人工合成SHH蛋白N末端基因的Pichia酵母表达及其多克隆抗体制备
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摘要
目的:人工合成SHH蛋白N末端基因的Pichia酵母表达,制备多克隆抗体。
方法:构建人工合成SHH蛋白N末端基因的Pichia酵母表达载体,获得酵母表达株,甲醇诱导表达目的蛋白,免疫Balb/c小鼠制备多克隆抗体,免疫印迹鉴定。
结果:构建人工合成SHH蛋白N末端基因的Pichia酵母表达载体,获得该基因酵母表达株,甲醇诱导表达目的蛋白,免疫动物制备出高效价的SHH多克隆抗体。
结论:获得SHH-N重组蛋白和SHH-N多克隆抗体制备的基因工程方法。为进一步研究Hedgehog信号通路在胰腺癌中的发病机制提供了有效工具。
AIM:To get the eucaryonic recombinate expression of sonic hedgehog in hedgehog signal path in human pancreatic cancer,and prepare the polyclonal antibody of SHH.
Methods: Construct the eukaryotic expression vector of the SHH-N,get the pichia pastoris expression strain, to induce the destination protein by using the methanol. Immune the Balb/c mouse to have the polyclonal antibody of SHH.
Results: The eukaryotic expression vector of the SHH-N was constructed successfully,get the pichia pastoris expression strain and the destination protein. Prepare the polyclonal antibody of SHH with high quality.
Conclusion:Recombinant plasmid vector of SHH-N was constructed and expressed protiein successfully.The SHH polyclonal antibody was made,which provide a useful tool in the further study of Hedgehog signal path in human pancreatic cancer.
方法:构建人工合成SHH蛋白N末端基因的Pichia酵母表达载体,获得酵母表达株,甲醇诱导表达目的蛋白,免疫Balb/c小鼠制备多克隆抗体,免疫印迹鉴定。
结果:构建人工合成SHH蛋白N末端基因的Pichia酵母表达载体,获得该基因酵母表达株,甲醇诱导表达目的蛋白,免疫动物制备出高效价的SHH多克隆抗体。
结论:获得SHH-N重组蛋白和SHH-N多克隆抗体制备的基因工程方法。为进一步研究Hedgehog信号通路在胰腺癌中的发病机制提供了有效工具。
AIM:To get the eucaryonic recombinate expression of sonic hedgehog in hedgehog signal path in human pancreatic cancer,and prepare the polyclonal antibody of SHH.
Methods: Construct the eukaryotic expression vector of the SHH-N,get the pichia pastoris expression strain, to induce the destination protein by using the methanol. Immune the Balb/c mouse to have the polyclonal antibody of SHH.
Results: The eukaryotic expression vector of the SHH-N was constructed successfully,get the pichia pastoris expression strain and the destination protein. Prepare the polyclonal antibody of SHH with high quality.
Conclusion:Recombinant plasmid vector of SHH-N was constructed and expressed protiein successfully.The SHH polyclonal antibody was made,which provide a useful tool in the further study of Hedgehog signal path in human pancreatic cancer.
引文
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1. 彭毅等. 《生物技术通报》,2000,(1):38
2. Clare JJ et al. Bio/Technology,1991,9:455—460
3. Fairlie WD , Zhang H , Brown P K, et al . Expression of a TGF-be2ta superfamily protein , macrophage inhibitory cytokines-1 , in the ye2ast Pichia pastoris. Gene , 2000 , 254 (1-2) : 67 - 761
4. Murphy K P Jr , Gagne P , Pazmany C , et al . Expression of human interleukin-17 in Pichia pastoris : purification and characterization. Pro Exp Puri , 1998 , 12(2) : 208 - 2141
5. Clare J J , Romanos MA , Rayment F B , et al . Production of mouse epidermal growth factor in yeast : high-level secetion using Pichia pastoris strains containing multiple gene copies. Gene , 1991 , 105 (2) :205 – 2121
6. Sreekrishna K, Nelles L , Potenz R , et al . High-level expression ,purification and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris . Bio2 chemistry , 1989 , 28 (9) : 4117 – 41251
7. Fairlie WD , Zhang H , Brown P K, et al . Expression of a TGF-be2ta superfamily protein , macrophage inhibitory cytokines-1 , in the ye2ast Pichia pastoris. Gene , 2000 , 254 (1-2) : 67 – 761
8. Petra V , Malte P , Marc E , et al . Yeast expression of the cytokinereceptor domain of the soluble interleukin-6 receptor. J Immunol Methods , 1996 , 199 : 47 – 541
