SEDT-PA致病基因WISP3突变体在软骨细胞中的表达及功能研究
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摘要
第一部分 晚发型脊柱骨骺发育不良伴进行性骨关节病(SEDT-PA)致病基因WISP3突变体的构建
目的：Wnt诱导分泌蛋白3(Wnt-inducible secreted protein 3，WISP3)是晚发型脊柱骨骺发育不良伴进行性骨关节病(spondyloepiphyseal dysplasia tarda with progressive arthropathy，SEDT-PA)的致病基因，构建WISP3基因致病型(1000T-C，840delT)的突变体，为研究突变基因的表达、定位和功能及SEDT-PA的发病机制奠定基础。
方法：用RT-PCR方法从正常人软骨细胞获得野生型WISP3基因的全长cDNA(WT-WISP3)，用定点突变方法获得我们已报道的两种WISP3基因突变类型的全长cDNA(MUT~(1000T/C)和MUT~(840delT))；经T载体克隆，测序证实后，将它们再分别亚克隆至真核表达载体pcDNA3.1(+)和带绿色荧光蛋白靶标的真核表达载体pEGFP-C2中，酶切和测序对插入片段进行分析和鉴定。
结果：构建的野生型WISP3基因和突变体的全长cDNA序列分别与文献及SEDT-PA患者WISP3基因突变类型一致；测序和酶切鉴定证实所有的插入片段序列正确。
结论：运用基因重组技术成功构建了SEDT-PA致病基因WISP3突变体，为进一步探讨SEDT-PA的发病机制创造条件。
Part One The Construction of Mutants of SEDT-PA's Pathogenic Gene
Objectives: Wnt-inducible secreted protein 3 (WISP3) was spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA)'s pathogenic gene.To construct two types of WISP3 gene's mutants (1000T-C,840delT) found in SEDT-PA patients.
Methods: Full-length cDNAs of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes (MUT~(1000T/C) and MUT~(840delT)) . The products were cloned into pEGM-T-easy vector and their sequences were analysed, then subcloned into eukaryotic expression vector pcDNA3.1 (+) and pEGFP-C2, the inserted fragments were analysed and reconfirmed by restriction endonuclease analysis and sequencing.
Results: By restriction endonuclease analysis and sequencing, the sequences of WT-WISP3, MUT~(1000T/C) and MUT~(840delT) were consistent with that in literature and mutated in SEDT-PA, and the open reading frames were matched with the vector sequence.
Conclusion: WISP3 gene's mutants of SEDT-PA were successfully
目的：Wnt诱导分泌蛋白3(Wnt-inducible secreted protein 3，WISP3)是晚发型脊柱骨骺发育不良伴进行性骨关节病(spondyloepiphyseal dysplasia tarda with progressive arthropathy，SEDT-PA)的致病基因，构建WISP3基因致病型(1000T-C，840delT)的突变体，为研究突变基因的表达、定位和功能及SEDT-PA的发病机制奠定基础。
方法：用RT-PCR方法从正常人软骨细胞获得野生型WISP3基因的全长cDNA(WT-WISP3)，用定点突变方法获得我们已报道的两种WISP3基因突变类型的全长cDNA(MUT~(1000T/C)和MUT~(840delT))；经T载体克隆，测序证实后，将它们再分别亚克隆至真核表达载体pcDNA3.1(+)和带绿色荧光蛋白靶标的真核表达载体pEGFP-C2中，酶切和测序对插入片段进行分析和鉴定。
结果：构建的野生型WISP3基因和突变体的全长cDNA序列分别与文献及SEDT-PA患者WISP3基因突变类型一致；测序和酶切鉴定证实所有的插入片段序列正确。
结论：运用基因重组技术成功构建了SEDT-PA致病基因WISP3突变体，为进一步探讨SEDT-PA的发病机制创造条件。
Part One The Construction of Mutants of SEDT-PA's Pathogenic Gene
Objectives: Wnt-inducible secreted protein 3 (WISP3) was spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA)'s pathogenic gene.To construct two types of WISP3 gene's mutants (1000T-C,840delT) found in SEDT-PA patients.
Methods: Full-length cDNAs of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes (MUT~(1000T/C) and MUT~(840delT)) . The products were cloned into pEGM-T-easy vector and their sequences were analysed, then subcloned into eukaryotic expression vector pcDNA3.1 (+) and pEGFP-C2, the inserted fragments were analysed and reconfirmed by restriction endonuclease analysis and sequencing.
