Notch配体Delta-like1、Delta-like4蛋白的表达及生物活性的测定
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摘要
Notch信号途径广泛存在于多种生物体内,且进化上高度保守,在胚胎发育、血细胞发育及肿瘤形成等多种病理生理过程中发挥重要作用。Notch信号在造血系统发育中起着十分重要的作用。近来发现,人造血干/祖细胞表面存在Notch受体的表达,而在骨髓基质细胞表面存在多种Notch配体的表达,提示Notch信号途径可能对造血干/祖细胞的自我更新与增殖起到重要作用。有研究表明Notch可以抑制造血干/祖细胞的分化,但促进其增殖。研究还发现,Notch信号途径能使共同淋巴样前体细胞向T细胞方向分化,抑制向B细胞方向的分化;在外周脾脏B细胞发育中,Notch信号激活使其向边缘带B细胞方向分化。此外,Notch信号的失调与血液系统的许多肿瘤相关,Notch信号与T细胞的恶性转化密切相关,Notch在其中起着癌基因的作用。
目前体外研究Notch信号对细胞增殖、分化和凋亡的作用多采用Notch配体刺激。Notch配体表达系统分真核表达系统和原核表达系统。真核表达绝大多数是哺乳动物细胞系,如CHO,COS7等,但由于哺乳动物细胞表达系统成本高、难操作、表达水平低,限制其广泛应用。而原核表达系统由于没有翻译后加工修饰系统,表达的真核细胞蛋白质多以包涵体形式表达,复性纯化方法复杂。而毕赤酵母表达系统兼有原核生物和真核生物的某些优点:有翻译后加工修饰系统,酵母能在简单廉价的培养基中快速高密度地繁殖,产量高,成本低,可进行工业化生产。因此在选择Notch配体的表达系统中我们首先选择毕赤酵母,其次是原核表达系统,但需要实现可溶性表达,以利于大规模应用。我们主要进行了如下几方面的研究:
1、毕赤酵母表达重组人Delta-like1ext-Fc(hDll1 ext-Fc)蛋白。PCR法扩增hDll1 ext片段,截取已有质粒pET32a-Fc中Fc片段,构建酵母表达载体pPIC9K- hDll1 ext -Fc,和pPIC9K-Fc,经过组氨酸营养缺陷,G418抗生素双重筛选阳性转化子,甲醇诱导表达。
2、重组hDll4的原核表达。构建原核表达载体pET32a-hdll4ext-26-217和pET32a-hdll4ext-93-217,转化大肠杆菌BL21,IPTG,30℃低温诱导表达,镍离子螯合珠(Invitrogen ProBondTM)一步纯化上清。
3、重组hDll4蛋白生物学功能的检测。通过报告基因实验检测重组hDll4蛋白的生物学活性。
结论:实现了hDll1 ext-Fc和人IgG1 Fc片段的在毕赤酵母中的表达;25℃低温诱导实现重组hDll4(His- hdll4ext -93-217)的可溶性表达及纯化;通过报告基因实验验证了重组hDll4(His- hdll4ext -93-217)蛋白的生物学活性。
The Notch signaling pathway, which is conserved during evolution from worm to man , has multiple and essential roles in embryogenesis, hematopoiesis and cancers. During mammalian hematopoiesis and lymphopoiesis, Notch signaling has multiple functions. Recently, it is discovered that Notch receptors and its ligands are expressed on the surface of the HSCs and bone marrow cells respectively, which denotes that Notch signaling plays a fundamental role in determining bone marrow hematopoietic precursor renewal and differentiation. Some researches show that Notch can not only inhibits HSCs differentiation but also promotes their expansion. These include regulation of T-cell commitment from a common lymphoid precursor and restrain the development of B-cell, regulation of T-cell development and marginal zone B-cell development. However, Notch signaling must be precisely regulated, as constitutive Notch signaling leads to T-cell malignancies.
In the study of cell proliferation, differentiation and apoptosis in vitro, researchers always activate Notch signaling by Notch ligand. And Notch ligands expression system can classify into eukaryotic and prokaryotic expression systems. The eukaryotic expression system is mostly mammalian cell lines, such as CHO, COS7. However, the mammalian expression system could not be applied extensively because of the expensive cost, strict manipulation and low expression. The prokaryotic expression system has no post-translational modification system and the exogenous proteins always emerge in the inclusion body, complicating the course of renaturation and purification. Pichia pastoris expression system has the advantages of mammalian and prokaryotic expression systems: post- translation system, low cost, high production, etc. Therefore, we choose the Pichia pastoris expression system firstly, then the prokaryotic expression system which comes to a soluble expression.