9. EasySelect Pichia Expression Kit,Catalog no.K1740-01,Invitrogen
10. Cregg J M, et al. Bio?Technology, 1993,11:905～910.
11. Cregg JM,Cereghino JL , Shi J , et al1Recombinant protein expression in Pichia pastoris [J ]1Mol Biotechnol ,2000 ,16(1) :23 – 521
12. Vassileva A ,Chugh DA ,Swaminathan S , et al1Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris [J ]1Protein Expr Purif ,2001 ,21(1) :71 - 801
13. Wegner GH1Emerging applications of the methylotrophic yeasts[J ]1FEMS Microbiol Rev ,1990 ,7(3 - 4) :279 - 2831
14. Scorer CA ,Clare JJ ,Mccombie , et al1Rapid selection using G418 of high copy number transformants of Pichia pastoris for high level foreign gene expres2 sion[J ]. BioP technology ,1994 ,12(2) :181 - 1841
15. Cregg JM,Tschopp JF ,Stillman C , et al1High level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast , Pichia pasto2 ris [J ]. BioP technology ,1987 ,5(4) :479 - 4861
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19. Cregg JM,Tschopp JF ,Stillman C , et al1High level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast , Pichia pastoris [J ]1BioP technology ,1987 ,5(4) :479 – 4861
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2. Hammerschmidt M, Brook A, McMahon AP. The world according to hedgehog. Trends Genet, 1997, 13: 14- 21.
3. Cohen MM Jr. The hedgehog signaling network. Am J Med Genet A,2003,123: 5- 28.
4. Ho KS, Scott MP. Sonic hedgehog in the nervous system: functions,modifications and mechanisms. Curr Opin Neurobiol, 2002, 12: 57-63.
5. Chen Y, Struhl G. Dual roles for patched in sequestering and transducing Hedgehog. Cell, 1996, 87: 553- 563.
6. Alcedo J, Ayzenzon M, Von Ohlen T, et al. The Drosophila smoothened gene encodes a seven - pass membrane protein, a putative receptor for the hedgehog signal. Cell, 1996, 86: 221- 232.
7. van den Heuvel M, Ingham PW. smoothened encodes a receptorlike serpentine protein required for hedgehog signalling. Nature,1996, 382: 547- 551.
8. Britto J , Tannahill D , Keynes R A. Critical role for sonic hedgehog signaling in the early expansion of the developing brain. Nat Neurosci , 2002 , 5 (2) : 87～88
9. Oro AE, Higgins KM, Hu Z, et al. Basal cell carcinomas in mice overexpressing sonic hedgehog. Science. 1997, 276: 817- 821.
10. Hahn H, Wicking C, Zaphiropoulous PG, et al. Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell, 1996, 85: 841- 851.
11. Nishimaki H , Kasai K, Kozaki K, et al . A role of activated Sonic hedgehog signaling for t he cellular proliferation of oral squamous cell carcinoma cell line [ J ] . Biochem Biophys Res Commun , 2004 , 314 (2) :3132320.
12. Ramalho2Santos M , Melton DA , McMahon AP. Hedgehog signals regulate multiple aspect s of gast rointestinal development [J ] . Development , 2000 , 127 (12) :276322772.
13. Hebrok M. Hedgehog signaling in pancreas development [J ] .Mech Dev , 2003 , 120 (1) :45247.
14. Van den Brink GR , Hardwick JC , Tytgat GN , et al . Sonic hedgehog regulates gast ric gland morphogenesis in man and mouse[J ] . Gast roenterology , 2001 , 121 (2) :3172328.
15. Thayer SP , Di Magliano MP , Heiser PW , et al . Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis [J ] . Nature , 2003 , 425 (6960) :8512856.
