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白细胞介素-10基因多拷贝表达盒的构建及在毕赤酵母中的表达 被引量：11

Construction of Multiple Copy Expression Cassette for Expression of Human Interleukin-10 in Pichia pastoris
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摘要 目的构建白细胞介素-10(IL-10)基因多拷贝表达盒,提高IL-10在毕赤酵母中的表达水平。方法体外构建重组表达载体αIL-10/pAO815,BamHⅠ和BglⅡ双酶切获得目的基因表达盒(AOX-αIL-10),再连接到BglⅡ酶切位点处,依次构建多拷贝重组载体n(AOX-αIL-10)/pAO815。从质粒pPIC9K上用NdeⅠ和SalⅠ双酶切获得Kan抗性基因,重组到多拷贝表达载体n(AOX-αIL-10)/pAO815上。重组载体n(AOX-αIL-10)/pAO815电转化毕赤酵母,PCR筛选含有IL-10基因的酵母转化子,甲醇诱导表达,对表达量高的转化子再次经重组载体n(AOX-αIL-10)/pAO815-Kan电转化,高浓度G418抗性筛选二次酵母转化子,使用甲醇诱导表达。ELISA测定IL-10含量,MC/9细胞测定IL-10活性。结果所构建的8拷贝表达盒的重组载体8(AOX-αIL-10)/pAO815和4拷贝表达盒4(AOX-αIL-10)/pAO815-Kan,转化子分泌表达IL-10水平最高,为(8.25±1.65)mg/L,比活性为1.465×105U/mg。结论已成功构建了高拷贝表达盒,并提高了IL-10在毕赤酵母中的表达水平。 Objective To construct the multiple copy expression cassette for human interleukin-10（IL-10） gene and improve the expression level of the gene in Pichia pastoris. Methods Construct recombinant plasmid αIL-10/pAO815 and digest with BamH Ⅰ and Bgl Ⅱ to obtain expression cassette AOX-αIL-10 for target gene, then link to Bgl Ⅱ site of αIL-10/pAO815 to construct multiple copy expression vector n（ AOX-αIL-10）/pAO815. Digest plasmid pPIC9K with Nde Ⅰ and Sal Ⅰ to obtain Kan-resistant gene and insert into n（ AOX-αIL-10）/pAO815. Transform expression vector n（ AOX-αIL-10）/pAO815 to Pichia pastoris by electrotransformation, and screen the transformants carrying IL-10 gene by PCR for expression under induction of methanol. The transformants with high expression level were further transformed to Pichia pastoris by electrotransformation using expression vector n （AOX-αIL-10）/pAO815- Kan,and new transformants were screened with G418 at a higher concentration for expression under induction of methanol. Determine the content of expressed IL-10 by ELISA,and the activity with MC/9 cells. Results Eight copy expression cassette 8（ AOX-αIL-10）/ pAOS15 and four copy expression cassette 4（ AOX-αIL-10）/pAO815-Kan were constructed. The highest expression level of IL-10 was （ 8. 25 ± 1.65 ） mg/L, and the specific activity was 1. 465 × 10^5 U/mg. Conclusion Multiple copy expression cassettes for IL-10 were successfully constructed, and the expression level of IL-10 in Pichia pastoris was significantly improved.
作者 井申荣 邹全明 洪愉 郭刚 毛旭虎
机构地区 第三军医大学医学检验系临床微生物及免疫学教研室
出处 《中国生物制品学杂志》 CAS CSCD 2007年第2期81-86,共6页 Chinese Journal of Biologicals
关键词 白细胞介素-10 毕赤酵母 多拷贝表达盒 Interleukin-10 Pichia pastoris Multiple copy expression cassette
分类号 R392.11 [医药卫生—免疫学] Q786 [生物学—分子生物学]
  • 相关文献

参考文献9

  • 1Asadullah K,Sterry W,Volk HD.Interleukin-10 therapy-review of a new approach.Pharmacol Rev,2003,55(2):241-269.
  • 2Moore KW,De Waal Malefyt R,Coffman RL,et al.Interleukin-10 and the interleukin-10 receptor.Annu Rev Immunol,2001,19 (3):683-765.
  • 3Lee SW,Hong YS,Chun CM,et al.Anti-inflammatory effects of IL-4 and IL-10 on human polymorphonuclear leukocytes.J Korean Med Sci,2002,17(1):7-14.
  • 4井申荣,邹全明,曾韦锟,毛旭虎,刘开云.重组人白细胞介素10在毕赤酵母中的表达及生物学活性测定[J].中华微生物学和免疫学杂志,2005,25(9):773-776. 被引量：5
  • 5Itvitrogen C.A manual of methods for the isolation and expression of recombinant proteins from Pichia pastoris strains containing multiple copies of a particular gene.2000.
  • 6Thompson-Snipes L,Dhar V,Bond MW,et al.Interleukin 10:a novel stimulatory factor for mast cells and their progenitors.J Exp Med,1991,173(2):507-510.
  • 7Vassileva A,Chugh DA,Swaminathan S,et al.Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris.Protein Expr Purif,2001,21(1):71-80.
  • 8Sreekrishna K,Brankamp RG,Kropp KE,et al.Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.Gene,1997,190(1):55-62.
  • 9Pans EJ,Willey J,Ridge RJ,et al.Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification.Protein Expr Purif,2002,26(2):202-210.

二级参考文献14

  • 1Moore KW, de Waal Malefyt R, Coffman RL, et al. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol, 2001, 19: 683-765.
  • 2Rosenblum IY, Johnson RC, Schmahai TJ. Preclinical safety evaluation of recombinant human interleukin-10. Regul Toxicol Pharmacol, 2002, 35:56-71.
  • 3Bondoc LL Jr, Varnerin JP, Tang JC. Resolution ofrecombinant human interleukin 10 from variants by recycling free flow focusing. Anal Biochem, 1997, 246: 234-238.
  • 4Menassa R, Kennette W, Nguyen V, et al. Subcellular targeting of human interleukin-10 in plants. J Biotechnol, 2004, 108: 179-183.
  • 5Invitrogen cooperation. A manual of methods for the isolation and expression of recombinant proteins from Pichia pastoris strains containing multiple copies of a particular gene. Version E. 2000.
  • 6Thompson-Snipes L, Dhar V, Bond MW, et al. Interleukin 10: a novel stimulatory factor for mast cells and their progenitors. J Exp Med 1991,173: 507-510.
  • 7Conti P, Kempuraj D, Kandere K, et al. IL-10, an inflammatory/inhibitory cytokine, but not always. mmuol Lett, 2003, 86: 123-129.
  • 8Asadullah K, Sterry W, Volk HD. Interleukin-10 therapy--review of a new approach. Pharmacol Rev, 2003, 55: 241-269.
  • 9Menassa R, Kennette W, Nguyen V, et al. Subcellular targeting of human interleukin-10 in plants. J Biotechnol,2004, 108(2): 179-183.
  • 10Kim TR, Goto Y, Hirota N, et al. High-level expression of bovine betalactoglobulin in Pichia pastoris and characterization of its physical properties. Protein Eng, 1997, 10: 1339-1345.

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同被引文献156

引证文献11

二级引证文献51

中国生物制品学杂志
2007年 第2期

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