AIF-1原核载体的构建及表达纯化
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摘要
人异体移植炎症因子1( human allograft inflammatory factor 1,hAIF-1,Daintain)是上世纪90年代中后期欧美几个研究小组从不同的研究目标中鉴定出的一个由巨噬细胞和活化的T淋巴细胞产生的细胞因子。全长147个氨基酸,分子量17kDa。分子内含有一个Ca2+结合的EF-手形结构域,推测在细胞钙代谢中起一定作用,另有一个由44个氨基酸残基片断组成的激素前体序列模式KR-KK-GKR,对胰岛素的分泌具有双向调节作用。不同的实验室研究发现,Daintain/AIF-1在肿瘤的发生和发展,心脏、肝脏的移植排斥,中枢神经系统损伤以及自身性免疫疾病中发挥重要作用。由于其含义深刻的结构和重要的生物学功能,已引起了生命科学和医学界的广泛关注。
由于Daintain/AIF-1在自然界中丰度很低,从天然产物中提取大量Daintain/AIF-1非常困难。所以本研究以Daintain/AIF-1氨基酸序列为依据,人工合成基因寡核苷酸片段,通过质粒抽提,酶切,将含Daintain/AIF-1的片断克隆到原核表达载体pET32-a(+)中,得到重组质粒pET32-a(+)-AIF-1,然后转化大肠杆菌BL21(DE3)。通过对诱导表达条件的优化,30℃,IPTG诱导2h后可溶性表达。经Sephrose-镍亲和层析柱纯化,并由SDS-PAGE鉴定产物的表观分子量约1.8×104Da。Western boltting进一步证明纯化产物含Daintain/AIF-1。说明Daintain/AIF-1-6His在大肠杆菌BL21(DE3)中表达成功。通过该方法来表达和纯化Daintain/AIF-1的突出优点是流程短,成本较低,技术成熟,可靠性高。
该工作为Daintain/AIF-1的结构和功能研究打下了基础,对以后用基因工程方法生产该蛋白,并用于临床具有一定意义。
Allograft inflammatory factor 1 (AIF-1)/ Daintain is a 147-aa residue polypeptide with a mass of 17kDa,characterd as a macrophage and T Lymphocyte factor by several European and American searth teams in the midanaphase of 90’s last century for different aims . Daintain/AIF-1 has an Ca2+-binding EF-hand suggested some function in Ca2+ metabolism,and an internal 44-residue segment with the sequence pattern–KR–KK–GKR–, a motif typical of peptide hormone precursors, regulating insulin secretion. Daintain/AIF-1 has been verified an important protein in tumor progress, heart/liver transplant rejection, central lesion, and autoimmune disease etc. For interesting structure and function, Daintain/AIF-1 is widespread investigated in biology and medical science.
Daintain/AIF-1 is too little to purify from wild material. So its oligo-nucleotide fragment was artificially synthetic based on the amino-acid sequence of Daintain/AIF-1 . After extracting plasmid, enzyme hydrolysis,the fragment was cloned into prokaryotic expression vector pET32-a(+). The recombinant expression plasmid pET32-a(+) -Daintain/AIF-1-6His was transformed into E.coli BL21(DE3). Then the conditions of inducement were optimized at 30℃, 2h. The fusion protein Daintain/AIF-1-6His was solubly expressed. The fusion protein was purified by Sephrose-NI affinity column. The molecular mass of the fraction determined by SDS-PAGE was about 18kDa, Existence of Daintain/AIF-1 was further identified by Western blotting .So the Daintain/AIF-1-6His fusion protein was successfully expressed in E. coli.
The work provides preparation for further study of the function of Daintain/AIF-1 and producing the protein with gene engineering for clinical application.
