表达纤溶酶抑制剂Textilinin-1的毕赤酵母工程菌遗传稳定性分析
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摘要
目的分析表达纤溶酶抑制剂Textilinin-1的毕赤酵母工程菌的遗传稳定性,为今后可能的大规模生产奠定基础。方法将分泌表达纤溶酶抑制剂Textilinin-1的毕赤酵母工程菌在YPD液体培养基中连续培养200代,对原始工程菌及第200代工程菌的菌落和细胞形态、分泌表达情况以及目的基因的拷贝数等进行检测。结果第200代工程菌在菌落特征、菌体形态、分泌表达情况、Textilinin-1基因序列及拷贝数等均与原始工程菌一致。结论该毕赤酵母工程菌具有良好的遗传稳定性,适合大规模生产。
Objective To analyze the genetic stability of recombinant Pichia pastoris strain for expression of Textilinin-1,a plasmin inhibitor,and lay a foundation of large-scale production in the future. Methods Recombinant P. pastroris strain for expression of Textilinin-1 was subcultured in YPD liquid medium for 200 passages. The original strain and the strain of passage 200 were observed for the morphology of colony and cells,and determined for secretory expression of target protein and copy number of target gene. Results The colony characters,cell morphology,secretory expression of target protein as well as sequence and copy number of Textilinin-1 gene of recombinant P. pastroris strain of passage 200 were consistent with those of original strain. Conclusion The recombinant P. pastroris recombinant strain for expression of Textilinin-1 showed high genetic stability,which was suitable for large-scale production.
Objective To analyze the genetic stability of recombinant Pichia pastoris strain for expression of Textilinin-1,a plasmin inhibitor,and lay a foundation of large-scale production in the future. Methods Recombinant P. pastroris strain for expression of Textilinin-1 was subcultured in YPD liquid medium for 200 passages. The original strain and the strain of passage 200 were observed for the morphology of colony and cells,and determined for secretory expression of target protein and copy number of target gene. Results The colony characters,cell morphology,secretory expression of target protein as well as sequence and copy number of Textilinin-1 gene of recombinant P. pastroris strain of passage 200 were consistent with those of original strain. Conclusion The recombinant P. pastroris recombinant strain for expression of Textilinin-1 showed high genetic stability,which was suitable for large-scale production.
引文
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[8]王军志.生物技术药物研究开发和质量控制[M]. 2版.北京:科学出版社,2007:168.
[9] LI J,FAN C S,ZHU N S. Study on the genetic stability of the recombinant Pichia pastoris strain expressing HBsAg binding protein[J]. Pharm Biotechnol,2014,21(1):18-21.(in Chinese)李津,范长胜,朱乃硕.表达乙肝表面抗原结合蛋白的毕赤酵母工程菌遗传稳定性研究[J].药物生物技术,2014,21(1):18-21.
[10] JIANG L M,TAN C Y,HU B,et al. Genetic and expression stability of recombinant Pichia pastoris expressing hepatitis B surface antigen containing PreS1,PreS2 and S epitopes[J].Chin J Biologicals,2008,21(6):486-488.蒋丽明,谭昌耀,胡波,等.乙型肝炎病毒前S1、前S2和S抗原重组毕赤酵母表达菌株的基因和表达稳定性[J].中国生物制品学杂志,2008,21(6):486-488.
[2] MILLERS E K,MASCI P P,LAVIN M F,et al. Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor,textilinin-1 from Pseudonaja textilis textilis[J]. Acta Cryst,2010,62(7):642-645.
[3] WANG H Y,LI N,LI X N,et al. Expression and application of textilinin-1,a plasmin inhibitor,in Pichia pastoris[J]. J SNAKE(Sci Nat),2014,26(3):273-277.(in Chinese)王宏英,李娜,李秀娜,等.纤溶酶抑制剂textilinin-1在毕氏酵母的表达及应用[J].蛇志,2014,26(3):273-277.
[4] CEREGHINO J L,CREGG J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMS Microbiol Rev,2000,24(1):45-66.
[5] LIN F L,ZHOU H B,ZHENG J,et al. Determination of gene copy numbers of exogenousβ-mannanase in Pichia pastoris by RT-PCR[J]. Chin J Bioproc Engi,2014,12(5):46-50.(in Chinese)林福来,周洪波,郑甲,等. RT-PCR检测毕赤酵母外源β-甘露聚糖酶基因拷贝数[J].生物加工过程,2014,12(5):46-50.
[6] XU L H,LIU C L,CHANG Y M,et al. Theory and method of double-standard curves method of relative quantification PCR[J]. Biotechnol Bull,2011,1:70-75.(in Chinese)徐丽华,刘春雷,常玉梅,等.双标准曲线相对定量PCR试验原理与方法[J].生物技术通报,2011,1:70-75.
[7] YAO J X,ZHOU X S,ZHANG Y X. Determination of copy number of foreign gene in genome of Pichia pastoris by real-time fluorescent quantitative PCR[J]. Chin J Biologicals,2009,22(12):1236-1243.(in Chinese)宣姚吉,周祥山,张元兴.实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数[J].中国生物制品学杂志,2009,22(12):1236-1243.
[8]王军志.生物技术药物研究开发和质量控制[M]. 2版.北京:科学出版社,2007:168.
[9] LI J,FAN C S,ZHU N S. Study on the genetic stability of the recombinant Pichia pastoris strain expressing HBsAg binding protein[J]. Pharm Biotechnol,2014,21(1):18-21.(in Chinese)李津,范长胜,朱乃硕.表达乙肝表面抗原结合蛋白的毕赤酵母工程菌遗传稳定性研究[J].药物生物技术,2014,21(1):18-21.
[10] JIANG L M,TAN C Y,HU B,et al. Genetic and expression stability of recombinant Pichia pastoris expressing hepatitis B surface antigen containing PreS1,PreS2 and S epitopes[J].Chin J Biologicals,2008,21(6):486-488.蒋丽明,谭昌耀,胡波,等.乙型肝炎病毒前S1、前S2和S抗原重组毕赤酵母表达菌株的基因和表达稳定性[J].中国生物制品学杂志,2008,21(6):486-488.