16. 基因工程原理 吴乃虎 北京 科学出版社 第二版 1998
17. EasySelect Pichia Expression Kit,Catalog no.K1740-01,Invitrogen
18. Cregg JM,Cereghino JL , Shi J , et al. Recombinant protein expression in Pichia pastoris [J ] Mol Biotechnol ,2000 ,16(1) :23 - 521
19. Vassileva 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 [J ]. Protein Expr Purif ,2001 ,21(1) :71 - 801
20. Wegner GH1Emerging applications of the methylotrophic yeasts[J ]. FEMS Microbiol Rev ,1990 ,7(3 - 4) :279 - 2831
21. Mount RC ,Jordan BE ,Hadfield C1Transformation of lithium - treated yeast cells and the selection of auxotrophic and dominant markers[J ], Methods Mol Biol ,1996 ,53(1) :139 – 1451
1. 彭毅等. 《生物技术通报》,2000,(1):38
2. Clare JJ et al. Bio/Technology,1991,9:455—460
3. Fairlie WD , Zhang H , Brown P K, et al . Expression of a TGF-be2ta superfamily protein , macrophage inhibitory cytokines-1 , in the ye2ast Pichia pastoris. Gene , 2000 , 254 (1-2) : 67 - 761
4. Murphy K P Jr , Gagne P , Pazmany C , et al . Expression of human interleukin-17 in Pichia pastoris : purification and characterization. Pro Exp Puri , 1998 , 12(2) : 208 - 2141
5. Clare J J , Romanos MA , Rayment F B , et al . Production of mouse epidermal growth factor in yeast : high-level secetion using Pichia pastoris strains containing multiple gene copies. Gene , 1991 , 105 (2) :205 – 2121
6. Sreekrishna K, Nelles L , Potenz R , et al . High-level expression ,purification and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris . Bio2 chemistry , 1989 , 28 (9) : 4117 – 41251
7. Fairlie WD , Zhang H , Brown P K, et al . Expression of a TGF-be2ta superfamily protein , macrophage inhibitory cytokines-1 , in the ye2ast Pichia pastoris. Gene , 2000 , 254 (1-2) : 67 – 761
8. Petra V , Malte P , Marc E , et al . Yeast expression of the cytokinereceptor domain of the soluble interleukin-6 receptor. J Immunol Methods , 1996 , 199 : 47 – 541
9. EasySelect Pichia Expression Kit,Catalog no.K1740-01,Invitrogen
10. Cregg J M, et al. Bio?Technology, 1993,11:905～910.
11. Cregg JM,Cereghino JL , Shi J , et al1Recombinant protein expression in Pichia pastoris [J ]1Mol Biotechnol ,2000 ,16(1) :23 – 521
12. Vassileva A ,Chugh DA ,Swaminathan S , et al1Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris [J ]1Protein Expr Purif ,2001 ,21(1) :71 - 801
13. Wegner GH1Emerging applications of the methylotrophic yeasts[J ]1FEMS Microbiol Rev ,1990 ,7(3 - 4) :279 - 2831
14. Scorer CA ,Clare JJ ,Mccombie , et al1Rapid selection using G418 of high copy number transformants of Pichia pastoris for high level foreign gene expres2 sion[J ]. BioP technology ,1994 ,12(2) :181 - 1841
15. Cregg JM,Tschopp JF ,Stillman C , et al1High level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast , Pichia pasto2 ris [J ]. BioP technology ,1987 ,5(4) :479 - 4861
16. Cereghino JL ,Cregg JM1Heterologous protein expression in the methylotro2 phic yeast Pichia pastoris [J ]. FEMS Microbiol Rev ,2000 ,24(1) :45 - 661
17. Mount RC ,Jordan BE ,Hadfield C1Transformation of lithium - treated yeast cells and the selection of auxotrophic and dominant markers[J ]1Methods Mol Biol ,1996 ,53(1) :139 – 1451
18. Scorer CA ,Clare JJ ,Mccombie , et al1Rapid selection using G418 of high copy number transformants of Pichia pastoris for high level foreign gene expres2 sion[J ]1BioP technology ,1994 ,12(2) :181 – 1841
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