Several experiments were included as follows:
1. Expression recombinant human Delta-like1ext-Fc (hDll1ext-Fc) in Pichia pastoris. Got hDll1ext fragments by PCR, the Fc fragment from pET32a -Fc. Constructed expression vectors pPIC9K- hDll1ext -Fc and pPIC9K-Fc. Screened the transformants by the histidine-deficiency and G418 media, and induced the expression of positive transformants with methanol.
2. Expression recombinant hDll4 in E.coli BL21. Constructed expression vectors pET32a-hdll4ext-26-217 and pET32a-hdll4ext -93-217. Induced expression with IPTG at 25℃.
3. Detected the recombinant hDll4 bioactivity though reporter assay in HeLa and COS7.
Results: We got recombinant hDll1ext-Fc and human IgG1 Fc proteins in Pichia pastoris; obtained and purified a soluble recombinant protein His- hdll4ext -93-217 in E.coli BL21 at 25℃; proved His- hdll4ext -93-217 bioactivity by reporter assay.
目前体外研究Notch信号对细胞增殖、分化和凋亡的作用多采用Notch配体刺激。Notch配体表达系统分真核表达系统和原核表达系统。真核表达绝大多数是哺乳动物细胞系,如CHO,COS7等,但由于哺乳动物细胞表达系统成本高、难操作、表达水平低,限制其广泛应用。而原核表达系统由于没有翻译后加工修饰系统,表达的真核细胞蛋白质多以包涵体形式表达,复性纯化方法复杂。而毕赤酵母表达系统兼有原核生物和真核生物的某些优点:有翻译后加工修饰系统,酵母能在简单廉价的培养基中快速高密度地繁殖,产量高,成本低,可进行工业化生产。因此在选择Notch配体的表达系统中我们首先选择毕赤酵母,其次是原核表达系统,但需要实现可溶性表达,以利于大规模应用。我们主要进行了如下几方面的研究:
1、毕赤酵母表达重组人Delta-like1ext-Fc(hDll1 ext-Fc)蛋白。PCR法扩增hDll1 ext片段,截取已有质粒pET32a-Fc中Fc片段,构建酵母表达载体pPIC9K- hDll1 ext -Fc,和pPIC9K-Fc,经过组氨酸营养缺陷,G418抗生素双重筛选阳性转化子,甲醇诱导表达。
2、重组hDll4的原核表达。构建原核表达载体pET32a-hdll4ext-26-217和pET32a-hdll4ext-93-217,转化大肠杆菌BL21,IPTG,30℃低温诱导表达,镍离子螯合珠(Invitrogen ProBondTM)一步纯化上清。
3、重组hDll4蛋白生物学功能的检测。通过报告基因实验检测重组hDll4蛋白的生物学活性。
结论:实现了hDll1 ext-Fc和人IgG1 Fc片段的在毕赤酵母中的表达;25℃低温诱导实现重组hDll4(His- hdll4ext -93-217)的可溶性表达及纯化;通过报告基因实验验证了重组hDll4(His- hdll4ext -93-217)蛋白的生物学活性。
The Notch signaling pathway, which is conserved during evolution from worm to man , has multiple and essential roles in embryogenesis, hematopoiesis and cancers. During mammalian hematopoiesis and lymphopoiesis, Notch signaling has multiple functions. Recently, it is discovered that Notch receptors and its ligands are expressed on the surface of the HSCs and bone marrow cells respectively, which denotes that Notch signaling plays a fundamental role in determining bone marrow hematopoietic precursor renewal and differentiation. Some researches show that Notch can not only inhibits HSCs differentiation but also promotes their expansion. These include regulation of T-cell commitment from a common lymphoid precursor and restrain the development of B-cell, regulation of T-cell development and marginal zone B-cell development. However, Notch signaling must be precisely regulated, as constitutive Notch signaling leads to T-cell malignancies.
In the study of cell proliferation, differentiation and apoptosis in vitro, researchers always activate Notch signaling by Notch ligand. And Notch ligands expression system can classify into eukaryotic and prokaryotic expression systems. The eukaryotic expression system is mostly mammalian cell lines, such as CHO, COS7. However, the mammalian expression system could not be applied extensively because of the expensive cost, strict manipulation and low expression. The prokaryotic expression system has no post-translational modification system and the exogenous proteins always emerge in the inclusion body, complicating the course of renaturation and purification. Pichia pastoris expression system has the advantages of mammalian and prokaryotic expression systems: post- translation system, low cost, high production, etc. Therefore, we choose the Pichia pastoris expression system firstly, then the prokaryotic expression system which comes to a soluble expression.
