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高比活人睫状神经营养因子突变体基因的构建及其在巴斯德毕赤酵母中的表达

Construction of superagonist mutein of human CNTF and its expression inPichia pastoris
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摘要 人睫状神经营养因子(hCNTF)及其突变体有望成为治疗肥胖症的新型药物。为了减少hCNTF的副反应,提高其疗效,在hCNTF四重突变体AX15 (R13K)的基础上引入S16 5D Q16 6H突变,构建了高比活的DH_AX15 (R13K)突变体。体外和体内实验表明DH_AX15 (R13K)的活性约是AX15 (R13K)的5倍。同时体内实验还发现DH_AX15(R13K)的作用比AX15 (R13K)更为持久。这种更为持久的作用可能是由于活性提高而非半衰期延长引起的。高比活的hCNTF突变体一方面可以在保证疗效的前提下减少蛋白用量,减少副反应;另一方面可以在不增加副反应的前提下增加最大耐受剂量,提高疗效。 Human ciliary neurotrophic factor (hCNTF) and its derivatives are promising therapeutics for obesity associated with diabetes. To reduce its side effects and increase its efficacy, superagonist mutein of human CNTF was constructed by the introduction of S165D/Q166H mutation into AX15(R13K), which is a mutein of naturally occurring hCNTF, with improved biological activity, stability, solubility and KEX2 resistance.In vitro TF_1 cell survival assay andin vivo antiobesity tests showed DH_AX(R13K) was about 5 fold more potent than AX15(R13K). It was further demonstrated that the antiobesity effect of DH_AX15(R13K) was more durable than that of AX15(R13K). The more durable effects of DH_AX15(R13K) is ascribed to its higher specific activity, but not to its prolonged half-life. The superagonist mutein of human CNTF would have an improved side effect profile and thus have superior therapeutic potential.
作者 赵洪亮 薛冲 熊向华 张伟 刘志敏
机构地区 军事医学科学院生物工程研究所
出处 《微生物学报》 CAS CSCD 北大核心 2005年第3期392-396,共5页 Acta Microbiologica Sinica
基金 国家"8 63计划"( 2 0 0 2AA2Z3 45B)~~
关键词 睫状神经营养因子 高比活突变体 巴斯德毕赤酵母 Superagonist mutein, Ciliary neurophic factor,Pichia pastoris
分类号 Q78 [生物学—分子生物学]
  • 相关文献

参考文献10

  • 1Sleeman M W, Anderson K D, Lambert P D, et al. The ciliary neurotrophic factor and its receptor, CNTFRα. Pharma Acta Helv,2000,74: 265 - 272.
  • 2Lambert P D, Anderson K D, Sleeman M W, et al. Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat,without cachexia or rebound weight gain, even in leptin-resistant obesity. Proc Natl Acad Sci USA, 2001,98(8) :4652- 4657.
  • 3Miller R G, Petajan J H, Bryan W W, et al. A placedo-controlled trial of recombinant human ciliary neurotrophic factor (rhCNTF) in Amylotrophic Lateral Sclerosis. Ann Neurol, 1996,39: 256 - 260.
  • 4Ettinger M P, Littlejohn T W, Schwartz S H, et al. Recombinant variant of ciliary neurotrophic factor for weight lose in obese adults.JAMA ,2003,289(14): 1826 - 1832.
  • 5赵洪亮,薛冲,熊向华,张伟,朱厚础,刘志敏.蛋白酶抗性人睫状神经营养因子突变体基因的构建及其在巴斯德毕赤酵母中的表达[J].生物工程学报,2004,20(3):394-397. 被引量：3
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二级参考文献13

  • 1Sleeman MW, Anderson KD, Lambert PD et al. The ciliary neurotrophic factor and its receptor, CNTFRα. Pharma Acta Helv, 2000,74(2) :265 - 272
  • 2Miller RG, Petajan JH, Bryan WW et al. A placedo-controlled trial of recombinant human ciliary neurotrophic factor (rhCNTF) in Amylotrophic Lateral Sclerosis. Ann Neurol, 1996,39(2):256-260
  • 3Lambert PD, Anderson KD, Sleeman MW et al. Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity. Proc Natl Acad Sci USA, 2001,98(8) :4652 -4657
  • 4Saggio I, Gloaguen I, Poiana G et al. CNTF variants with increased biological potency and receptor selectivity defined a function site of receptor interaction. The EMBO J, 1995,14(13) :3045 - 3054
  • 5Werten MWT, Bosch TTVD, Wind RD et al. High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast, 1999,15(6): 1087 -1096
  • 6Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Review,2000,24( 1 ) :45 -66
  • 7Boehm T, Pirie SS, Trinh LB et al. Disruption of the KEX1 gene in Pichia pastoris allows expression of full-length murine and human endostatin. Yeast, 1999,15(3) :563 - 572
  • 8Deeley, Michael C, Price et al. DNAs encoding analog GM-CSF molecules displaying resistance to proteases which cleave at adjacent dibasic residues. United States Patent 5,391,485
  • 9Lin LFH, Armes LG, Sommert A et al. Isolation and characterization of ciliary neurotrophic factor from rabbit sciatic nerves. J Biol Chem,1990,265(15) :8942 - 8947
  • 10Jordan GL, Harcum SW. Characterization of up-regulated protease in an industrial recombinant Escherichia coli fermentation. J Ind Microb Biotech, 2002,28: 74 - 80

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微生物学报
2005年 第3期

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