由于Daintain/AIF-1在自然界中丰度很低,从天然产物中提取大量Daintain/AIF-1非常困难。所以本研究以Daintain/AIF-1氨基酸序列为依据,人工合成基因寡核苷酸片段,通过质粒抽提,酶切,将含Daintain/AIF-1的片断克隆到原核表达载体pET32-a(+)中,得到重组质粒pET32-a(+)-AIF-1,然后转化大肠杆菌BL21(DE3)。通过对诱导表达条件的优化,30℃,IPTG诱导2h后可溶性表达。经Sephrose-镍亲和层析柱纯化,并由SDS-PAGE鉴定产物的表观分子量约1.8×104Da。Western boltting进一步证明纯化产物含Daintain/AIF-1。说明Daintain/AIF-1-6His在大肠杆菌BL21(DE3)中表达成功。通过该方法来表达和纯化Daintain/AIF-1的突出优点是流程短,成本较低,技术成熟,可靠性高。
该工作为Daintain/AIF-1的结构和功能研究打下了基础,对以后用基因工程方法生产该蛋白,并用于临床具有一定意义。
Allograft inflammatory factor 1 (AIF-1)/ Daintain is a 147-aa residue polypeptide with a mass of 17kDa,characterd as a macrophage and T Lymphocyte factor by several European and American searth teams in the midanaphase of 90’s last century for different aims . Daintain/AIF-1 has an Ca2+-binding EF-hand suggested some function in Ca2+ metabolism,and an internal 44-residue segment with the sequence pattern–KR–KK–GKR–, a motif typical of peptide hormone precursors, regulating insulin secretion. Daintain/AIF-1 has been verified an important protein in tumor progress, heart/liver transplant rejection, central lesion, and autoimmune disease etc. For interesting structure and function, Daintain/AIF-1 is widespread investigated in biology and medical science.
Daintain/AIF-1 is too little to purify from wild material. So its oligo-nucleotide fragment was artificially synthetic based on the amino-acid sequence of Daintain/AIF-1 . After extracting plasmid, enzyme hydrolysis,the fragment was cloned into prokaryotic expression vector pET32-a(+). The recombinant expression plasmid pET32-a(+) -Daintain/AIF-1-6His was transformed into E.coli BL21(DE3). Then the conditions of inducement were optimized at 30℃, 2h. The fusion protein Daintain/AIF-1-6His was solubly expressed. The fusion protein was purified by Sephrose-NI affinity column. The molecular mass of the fraction determined by SDS-PAGE was about 18kDa, Existence of Daintain/AIF-1 was further identified by Western blotting .So the Daintain/AIF-1-6His fusion protein was successfully expressed in E. coli.
The work provides preparation for further study of the function of Daintain/AIF-1 and producing the protein with gene engineering for clinical application.
引文
[1] Utans U, Arceci RJ, Yamashita Y, et al. Cloning and characterization of allograft inflammatory factor-1: a novel macrophage factor identified in rat cardiac allograft with chronic rejection. J. Clin. Invest, 1995, 6:2954~62
[2] Utans U, Quist WC, Mc Manus BM, et al. Allograft inflammatory factory-1. A cytokine-responsive macrophage molecule expressed in transplanted human hearts. Transplantation, 1996, 61:1387~1392
[3] Autieri MV. cDNA cloning of human allograft inflammatory factor-1: tissue distribution, cytokine induction, and mRNA expression in injured rat carotid arteries. Biochem Biophys Res Commun, 1996, 1;228 (1):29~37
[4] Chen ZW, Ahren B, Ostenson CG, et al. Identification, isolation, and characterization of daintain (allograft inflammatory factor1), a macrophage polypeptide with effects on insulin secretion and abundantly present in the pancreas of prediabetic BB rats. Proc Natl Acad Sci, 1997, 94:13879~13884
[5] Kruse M, Steffen R, Batel R, et al. Differential expression of allograft inflammatory factor 1 and of glutathione peroxidase during auto- and allograft response in marine sponges. J Cell Sci, 1999, 112 ( Pt 23):4305~13
[6] Muller WE, Krasko A, Skorokhod A, et al. Histocompatibility reaction in tissue and cells of the marine sponge Suberites domuncula in vitro and in vivo: central role of the allograft inflammatory factor 1. Immunogenetics, 2002, 54(1):48~58