Several experiments were included as follows:
1. Expression recombinant human Delta-like1ext-Fc (hDll1ext-Fc) in Pichia pastoris. Got hDll1ext fragments by PCR, the Fc fragment from pET32a -Fc. Constructed expression vectors pPIC9K- hDll1ext -Fc and pPIC9K-Fc. Screened the transformants by the histidine-deficiency and G418 media, and induced the expression of positive transformants with methanol.
2. Expression recombinant hDll4 in E.coli BL21. Constructed expression vectors pET32a-hdll4ext-26-217 and pET32a-hdll4ext -93-217. Induced expression with IPTG at 25℃.
3. Detected the recombinant hDll4 bioactivity though reporter assay in HeLa and COS7.
Results: We got recombinant hDll1ext-Fc and human IgG1 Fc proteins in Pichia pastoris; obtained and purified a soluble recombinant protein His- hdll4ext -93-217 in E.coli BL21 at 25℃; proved His- hdll4ext -93-217 bioactivity by reporter assay.
引文
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[5] J.M. Ascano, L.J. Beverly and A.J. Capobianco, The C-terminal PDZ-Ligand of JAGGED1 is essential for cellular transformation. J Biol Chem., 2003, 278: 8771-8779.
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[2] Scadden DT. T-cell differentiation:Notch another step. Blood, 2003, 102(7):23l6.
[3] Freddy Radtke, Francois Schweisguth, Warren Pear. The Notch‘gospel’. EMBO reports, 2005, 6:1120-1125.
[4] Sabine Pfister,Gerhard K. H. Przemeck, Josef-Karl Gerber,Johannes Beckers, Jerzy Adamski and Martin Hrabéde Angelis. Interaction of the MAGUK Family Member Acvrinp1 and the Cytoplasmic Domain of the Notch Ligand Delta1. J Mol Biol., 2003, 333(2):229-35.
[5] J.M. Ascano, L.J. Beverly and A.J. Capobianco, The C-terminal PDZ-Ligand of JAGGED1 is essential for cellular transformation. J Biol Chem., 2003, 278: 8771-8779.
[6] Martin Baron. An overview of the Notch signalling pathway. Semin Cell Dev Biol., 2003, 14 (2):113-9.
[7] Maillard I, Fang T, Pear WS. Regulation of lymphoid development, different- tiation, and function by the Notch pathway. Annu Rev Immunol., 2005, 23: 945-74.
[8] Tun, T., Hamaguchi, Y., Matsunami, N., Furukawa, T., Honjo, T. and Kawaichi, M. Recognition sequence of a highly conserved DNA binding protein RBP-J kappa. Nucl Acids Res., 1994, 22:965-971.
[9] Iso T, Kedes L , Hamamori Y. HES and HERP families: Multipleeffectors of the Notch signaling pathway. J Cell Physiol., 2003,194 (3):37-255.
[10] Stier S, Cheng T, Dombkowski D, Carlesso N, Scadden DT. Notchl activation increases hematopoietic stem cell self-renewal in vivo and favors lymphoid over myeloid lineage outcome. Blood, 2002, 99(7):2369-2378.
[11] Hadland BK, Huppert SS, Kanungo J, Xue Y, Jiang R, et al. A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development. Blood, 2004, 104(10):3097-105.
[12] Kohshi Ohishi, Barbara Varnum-Finney, and Irwin D. Bernstein. Delta-1 enhances marrow and thymus repopulating ability of human CD34+CD38– cord blood cells. J Clin Invest., 2002, 110:1165-1174.
[13] Wei Han, Qian Ye, and Malcolm A. S. Moore. A soluble form of human Delta-like-1 inhibits differentiation of hematopoietic progenitor cells. Blood, 2000, 95(5):1616-1625.
[14] Radtke F, Wilson A, Stark G, et al. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity, 1999, 10(5):547-58.
[15] Han H, Tanigaki K, Yamamoto N, et al. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int Immunol., 2002, 14(6):637-645.
[16] Pui JC, Allman D, Xu L, et al. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity, 1999, 11(3):299-308.
[17] Morimura T, Miyatani S, Kitamura D,et al. Notch signaling suppresses IgH gene expression in chicken B cells: implication in spatially restricted expression of Serrate2/Notch1 in the bursa of Fabricius. J Immunol., 2001,166(5): 3277- 3283.
[18] Hoffmann R, Seidl T, Neeb M,et al. Changes in gene expression profiles in developing B cells of murine bone marrow.Genome Res.,2002, 12(1):98-111.
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