山,苏志国.重组人血清白蛋白生产工艺研究进展[J].微生物学通报, 2003, 30(5): 128-132.
2 Kitabchi AE. The biological and immunological properties of pork and beef insulin, proinsulin and connecting peptides. J Clin Invest , 1970, 49:979-987.
3王志珍,梁栋材.胰岛素分子结构与功能关系的复杂性.生物化学杂志(J Biochem), 1985, 1(1):9-21.
4岳国华,朱尚权(Yue G H, Zhu S Q).胰岛素受体结构与功能研究概况.生物化学与生物物理进展(Prog Biochem Biophys), 1992, 19(1): 1-5.
5 Munte CE, Vilela L, Kalbitzer HR. Garratt RC Solution structure of human proinsulin C-peptide. FEBS J. 2005 (272) 4284-4293.
6 Aladdin Pramanik, Karin Ekberg. C-Peptide Binding to Human Cell Membranes: Importance of Glu27. Biochemical and Biophysical Research Communications . 2001, 284:94-98.
7王先令,陆菊明. C肽作用的研究进展.国外医学内分泌学分册, 2007, 24:4276-278.
8 Johansson BL, Kernell A, Sjoberg S, et al. Influence of combined C-peptide and insulin administration on renal-function and metabolic control in diabetes type-1. Journal of Clinical Endocrinology and Metabolism, 1993, 77(4):976-981.
9 Ido Y, Vindigni A,Chang K , et al. Prevention of vascular and neural dysfunction in diabetic rats by C-peptide. Science, 1997, 277(5325):563-566.
10赵晓龙,高鑫,刘智慧,等.C肽联合胰岛素干预治疗对大鼠早期糖尿病肾病的干预[J].中华内分泌代谢杂志, 2002, 18(4):272-273.
11 Sjoquist M, Huang W, Johansson BL. Effects of C-pep tide on renal function at early stage of experimental diabetes. Kidney Int, 1998, 54 (3) : 758.
12 Rizk N, Dunbar JC. Insulin-mediated increase in sympathetic nerve activity is attenuated by C-peptide in diabetic rats. Exp BiolMed (Maywood) , 2004, 229 (1) : 80.
13孙蔚,高鑫,刘智慧. C肽对链脲佐菌糖尿病大鼠的糖尿病肾病的干预作用[J].中华内分泌代谢杂志, 2003, 4(2):143-144.
15 Samnegard B, Jacobson SH, Jaremko G, et al. Effects of C-peptide on glomerular and renal size and renal function in diabetic rats[J]. Kindey Int, 2001, 60(4):1258-1265.
16 Johansson B L, Brog K, Fernqvist-Forbes E, et al. Beneficial effects of C-peptide on incipient nephropathy and nephropathy in patients with typeⅠdiabetes mellitus[J]. Diabete Medcine, 2000, 17:181-189.
17 Karin Ekberg, Bjorn Jonsson, John Wahren. Amelioration of Sensory Nerve Dysfunction by C-Peptide in Patients With Type 1 Diabetes[J]. DIABETES, 2003 52:536-541.
18田丽萍,张育坚,龚毅,等.人胰岛素原C肽的表达、纯化及其细胞活性研究.生物工程学报, 22(6):928-931.
19 Li ZG, Zhang WX, Sima AAF. C-peptide enhaences insulin mediated cell growth and protection against high glucose induced apoptosis in SH-Sy(5) cells. Diatetes Metabolism Research and Reviews, 2003, 19(5):375-385.
20 Li L, Oshida Y, Kusunnoki M, et al. Rats C-peptideⅠandⅡstimulate glucose utilization in STZ-induced diabetic rat [J]. Diabetologia, 1999, 42:958-964.
21 Wu W, Oshida Y, Wang W P, et al. Effect of C-peptide admistration on whole body glucose utilization in STZ-induced diabetic rats[J]. Acta Physiol Scand, 1996, 157:253-265 .
22 Forst T,Rave K, P fuetzer A, et al, Effect of C-peptide on glucose metabolism in patients with typeⅠdiabetes[J]. Diabetes Care, 2002, 25(6):1096-1097.
23 ZG L i, Qiang X, Sima AA, et al. C-peptide attenuates protein tyrosine phosphatase activity and enhances glycogen synthesis in L6 myoblasts. Biochem Biophys Res Communi, 2001, 28 (3) : 615.
24 Rigler R,Pramani kA, Jonasson P, et al. Specific binding of proinsulin C-peptide to human cell membranes[J]. Proc Natl Acad Sci USA, 1999, 96:13318-23.
25 Wahren J, Ekberg K, Johansson J, et al. Role of C-peptide in human physiology[J]. Am J Physiol Endocrinol Metab, 2000, 78:759-68.
26孟东,于德民. C肽对高糖状态下人脐静脉内皮细胞体外表达eNOS的影响.天津医药, 2004, 32(7):389-391.
27 Jan Johasson, Jawed Shafqat. Molecular effects of proinsulin C-peptide. Biochemical and Biophysical Research Communications, 2002, 295:1035-1040.
28戚楠,马清钧.长效重组蛋白药物的研究进展[J].中国生物工程杂志, 2006, 26(2): 79-82.
29王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志, 2003, 23(10): 23-27.
30 Paulina Balbás, Argelia Lorence. Recombinant Gene Expression: Reviews and Protocols [M]. 2nd ed. Totowa: Humana Press Inc.
31李育阳主编.基因表达技术[M].北京:科学出版社, 2001.77-91.
32张惠展主编.基因工程概论[M].上海:华东理工大学出版社, 1999. 197-485.
33 Romanos M. Advances in the use of Pichia pastoris for high-level gene expression [J].Current Opinion in Biotechnology, 1995, 6(5): 527-533.
34 Peter E Sudbery. The expression of recombinant proteins in yeasts [J]. Current Opinion in Biotechnology, 1996, 7: 517-524.
35 Joan LC, James MC. Heterologous protein expression in the methylotrophic yeast Pichia pastoris [J]. FEMS Microbiology Reviews, 2000, 24:45-66.
36张伍魁,范清林,宋礼华.毕赤酵母表达系统在外源基因表达中的研究进展及应用[J].中国生物工程杂志, 2006, 26(1): 87-91.
37于平.巴斯德毕赤酵母表达系统研究进展[J].工业微生物, 2005, 35(3): 50-54.
38黄石,邹民吉,徐东刚.毕赤酵母分泌表达系统的研究进展[J].医学研究通讯, 2004, 33(7): 34-36.
39李健仔.巴斯德毕赤酵母外源基因表达系统[J].生物学通报, 2005, 40(3): 21-23.
40 Joan LC, James MC. Heterologous protein expression in the methylotrophic yeast Pichia pastoris [J]. FEMS Microbiology Reviews, 2000, 24:45-66.
41赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析.生物工程学报. 2003, 16(3):308-311.
42 Invitrogen. Pichia Expression Kit: A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris.
43 Koti Sreekrinshna, Robert G, Brankamp, et al. Strategies for optimal protein expression and secretion in the methylotrophic yeast Pichia [J]. gene, 1997, 190: 55-62.
44聂东宋,梁宋平.外源蛋白在巴氏毕赤酵母中高效表达的策略[J].吉林大学学报, 2001, 22(3): 40-44.
45李洪钊,李亮助.巴斯德毕赤酵母表达系统优化策略[J].微生物学报, 2003, 43(2): 288-292.
46李欣,郭树华.影响外源基因在巴氏毕赤酵母中表达的因素[J].生物技术通讯, 2000, 11(2): 132-134,140.
47邬小兵,乐国伟.影响毕赤酵母高效表达外源蛋白的因素[J].生物技术, 2002, 12(4): 44-46.
48 Romanos M. Advances in the use of Pichia pastoris for high-level gene expression [J]. Current Opinion in Biotechnology, 1995, 6(5): 527-533.
49 Vassileva A, Chugh DA, Swaminathan S, etc. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris [J]. Protein expression and purification, 2001, 21(1): 71-80.
50马兴元,谭建华,朱平,等.巴斯德毕赤酵母表达系统及其在外源蛋白生产中的优势与应用前景[J].中国兽医学报. 2003, 23(1):98-101.
51雷楗勇,张莲芬,金坚,等.人β干扰素-血清白蛋白融合蛋白在毕赤酵母中的分泌表达.中国生物工程杂志. 2007, 26(7):13-18.
52 Koti Sreekrinshna, Robert G, Brankamp, et al. Strategies for optimal protein expression and secretion in the methylotrophic yeast Pichia [J]. gene, 1997, 190: 55-62.
53孙卫民,王惠琴主编.细胞因子研究方法学[M].北京:人民卫生出版社, 1999. 76-78.
54彭彦,王勇,宋方洲,等. IL-18在毕赤酵母中的表达和纯化[J].第四军医大学学报, 2005, 26(22):2057-2061.
55张耀东,杨伯伦.强阴离子色谱法从毕赤酵母培养液中分离纯化重组巴西日圆线虫乙酰胆碱酯酶.色谱, 2006, 24(1): 39-41.
56刘战民,陆兆新,吕凤霞,等.毕赤酵母工程菌原果胶酶的分离纯化[J].西北农林科技大学学报, 2006, 34(1): 79-83,92.
57张伟,陈劲春.蛇神经毒素在毕赤酵母中的分泌表达及其分离纯化[J].北京化工大学学报:自然科学版, 2005, 32(6): 20-22.
58刘永东,王云山,苏志国.重组人血清白蛋白生产工艺研究进展[J].微生物学通报, 2003, 30(5): 128-132.