[7] Deininger MH, Meyermann R, Schluesener HJ. The allograft inflammatory factor-1 family of proteins. FEBS, 2002, 514:115~121
[8] Imai Y, Ibata I, Ito D, et al. A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem. Biophys Res. Commun, 1996, 224:855~862
[9] Ito D, Imai Y, Ohsawa K, et al. Microglia-specific localisation of a novel calcium binding protein, Iba1. Mol Brain Res, 1998, 57:1~9
[10] Tanaka S, Suzuki K, Watanabe M, et al. Up-regulation of a new microglial gene, mrf-1, in response to programmed neuronal cell death and degeneration. J. Neurosci,1998,18:6358~6369
[11] Autieri MV,Agrawal N. IRT-1, a novel interferon-gamma-responsive transcript encoding a growth-suppressing basic leucine zipper protein. J. Biol. Chem, 1998, 273:14731~ 14737
[12] Autieri MV, Prystowsky MB, Ohlstein EH. Isolation and characterization of BART-1: A novel balloon angioplasty responsive transcript in rat carotid arteries. DNA Cell Biol, 1996, 15: 297~304
[13] Iris FJ, Bougueleret L, Prieur S, et al. Dense Alu clustering and a potential new member of the NF kappa B family within a 90 kilobase HLA class III segment. Nat Genet, 1993, 3(2):137~45
[14] Hara H, Ohta M, Ohta K, et al. Isolation of Two Novel Alternative Splicing Variants of Allograft Inflammatory Factor-1. Biol. Chem, 1999, 380:1333~1336
[15] Neville MJ, Campbell RD. A New Member of the Ig Superfamily and a V-ATPase G Subunit Are Among the Predicted Products of Novel Genes Close to the TNF Locus in the Human MHC. The Journal of Immunology, 1999, 162: 4745~4754
[16] Gruen JR, Weissman SM. Human MHC class III and IV genes and disease associations. Front Biosci, 2001, 6: 960~72
[17] Glover MD, Seidel GE Jr. Increased messenger RNA for allograft inflammatory factor-1, LERK-5, and a novel gene in 17.5-day relative to 15.5-day bovine embryos. Biol Reprod, 2003, 69(3):1002~12
[18] Autieri MV, Carbone C, Mu A. Expression of allograft inflammatory factor-1 (AIF1) is a marker of activated human VSMC and arterial injury. Arterioscler Thromb Vasc Biol, 2000, 20:1737~1744
[19] Schwab JM, Frei E, Klusman I, et al. AIF-1 expression defines a proliferating and alert microglial/macrophage phenotype following spinal cord injury in rats. J. Neuroimmunol, 2001, 119:214~222
[20] Mittelbronn M, Dietz K, Schluesener HJ et al. Local distribution of microglia in the normal adult human central nervous system differs by up to one order of magnitude. Acta. Neuropathol, 2001,101:249~255
[21] Postler E, Rimner A, Beschorner R, et al. Allograft-inflammatory-factor-1 is upregulated in microglial cells in human cerebral infarctions. J. Neuroimmunol, 2000,104:85~91
[22] Beschorner R, Engel S, Mittelbronn M, et al. Differential regulation of the monocytic calcium-binding peptides macrophage-inhibiting factor related protein-8 (MRP8/S100A8) and allograft inflammatory factor-1 (AIF-1) following human traumatic brain injury. Acta Neuropathol, 2000,100:627~634
[23] Deininger MH, Seid K, Engel S, et al. Allograft inflammatory factor-1 defines a distinct subset of infiltrating macrophages/microglial cells in rat and human gliomas. Acta Neuropathol, 2000,100:673~680
[24] Fauser S, Nguyen TD, Bekure K, et al. Differential activation of microglial cells in local and remote areas of IRBP1169-1191-induced rat uveitis. Acta Neuropathol, 2001,101:565~571
[25] Schluesener HJ, Seid K, Kretzschmar J et al. Allograft-inflammatory factor-1 in rat experimental autoimmune encephalomyelitis, neuritis, and uveitis: expression by activated macrophages and microglial cells. Glia, 1998, 24:244~251