Results: By restriction endonuclease analysis and sequencing, the sequences of WT-WISP3, MUT~(1000T/C) and MUT~(840delT) were consistent with that in literature and mutated in SEDT-PA, and the open reading frames were matched with the vector sequence.
Conclusion: WISP3 gene's mutants of SEDT-PA were successfully
引文
[1] Wynne-Davies R,Hall C,Ansell BM.Spodyloepiphyseal dysplasia tarda with progressive arthropathy1 A"new"disorder of autosomal recessive inheritance1.J Bone Joint Surg Br, 1982,64:442-445.
[2] Byrne PA,Rajan KT.Spondyloepiphyseal dysplasia tarda with progressive arthropathy mimicking juvenile chronic arthritis.Br J Rheumatol ,1998 ,37:233-234.
[3] Kocyigit H,Arkun R,Ozkinay F,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.Clin Rheumatol,2000,19:238-241.
[4] Van Buggenhout G, de Smet L, Maroteaux P, et al.Progressive pseudorheumatoid dysplasia: report of a patient with symptoms present at birth.Genet Couns, 1998, 9:277-281.
[5] Teebi AS, Al Awadi SA.Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed among Arabs.J Med Genet,1986,23:189-191.
[6] Balci S,Aypar E,Kasapcopur O,et al.An eleven-year-old female Turkish patient with progressive pseudorheumatoid dysplasia mimicking juvenile idiopathic arthritis.Clin Exp Rheumatol,2001,19:759.
[7] El-Shanti HE, Omari HZ, Qubain HI.Progressive pseudorheumatoid dysplasia: report of a family and review.J Med Genet, 1997, 34:559-563.
[8] Al-Awadi SA,Farag TI,Naguib K,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.J Med Genet,1984,21:193-196.
[9] Spranger J,Albert C,Schilling F,et al.Progressive pseudorheumatoid arthritis of childhood (PPAC).A hereditary disorder simulating rheumatoid arthritis.Eur J Pediatr,1983,14:34-40.
[10] Rezai-Delui H,Mamoori G,Sadri-Mahvelati E,et al.Progressive pseudorheumatoid chondrodysplasia:a report of nine cases in three families.Skeletal Radiol, 1994,23:411 -419.
[11] Hurvitz JR,Suwairi WM,Van Hul W,et al.Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia.Nature Genet 1999;23:94-98.
[12] Valerie Delague,Eliane Chouery,Sandra Corbani,et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia:Identification of two novel mutations,and description of a founder effect.Am J Med Genet A.2005;138(2):118-126.
[13] Ehl S,Uhl M,Berner R,Bonafe L,et al.Clinical,radiographic,and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy.Rheumatol Int 2004;24: 53-56.
[14] Perbal B,Brigstock DR,Lau LF.Report on the second international workshop on the CCN family of genes.Mol Pathol,2003,56:80-85.
[15] Perbal B.NOV (nephroblastoma overexpressed) and CCN family of genes:structural and functional issues.Mol Pathol, 2001, 54:57-79.
[16] Brigstock DR.The CCN family: a new stimulus package.J Endocrinol, 2003,178:169-175.
[17] Liao EY, Peng YQ, Zhou HD, et al.Gene symbol: WISP3. Disease:spondyloepihyseal dysplasia tarda with progressive arthropathy.Hum Genet.2004 Jul;115(2):169.
[18] Liao EY,Peng YQ,Zhou HD,et al.Gene symbol: WISP3. Disease:spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul；115(2)：174．
[19] 彭依群，廖二元，顾慧敏等．复合杂合性CCN6基因突变致晚发型脊柱骨骺发育不良伴进行性骨关节病的关节软骨病理和分子病因研究．中华医学杂志，2004；84：1796-1803．
[20] 周后德，彭依群，谷卫等。SEDT-PA患者关节软骨细胞生物学特性及基因表达谱的改变。中华内科杂志，2005，44：16-21．
[1] Wynne-Davies R, Hall C, Ansell BM. Spodyloepiphyseal dysplasia tarda with progressive arthropathy1 A"new"disorder of autosomal recessive inheritance1. J Bone Joint Surg Br, 1982, 64: 442-445.
[2] Spranger J, Albert C, