重组chi46在毕赤酵母中的表达及抗病原菌初步研究
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摘要
球毛壳菌(Chaetomium globosum)是一种应用前景广阔的植物病害生物防治菌,含有很多优良的生物防治基因,球毛壳菌46kD几丁质酶(chi46)就是其中之一。几丁质酶(chitinase Ec.3.2.14)能够催化病原真菌细胞壁几丁质水解为几丁寡糖和N-乙酰氨基葡萄糖,广泛存在于各种微生物、植物及动物细胞和组织中,参与多种生理过程。几丁质酶在生物防治和几丁质资源的利用等方面具有巨大的应用潜力,因而受到了广泛的重视。
为了利用重组几丁质酶生产广谱抗性的生物农药,鉴于毕赤酵母(Pichia pastoris)易于培养、外源蛋白表达量高等特点,故本研究采用毕赤酵母分泌表达系统作为表达生产工具,从已构建好的质粒pGEX-chi中通过设计的引物扩增出球毛壳菌chi46基因,将经EcoR I和Not I双酶切的chi46基因和酵母表达载体pPIC9进行体外连接,构建酵母重组表达载体,经过菌液PCR检测,双酶切检测并测序,得到有正确阅读框的重组表达质粒pPIC9-chi6。
将pPIC9-chi6和pPIC9空质粒分别用限制性内切酶Stu I线性化后,采用电转化的方法分别转化GS115和KM71酵母感受态细胞,经过甲醇利用表型的筛选和PCR检测筛选得到2株高效利用甲醇GS115型和3株低效利用甲醇KM71型阳性转化子。通过检测各转化子发酵液上清每隔24h的蛋白表达情况和酶活情况筛选出高效表达CHI46的工程菌株G6-5和K6-9。
对两株工程菌株分泌的重组CHI46粗酶液进行了酶活测定与酶学特性研究,同时对于重组CHI46的抑菌性进行了初步摸索。发现GS115转化子G6-5比KM71转化子K6-9的产酶高峰出现晚3d左右,产酶活力较后者高8.24%。重组CHI46在35℃-65℃的温度范围内,保持着相对稳定的酶活性;重组CHI46亦有很广的pH耐受范围,在pH3-10有较稳定的酶活力。这揭示出它生防领域中良好的应用前景。确定酶系统发挥活性的最佳组合,即反应体系Cu2+ 5mmol/L,温度45℃,pH值5.0,依照该体系以N.O-羧甲基壳聚糖为底物测得的重组CHI46发酵液酶活能达到30.19U。对不同底物的酶活测定结果表明,重组CHI46对于真菌细胞壁的酶活普遍较化合物高。
重组CHI46粗酶液对立枯丝核菌(Rhizoctonia solani)、杨树叶枯病菌(Alternaria alternata)、杨树烂皮病菌(Cytospora chrysosperma)的生长具有一定抑制作用,从而证明了重组CHI46具有降解病原真菌细胞壁的能力。
几丁质酶的研究是一项具有重要经济价值与理论价值的研究课题。球毛壳几丁质酶能在毕赤酵母中分泌出具生物活性的目的蛋白,而且表达产物同样具有几丁质酶抑制真菌生长的优良特性,这就预示了表达几丁质酶在几丁质工业和生物学领域的广阔应用前景。因此,期望能对该基因进行改造,寻找合适的发酵工艺、探索使酶活稳定的方法,获得真正有工业应用价值的酵母工程菌株,生产针对植物真菌病害防治的生物农药。
Chaetomium globosum is an important plant biocontrol fungus with wide application. C. globosum possesses many important enzymes genes that have tremendous value in biocontrol, one of which is a 46kD endochitinase gene (chi46). Chitinase (Ec.3.2.14), a group of enzymes capable of degrading pathogenic fungi chitin directly to chitin oligosaccharides or N-acetylglucosamine, are produced by a wide variety of organisms including plants, microorganism and animals, and contribute to many physiological reaction. Chitinase have received increased attention because of their potential application in biocontrol of plant-pathogenic fungi and pests, as well as in bioconversion of shellfish chitin wastes.
In order to use the biocontrol potential of chi46 to produce broad-spectrum resistant Biopesticide, this study used Pichia pastoris as expression system,which was easy to culture and expressed high-level recombinant proteins. After amplifying the chitinase gene from pGEX-chi with designed primers, The chi46 gene and expression vector pPIC9 were digested with EcoR I and Not I,and ligation was carried out in vitro. Detected by PCR, double enzyme digestion and sequence, the recombinant expression plasmid with correct reading frame pPIC9-chi6 was constructed.
The constructed plasmid pPIC9-chi6 was linearized with a restriction enzyme Stu I and transformed into Pichia GS115 and KM71 competent cell by electransformation method. Detected by Methanol-utilizing phenotype screening and PCR analysis, two GS115 (Mut+) transformants and three KM71 (Muts) transformants was got. The parent vector was linearized with the same restriction enzyme and transformed to GS115 and KM71 as control. Induced by methanol, these integrants were analyzed expression levels every 24 hours of interest protein by SDS-PAGE and the engineering strains with high chitinase expression level, such as G6-5 and K6-9 was determined.
We carried out some study on bioactivities of chitinase including enzyme activity, some enzyme characteristics and fungi-repressing test. Research found that the optimum fermentation days for GS115 transformant of G6-5 was 3 days later than that for KM71transformant of K6-9, but enzyme activity of G6-5 was 8.24% higher than K6-9. Recombinant CHI46 remains relatively stable enzyme activity at 35℃-65℃and pH3-10, which indicates its good prospect of application in biocontrol.The best combination of enzyme system was gotten, that was Cu2+ 5mmol/L, 45℃, pH 5.0, and in the condition of which the recombinant CHI46 activity with N,O-carboxymethyl chitosan as the substrate was 30.19U Measureing enzyme cativity with different substrates, we found that recombinant CHI46 activities with fungi cell wall as the substrate were much higher than those with compounds.
Recombinant CHI46 inhibited the growth of Rhizoctonia solani, Alternaria alternata and Cytospora chrysosperma, that indicatd the recombinant CHI46 had the capability of degrading cell wall of fungal pathogen.
The research of CHI46 has great value in economy and theory. The Chaetomium globosum chitinase was secreted into the culture medium by the yeast Pichia pastoris in a functionally activity form, and the expression chitinase can inhibit the growth of some fungi. It implied the expression chitinase could be applied widely for the chitin industry and biological fields. Therefore, we hope to reconstruct the chi46 gene, exploration of appropriate methord of fermentation and enzyme activity stabilization to obtain the yeast engineering strains suitable for industrial application and produce bio-pesticide for biological control of plant diseases.
为了利用重组几丁质酶生产广谱抗性的生物农药,鉴于毕赤酵母(Pichia pastoris)易于培养、外源蛋白表达量高等特点,故本研究采用毕赤酵母分泌表达系统作为表达生产工具,从已构建好的质粒pGEX-chi中通过设计的引物扩增出球毛壳菌chi46基因,将经EcoR I和Not I双酶切的chi46基因和酵母表达载体pPIC9进行体外连接,构建酵母重组表达载体,经过菌液PCR检测,双酶切检测并测序,得到有正确阅读框的重组表达质粒pPIC9-chi6。
将pPIC9-chi6和pPIC9空质粒分别用限制性内切酶Stu I线性化后,采用电转化的方法分别转化GS115和KM71酵母感受态细胞,经过甲醇利用表型的筛选和PCR检测筛选得到2株高效利用甲醇GS115型和3株低效利用甲醇KM71型阳性转化子。通过检测各转化子发酵液上清每隔24h的蛋白表达情况和酶活情况筛选出高效表达CHI46的工程菌株G6-5和K6-9。
对两株工程菌株分泌的重组CHI46粗酶液进行了酶活测定与酶学特性研究,同时对于重组CHI46的抑菌性进行了初步摸索。发现GS115转化子G6-5比KM71转化子K6-9的产酶高峰出现晚3d左右,产酶活力较后者高8.24%。重组CHI46在35℃-65℃的温度范围内,保持着相对稳定的酶活性;重组CHI46亦有很广的pH耐受范围,在pH3-10有较稳定的酶活力。这揭示出它生防领域中良好的应用前景。确定酶系统发挥活性的最佳组合,即反应体系Cu2+ 5mmol/L,温度45℃,pH值5.0,依照该体系以N.O-羧甲基壳聚糖为底物测得的重组CHI46发酵液酶活能达到30.19U。对不同底物的酶活测定结果表明,重组CHI46对于真菌细胞壁的酶活普遍较化合物高。
重组CHI46粗酶液对立枯丝核菌(Rhizoctonia solani)、杨树叶枯病菌(Alternaria alternata)、杨树烂皮病菌(Cytospora chrysosperma)的生长具有一定抑制作用,从而证明了重组CHI46具有降解病原真菌细胞壁的能力。
几丁质酶的研究是一项具有重要经济价值与理论价值的研究课题。球毛壳几丁质酶能在毕赤酵母中分泌出具生物活性的目的蛋白,而且表达产物同样具有几丁质酶抑制真菌生长的优良特性,这就预示了表达几丁质酶在几丁质工业和生物学领域的广阔应用前景。因此,期望能对该基因进行改造,寻找合适的发酵工艺、探索使酶活稳定的方法,获得真正有工业应用价值的酵母工程菌株,生产针对植物真菌病害防治的生物农药。
Chaetomium globosum is an important plant biocontrol fungus with wide application. C. globosum possesses many important enzymes genes that have tremendous value in biocontrol, one of which is a 46kD endochitinase gene (chi46). Chitinase (Ec.3.2.14), a group of enzymes capable of degrading pathogenic fungi chitin directly to chitin oligosaccharides or N-acetylglucosamine, are produced by a wide variety of organisms including plants, microorganism and animals, and contribute to many physiological reaction. Chitinase have received increased attention because of their potential application in biocontrol of plant-pathogenic fungi and pests, as well as in bioconversion of shellfish chitin wastes.
In order to use the biocontrol potential of chi46 to produce broad-spectrum resistant Biopesticide, this study used Pichia pastoris as expression system,which was easy to culture and expressed high-level recombinant proteins. After amplifying the chitinase gene from pGEX-chi with designed primers, The chi46 gene and expression vector pPIC9 were digested with EcoR I and Not I,and ligation was carried out in vitro. Detected by PCR, double enzyme digestion and sequence, the recombinant expression plasmid with correct reading frame pPIC9-chi6 was constructed.
The constructed plasmid pPIC9-chi6 was linearized with a restriction enzyme Stu I and transformed into Pichia GS115 and KM71 competent cell by electransformation method. Detected by Methanol-utilizing phenotype screening and PCR analysis, two GS115 (Mut+) transformants and three KM71 (Muts) transformants was got. The parent vector was linearized with the same restriction enzyme and transformed to GS115 and KM71 as control. Induced by methanol, these integrants were analyzed expression levels every 24 hours of interest protein by SDS-PAGE and the engineering strains with high chitinase expression level, such as G6-5 and K6-9 was determined.
We carried out some study on bioactivities of chitinase including enzyme activity, some enzyme characteristics and fungi-repressing test. Research found that the optimum fermentation days for GS115 transformant of G6-5 was 3 days later than that for KM71transformant of K6-9, but enzyme activity of G6-5 was 8.24% higher than K6-9. Recombinant CHI46 remains relatively stable enzyme activity at 35℃-65℃and pH3-10, which indicates its good prospect of application in biocontrol.The best combination of enzyme system was gotten, that was Cu2+ 5mmol/L, 45℃, pH 5.0, and in the condition of which the recombinant CHI46 activity with N,O-carboxymethyl chitosan as the substrate was 30.19U Measureing enzyme cativity with different substrates, we found that recombinant CHI46 activities with fungi cell wall as the substrate were much higher than those with compounds.
Recombinant CHI46 inhibited the growth of Rhizoctonia solani, Alternaria alternata and Cytospora chrysosperma, that indicatd the recombinant CHI46 had the capability of degrading cell wall of fungal pathogen.
The research of CHI46 has great value in economy and theory. The Chaetomium globosum chitinase was secreted into the culture medium by the yeast Pichia pastoris in a functionally activity form, and the expression chitinase can inhibit the growth of some fungi. It implied the expression chitinase could be applied widely for the chitin industry and biological fields. Therefore, we hope to reconstruct the chi46 gene, exploration of appropriate methord of fermentation and enzyme activity stabilization to obtain the yeast engineering strains suitable for industrial application and produce bio-pesticide for biological control of plant diseases.
引文
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61 S. Zeilinger, C. Galhaup, K. Payer. Chitinase Gene Expression during Mycoparasitic Interaction of Trichoderma harzianum with Its Host. Fungal Genet Biol. 1999, 26(2):131-140
62 Y. Zhu, E. A. Maher, S. Masoud, et al. Enhanced Protection Against Fungal Attack by Constitutive Coexpression of Chitinase and Glucanase Genes in Transgenic Tobacco. Bio/Technology. 1994, 12(8):807–812
63 N. Rattanakit, A. Plikomol, S. Yano, et al. Utilization of Shrimp Shellfish Waste as a Substrate for Solid-state Cultivation of Aspergillus sp. S1-13: Evaluation of a Culture Based on Chitinase Formation Which is Necessary for Chitin-Assimilation. J Biosci Bioeng. 2002, 93(6):550-556
64 J. K Sharp, B. Valent, P. Albersheim. Purification and Partial Charactrization of aβ–glucan Fragment that Elicits Phytoalexin Accumulation in Soybean. J Biol Chem. 1984, 259(18):11312-11320
65 K. Tsukada, M. Ishizaka, T. Yamaguchi, et a1. Rice Receptor for Chitin Oligosaccharide Elicitor does not Couple to Heterotrimeric G-protein: Elicitor Responses of Suspension Cultured Rice Cells from Daikoku Dwarf(d1) Mutants Lacking a Functional G-protein a Subunit. Physiol Plant. 2002, 166(10):373-382
66张虎,杜昱光,虞星炬.几丁寡糖与壳寡糖的制备和功能.中国生化药物杂志. 1999, 20(2):99-101
67竺国芳,赵鲁杭.几丁寡糖和壳聚糖的研究进展.中国海洋药物. 2000, 19(1): 43-46
68 J. J. Clare, F. B. Rayment, S. P. Ballantine, et al. Hight– level Expression of Tetanus Toxin Fragment C in pichia pastoris Strains Containing Multiple Tandem Integration of the Gene. Bil/technology. 1991, 9:455-460
69 M. Hasslacher, M. Schall, M. Hayn, et al. High-level Intreacllular Expression of Hydroxynnitrile Lyase from the Tropical Rubber Tree Hevea brasiliensis in Microbial Hosts. Protein Expre Purif. 1997, 11(1):61-71
70 J. M. Cregg, J. L. Cereghino, J. Shi, et al. Recombinant Protein Expression in Pichia pastoris. Mol Biotechnol. 2000, 16(1):23-52
71 J. L. Cereghino, J. M. Cregg. Heterologous Protein Expression in the Methylotrop- hic Yeast Pichia pastoris. FEMS Microbiol Rev. 2000, 24(1):45-66
72 C. P. Hollenberg, G. Gellissen. Production of Recombinant Proteins by Methlotrophic Yeasts. Curr Opin Biotechnol. 1997, 8(5):554-560
73 I. J. Clare, M. A. Romanos, F. B. Rayment, et al. Production of Epidermal Growth Factor in Yeast: High Level Secretion Using Pichia pastoris Strains Containing Multiple Gene Copies. Gene. 1991, 105(2): 205-212
74 M. A. Romanos, I. J. Clare, K. M. Beesley, et al. Recombinant Bordetella Pertussispertactin p69 from the Yeast Pichia pastoris High Level Production and Immunological Properties. Vaccine. 1991, 9:901-906
75 J. M. Cregg, J. F. Tschopp, C. Stillman, et al. High-level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast Pichia pastoris. Bio/Technology. 1987, 5:479-485
76 J. J. Clare, M. A. Romanos , F. B. Rayment, et al. Production of Mouse Epidermal Growth Factor in Yeast: High-level Secretion Using Pichia pastoris Strains Containing Multiple Gene Copies. Gene. 1991, 105(2):205-212
77 M. E.Digan, S. V. Lair, R. A. Brierley, et al. Continuous Production of a Novel Lysozyme via Secretion from the Yeast Pichia pastoris. Bio/Technology. 1989, 7:160-164
78 E. Paifer, E. Margolles, J. Cremate, et al. Efficient Expression and Secretion of Recombinant Alpha Mmylase in Pichia pastoris Using Two Signal Sequences. Yeast. 1994, 10(11):1415-1419.
79 Guo W, L. Gonzalez-Candelas, P. E. Kolattukudy. Cloning of a Novel Constitutively Expressed Pectate Lyase Gene pelB from Fusarium solanif sp. Pisi and Characterization of the Gene Product Expressed in Pichia pastoris. J Bacteriol. 1995, 177(24):7070-7077
80 N. V.Paige, H. Frantisek. High Level Expression of Human Liver Monoamine Oxidase Bin Pichia pastoris. Protein Expression Purification. 2000, 20(2): 334-345
81 T. Kjeldsen. Yeast Secretory Expression of Insulin Precursors. Appl Microbiol Biotechnol. 2000, 54 (3):277-286
82 G. C. Sen, R. R. Dighe. Hyper Expression of Biologically Active Human Chorionic Gonadotrop in Using the Methylotropic Yeast Pichia pastoris. J Mol Endocrinol.1999, 22(3):273-283
83吴玉水,任君萍,黄庆生,等.乙型脑炎病毒E蛋白在毕赤酵母中的表达.细胞与分子免疫学杂志. 2003, 19(2):130-135
84覃雅丽,陈焕春,唐勇,等.伪狂犬病病毒Ea株蟒基因主要抗原区在毕赤酵母中的表达.微生物学报. 2002, 42(5):543-549
85钱平,周艳君,童光志,等.猪繁殖与呼吸综合征病毒ORF6基因在毕赤酵母中的表达.中国预防兽医学报. 2002, 24(3):161-164
86苏畅,夏文水,惠源.氨基葡萄糖和乙酰氨基葡萄糖的测定方法.食品工业科技. 2003, 24(6):74-77
87 F. Villatte, A. S. Hussein, T. T. Bachmano. Expression Level of Heterologous Proteins in Pichia Pastoris is Influenced by Flask Design. Appl Microbiol Biotechnol. 2001, 55(4):463-465
88 J. Xie, L. Zhang, Q. Ye, et al. Angiostatin Production in Cultivation of Recombinant Pichia pastoris Fed with Mixed Carbon Sources. Biotechnol Lett. 2003, 25(2):173-177
89 M. Romanos. Advances in the Use of Pichia pastoris for High-level Gene Expression.Current Opinion in Biotechnology. 1995, 6(5):527-533
90唐元家,余柏松.巴斯德毕赤酵母表达系统.国外医药抗生素分册. 2002, 23(6):246
91王书.白假丝酵母几丁质酶cDNA的克隆及其在大肠杆菌和毕赤酵母中表达的研究.辽宁师范大学硕士学位论文. 2000:38-39
92 H. Hohenblum, B. Gasser, M. Maurer, et al. Effects of Gene Dosage, Promoters, and Substrates on Unfolded Protein Stress of Recombinant Pichia pastoris. Biotechnol. Bioeng. 2004, 85(4):367-375
93陶刚,刘杏忠,王革,等.木霉几丁质酶对烟草赤星病菌的作用.中国生物防治. 2004, 20(4):252-255
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55李华,刘开启,王革.微生物几丁质酶在植物病害防治中的应用.仲恺农业技术学院学报. 2003, 16(1):54-60
56徐同,柳良好.木霉几丁质酶及其对植物病源真菌的拮抗作用.植物病理学报. 2002, 32(2):97-102
57 H. M. Toyoda, T. Yamaga, S. Ikeda, et al. Suppression of the Powdery Mildew Pathogen by Chitinase Microinjected into Barley Coleoptile Epidermal Cells. Plant Cell Reports. 1991, 10:217-2120
58 R. Shapira. Control of Plant Diseases by Chitinase Expression from Cloned DNA in Escherichia coli. Phytopathology. 1999, 79:1240-1249
59 Lorito Mass. Microbial genes expressed in transgenic plants to improvedesease resistance. Journal of Plant Pathology. 1999, 81 (2):73-88
60 T. Terakawa, H. Horiuchi, M. Koike. A Fungal Chitinase Gene from Rhizopus Oligosporus Confers Antifungal Activity to Transgenic Tobacco. Plant Cell Reports. 1997, 16:439-443
61 S. Zeilinger, C. Galhaup, K. Payer. Chitinase Gene Expression during Mycoparasitic Interaction of Trichoderma harzianum with Its Host. Fungal Genet Biol. 1999, 26(2):131-140
62 Y. Zhu, E. A. Maher, S. Masoud, et al. Enhanced Protection Against Fungal Attack by Constitutive Coexpression of Chitinase and Glucanase Genes in Transgenic Tobacco. Bio/Technology. 1994, 12(8):807–812
63 N. Rattanakit, A. Plikomol, S. Yano, et al. Utilization of Shrimp Shellfish Waste as a Substrate for Solid-state Cultivation of Aspergillus sp. S1-13: Evaluation of a Culture Based on Chitinase Formation Which is Necessary for Chitin-Assimilation. J Biosci Bioeng. 2002, 93(6):550-556
64 J. K Sharp, B. Valent, P. Albersheim. Purification and Partial Charactrization of aβ–glucan Fragment that Elicits Phytoalexin Accumulation in Soybean. J Biol Chem. 1984, 259(18):11312-11320
65 K. Tsukada, M. Ishizaka, T. Yamaguchi, et a1. Rice Receptor for Chitin Oligosaccharide Elicitor does not Couple to Heterotrimeric G-protein: Elicitor Responses of Suspension Cultured Rice Cells from Daikoku Dwarf(d1) Mutants Lacking a Functional G-protein a Subunit. Physiol Plant. 2002, 166(10):373-382
66张虎,杜昱光,虞星炬.几丁寡糖与壳寡糖的制备和功能.中国生化药物杂志. 1999, 20(2):99-101
67竺国芳,赵鲁杭.几丁寡糖和壳聚糖的研究进展.中国海洋药物. 2000, 19(1): 43-46
68 J. J. Clare, F. B. Rayment, S. P. Ballantine, et al. Hight– level Expression of Tetanus Toxin Fragment C in pichia pastoris Strains Containing Multiple Tandem Integration of the Gene. Bil/technology. 1991, 9:455-460
69 M. Hasslacher, M. Schall, M. Hayn, et al. High-level Intreacllular Expression of Hydroxynnitrile Lyase from the Tropical Rubber Tree Hevea brasiliensis in Microbial Hosts. Protein Expre Purif. 1997, 11(1):61-71
70 J. M. Cregg, J. L. Cereghino, J. Shi, et al. Recombinant Protein Expression in Pichia pastoris. Mol Biotechnol. 2000, 16(1):23-52
71 J. L. Cereghino, J. M. Cregg. Heterologous Protein Expression in the Methylotrop- hic Yeast Pichia pastoris. FEMS Microbiol Rev. 2000, 24(1):45-66
72 C. P. Hollenberg, G. Gellissen. Production of Recombinant Proteins by Methlotrophic Yeasts. Curr Opin Biotechnol. 1997, 8(5):554-560
73 I. J. Clare, M. A. Romanos, F. B. Rayment, et al. Production of Epidermal Growth Factor in Yeast: High Level Secretion Using Pichia pastoris Strains Containing Multiple Gene Copies. Gene. 1991, 105(2): 205-212
74 M. A. Romanos, I. J. Clare, K. M. Beesley, et al. Recombinant Bordetella Pertussispertactin p69 from the Yeast Pichia pastoris High Level Production and Immunological Properties. Vaccine. 1991, 9:901-906
75 J. M. Cregg, J. F. Tschopp, C. Stillman, et al. High-level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast Pichia pastoris. Bio/Technology. 1987, 5:479-485
76 J. J. Clare, M. A. Romanos , F. B. Rayment, et al. Production of Mouse Epidermal Growth Factor in Yeast: High-level Secretion Using Pichia pastoris Strains Containing Multiple Gene Copies. Gene. 1991, 105(2):205-212
77 M. E.Digan, S. V. Lair, R. A. Brierley, et al. Continuous Production of a Novel Lysozyme via Secretion from the Yeast Pichia pastoris. Bio/Technology. 1989, 7:160-164
78 E. Paifer, E. Margolles, J. Cremate, et al. Efficient Expression and Secretion of Recombinant Alpha Mmylase in Pichia pastoris Using Two Signal Sequences. Yeast. 1994, 10(11):1415-1419.
79 Guo W, L. Gonzalez-Candelas, P. E. Kolattukudy. Cloning of a Novel Constitutively Expressed Pectate Lyase Gene pelB from Fusarium solanif sp. Pisi and Characterization of the Gene Product Expressed in Pichia pastoris. J Bacteriol. 1995, 177(24):7070-7077
80 N. V.Paige, H. Frantisek. High Level Expression of Human Liver Monoamine Oxidase Bin Pichia pastoris. Protein Expression Purification. 2000, 20(2): 334-345
81 T. Kjeldsen. Yeast Secretory Expression of Insulin Precursors. Appl Microbiol Biotechnol. 2000, 54 (3):277-286
82 G. C. Sen, R. R. Dighe. Hyper Expression of Biologically Active Human Chorionic Gonadotrop in Using the Methylotropic Yeast Pichia pastoris. J Mol Endocrinol.1999, 22(3):273-283
83吴玉水,任君萍,黄庆生,等.乙型脑炎病毒E蛋白在毕赤酵母中的表达.细胞与分子免疫学杂志. 2003, 19(2):130-135
84覃雅丽,陈焕春,唐勇,等.伪狂犬病病毒Ea株蟒基因主要抗原区在毕赤酵母中的表达.微生物学报. 2002, 42(5):543-549
85钱平,周艳君,童光志,等.猪繁殖与呼吸综合征病毒ORF6基因在毕赤酵母中的表达.中国预防兽医学报. 2002, 24(3):161-164
86苏畅,夏文水,惠源.氨基葡萄糖和乙酰氨基葡萄糖的测定方法.食品工业科技. 2003, 24(6):74-77
87 F. Villatte, A. S. Hussein, T. T. Bachmano. Expression Level of Heterologous Proteins in Pichia Pastoris is Influenced by Flask Design. Appl Microbiol Biotechnol. 2001, 55(4):463-465
88 J. Xie, L. Zhang, Q. Ye, et al. Angiostatin Production in Cultivation of Recombinant Pichia pastoris Fed with Mixed Carbon Sources. Biotechnol Lett. 2003, 25(2):173-177
89 M. Romanos. Advances in the Use of Pichia pastoris for High-level Gene Expression.Current Opinion in Biotechnology. 1995, 6(5):527-533
90唐元家,余柏松.巴斯德毕赤酵母表达系统.国外医药抗生素分册. 2002, 23(6):246
91王书.白假丝酵母几丁质酶cDNA的克隆及其在大肠杆菌和毕赤酵母中表达的研究.辽宁师范大学硕士学位论文. 2000:38-39
92 H. Hohenblum, B. Gasser, M. Maurer, et al. Effects of Gene Dosage, Promoters, and Substrates on Unfolded Protein Stress of Recombinant Pichia pastoris. Biotechnol. Bioeng. 2004, 85(4):367-375
93陶刚,刘杏忠,王革,等.木霉几丁质酶对烟草赤星病菌的作用.中国生物防治. 2004, 20(4):252-255
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