[26] Watano K, Iwabuchi K, Fujii S, et al. Allograft inflammatory factor-1 augments production of interleukin-6, -10 and -12 by a mouse macrophage line. Immunology, 2001,104(3):307~16
[27] Beiter T, Artelt MR, Trautmann K, et al. Experimental autoimmune neuritis induces differential microglia activation in the rat spinal cord. J Neuroimmunol, 2005, 160(1-2):25~31
[28] Pashenkov M, Efendic S, Zhu J, et al. Augmented expression of daintain/allograft inflammatory factor-1 is associated with clinical disease: dynamics of daintain/allograft inflammatory factor-1 expression in spleen, peripheral nerves and sera during experimental autoimmune neuritis. Scand. J. Immunol, 2000, 52:117~122
[29] Eltze E, Bettendorf O, Rody A, et al. Influence of local complications on capsule formation around model implants in a rat model. J Biomed Mater Res A. J. Biomed Mater Res, 2003, 64(1):12~9
[30] Mentschel J, Deininger MH, Schluesener HJ, et al. Effects of Malnutrition on the Expression of Daintain/AIF-1 in the Gut Mucosa of Pigs. J. Vet. Med. A Physiol Pathol Clin Med , 2002,49(4)
[31] Kuschel R, Deininger MH, Meyermann R, et al. Allograft inflammatory factor-1 is expressed by macrophages in injured skeletal muscle and abrogates proliferation anddifferentiation of satellite cells. J Neuropathol Exp Neurol, 2000, 59(4):323~32
[32] Hazan RB, Phillips GR, Qiao RF, et al. Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol, 2000, 21;148(4):779~90
[33] Autieri MV, Christopher M. Over expression of Allograft Inflammatory Factor-1 Promotes Proliferation of Vascular Smooth Muscle Cells by Cell Cycle Deregulation. Arteriosclerosis, Thrombosis, and Vascular Biology, 2001, 21:1421
[34] Autieri MV, Carbone CJ, Eisen H. The growth enhancing effects of allograft inflammatory Factor-1 (AIF-1) in VSMC are dose-dependent and mediated by its ability to bind calcium. J Heart Lung Transplant, 2001, 20(2):198
[35] LabrouN,ClonisYD1 The affinity technology in down 2 stream processing 1 J Biotechnol,1994,36:95
[36] Kane J F, Hartiey D L. Formation of recombinant protein inclusion bodies in Eschericha coli. Tibtech, 1988,6: 95
[37] 白东亭,许丽锋. 重组大肠杆菌的包涵体. 中国生物制品学杂志,1996, 9(4): 188~191
[38] Georgiou G, Telford J N, Shuler M L, et al . Localization of inclusion bodies in Escherichia coli overproducing β-lactamase or alkaline phosphatase. Appl Environ Microbiol, 1986, 52: 1157~1161
[39] Oswald T, Wende W, Pingoud A, et al. Comparison of N2terminal affinity fusion domains :effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoR Ⅴ . Appl Microbiol Biotechnol, 1994, 42: 73~77
[40] Boshai WR, Rappuoli R, Murphy JR. High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli. J Bacteriol,1987,169(11): 5140~51
[41] Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual,2nd ed. New York: Cold Spring Harbor Laboratory Press,1989. 888
[42] HALL R E, AGARWAL S, KESTLER D P, et al. cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product. Biochem J, 1996, 319: 919~927
[43] Teng H, Pi?ón J D, Weiss S R, et al. Expression of Murine CoronavirusRecombinant Papain-Like Proteinase: Efficient Cleavage Is Dependent on the Lengths of both the Substrate and the Proteinase Polypeptides. Journal of Virology, 1999, 73(4): 2658~2666
[44] O'Riordain G, Radauer C, Hoffmann-Sommergruber K, et al. Cloning and molecular characterization of the Hevea brasiliensis allergen Hev b 11, a class I chitinase. Clin Exp Allergy, 2002, 32(3): 455~62
[45] 岳国华, 朱尚权. 胰岛素受体结构与功能研究概况. 生物化学与生物物理进展,1992,19(1):1~5
[46] 吴乃虎. 基因工程原理[M] . 北京: 科学出版社,1998.34~90
[47] Steven H. Strauss. Genomics ,Genetic Engineering , and Domestication of Crops. Science , 2003 , 300(1) :61~62.
[2] Utans U, Quist WC, Mc Manus BM, et al. Allograft inflammatory factory-1. A cytokine-responsive macrophage molecule expressed in transplanted human hearts. Transplantation, 1996, 61:1387~1392
[3] Autieri MV. cDNA cloning of human allograft inflammatory factor-1: tissue distribution, cytokine induction, and mRNA expression in injured rat carotid arteries. Biochem Biophys Res Commun, 1996, 1;228 (1):29~37
[4] Chen ZW, Ahren B, Ostenson CG, et al. Identification, isolation, and characterization of daintain (allograft inflammatory factor1), a macrophage polypeptide with effects on insulin secretion and abundantly present in the pancreas of prediabetic BB rats. Proc Natl Acad Sci, 1997, 94:13879~13884
[5] Kruse M, Steffen R, Batel R, et al. Differential expression of allograft inflammatory factor 1 and of glutathione peroxidase during auto- and allograft response in marine sponges. J Cell Sci, 1999, 112 ( Pt 23):4305~13
[6] Muller WE, Krasko A, Skorokhod A, et al. Histocompatibility reaction in tissue and cells of the marine sponge Suberites domuncula in vitro and in vivo: central role of the allograft inflammatory factor 1. Immunogenetics, 2002, 54(1):48~58
[7] Deininger MH, Meyermann R, Schluesener HJ. The allograft inflammatory factor-1 family of proteins. FEBS, 2002, 514:115~121
[8] Imai Y, Ibata I, Ito D, et al. A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem. Biophys Res. Commun, 1996, 224:855~862
[9] Ito D, Imai Y, Ohsawa K, et al. Microglia-specific localisation of a novel calcium binding protein, Iba1. Mol Brain Res, 1998, 57:1~9
[10] Tanaka S, Suzuki K, Watanabe M, et al. Up-regulation of a new microglial gene, mrf-1, in response to programmed neuronal cell death and degeneration. J. Neurosci,1998,18:6358~6369
[11] Autieri MV,Agrawal N. IRT-1, a novel interferon-gamma-responsive transcript encoding a growth-suppressing basic leucine zipper protein. J. Biol. Chem, 1998, 273:14731~ 14737
[12] Autieri MV, Prystowsky MB, Ohlstein EH. Isolation and characterization of BART-1: A novel balloon angioplasty responsive transcript in rat carotid arteries. DNA Cell Biol, 1996, 15: 297~304
[13] Iris FJ, Bougueleret L, Prieur S, et al. Dense Alu clustering and a potential new member of the NF kappa B family within a 90 kilobase HLA class III segment. Nat Genet, 1993, 3(2):137~45
[14] Hara H, Ohta M, Ohta K, et al. Isolation of Two Novel Alternative Splicing Variants of Allograft Inflammatory Factor-1. Biol. Chem, 1999, 380:1333~1336
[15] Neville MJ, Campbell RD. A New Member of the Ig Superfamily and a V-ATPase G Subunit Are Among the Predicted Products of Novel Genes Close to the TNF Locus in the Human MHC. The Journal of Immunology, 1999, 162: 4745~4754
[16] Gruen JR, Weissman SM. Human MHC class III and IV genes and disease associations. Front Biosci, 2001, 6: 960~72
[17] Glover MD, Seidel GE Jr. Increased messenger RNA for allograft inflammatory factor-1, LERK-5, and a novel gene in 17.5-day relative to 15.5-day bovine embryos. Biol Reprod, 2003, 69(3):1002~12
[18] Autieri MV, Carbone C, Mu A. Expression of allograft inflammatory factor-1 (AIF1) is a marker of activated human VSMC and arterial injury. Arterioscler Thromb Vasc Biol, 2000, 20:1737~1744
[19] Schwab JM, Frei E, Klusman I, et al. AIF-1 expression defines a proliferating and alert microglial/macrophage phenotype following spinal cord injury in rats. J. Neuroimmunol, 2001, 119:214~222
[20] Mittelbronn M, Dietz K, Schluesener HJ et al. Local distribution of microglia in the normal adult human central nervous system differs by up to one order of magnitude. Acta. Neuropathol, 2001,101:249~255
[21] Postler E, Rimner A, Beschorner R, et al. Allograft-inflammatory-factor-1 is upregulated in microglial cells in human cerebral infarctions. J. Neuroimmunol, 2000,104:85~91
[22] Beschorner R, Engel S, Mittelbronn M, et al. Differential regulation of the monocytic calcium-binding peptides macrophage-inhibiting factor related protein-8 (MRP8/S100A8) and allograft inflammatory factor-1 (AIF-1) following human traumatic brain injury. Acta Neuropathol, 2000,100:627~634
[23] Deininger MH, Seid K, Engel S, et al. Allograft inflammatory factor-1 defines a distinct subset of infiltrating macrophages/microglial cells in rat and human gliomas. Acta Neuropathol, 2000,100:673~680
[24] Fauser S, Nguyen TD, Bekure K, et al. Differential activation of microglial cells in local and remote areas of IRBP1169-1191-induced rat uveitis. Acta Neuropathol, 2001,101:565~571
[25] Schluesener HJ, Seid K, Kretzschmar J et al. Allograft-inflammatory factor-1 in rat experimental autoimmune encephalomyelitis, neuritis, and uveitis: expression by activated macrophages and microglial cells. Glia, 1998, 24:244~251
[26] Watano K, Iwabuchi K, Fujii S, et al. Allograft inflammatory factor-1 augments production of interleukin-6, -10 and -12 by a mouse macrophage line. Immunology, 2001,104(3):307~16
[27] Beiter T, Artelt MR, Trautmann K, et al. Experimental autoimmune neuritis induces differential microglia activation in the rat spinal cord. J Neuroimmunol, 2005, 160(1-2):25~31
[28] Pashenkov M, Efendic S, Zhu J, et al. Augmented expression of daintain/allograft inflammatory factor-1 is associated with clinical disease: dynamics of daintain/allograft inflammatory factor-1 expression in spleen, peripheral nerves and sera during experimental autoimmune neuritis. Scand. J. Immunol, 2000, 52:117~122
[29] Eltze E, Bettendorf O, Rody A, et al. Influence of local complications on capsule formation around model implants in a rat model. J Biomed Mater Res A. J. Biomed Mater Res, 2003, 64(1):12~9
[30] Mentschel J, Deininger MH, Schluesener HJ, et al. Effects of Malnutrition on the Expression of Daintain/AIF-1 in the Gut Mucosa of Pigs. J. Vet. Med. A Physiol Pathol Clin Med , 2002,49(4)
[31] Kuschel R, Deininger MH, Meyermann R, et al. Allograft inflammatory factor-1 is expressed by macrophages in injured skeletal muscle and abrogates proliferation anddifferentiation of satellite cells. J Neuropathol Exp Neurol, 2000, 59(4):323~32
[32] Hazan RB, Phillips GR, Qiao RF, et al. Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol, 2000, 21;148(4):779~90
[33] Autieri MV, Christopher M. Over expression of Allograft Inflammatory Factor-1 Promotes Proliferation of Vascular Smooth Muscle Cells by Cell Cycle Deregulation. Arteriosclerosis, Thrombosis, and Vascular Biology, 2001, 21:1421
[34] Autieri MV, Carbone CJ, Eisen H. The growth enhancing effects of allograft inflammatory Factor-1 (AIF-1) in VSMC are dose-dependent and mediated by its ability to bind calcium. J Heart Lung Transplant, 2001, 20(2):198
[35] LabrouN,ClonisYD1 The affinity technology in down 2 stream processing 1 J Biotechnol,1994,36:95
[36] Kane J F, Hartiey D L. Formation of recombinant protein inclusion bodies in Eschericha coli. Tibtech, 1988,6: 95
[37] 白东亭,许丽锋. 重组大肠杆菌的包涵体. 中国生物制品学杂志,1996, 9(4): 188~191
[38] Georgiou G, Telford J N, Shuler M L, et al . Localization of inclusion bodies in Escherichia coli overproducing β-lactamase or alkaline phosphatase. Appl Environ Microbiol, 1986, 52: 1157~1161
[39] Oswald T, Wende W, Pingoud A, et al. Comparison of N2terminal affinity fusion domains :effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoR Ⅴ . Appl Microbiol Biotechnol, 1994, 42: 73~77
[40] Boshai WR, Rappuoli R, Murphy JR. High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli. J Bacteriol,1987,169(11): 5140~51
[41] Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual,2nd ed. New York: Cold Spring Harbor Laboratory Press,1989. 888
[42] HALL R E, AGARWAL S, KESTLER D P, et al. cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product. Biochem J, 1996, 319: 919~927
[43] Teng H, Pi?ón J D, Weiss S R, et al. Expression of Murine CoronavirusRecombinant Papain-Like Proteinase: Efficient Cleavage Is Dependent on the Lengths of both the Substrate and the Proteinase Polypeptides. Journal of Virology, 1999, 73(4): 2658~2666
[44] O'Riordain G, Radauer C, Hoffmann-Sommergruber K, et al. Cloning and molecular characterization of the Hevea brasiliensis allergen Hev b 11, a class I chitinase. Clin Exp Allergy, 2002, 32(3): 455~62
[45] 岳国华, 朱尚权. 胰岛素受体结构与功能研究概况. 生物化学与生物物理进展,1992,19(1):1~5
[46] 吴乃虎. 基因工程原理[M] . 北京: 科学出版社,1998.34~90
[47] Steven H. Strauss. Genomics ,Genetic Engineering , and Domestication of Crops. Science , 2003 , 300(1) :61~62.
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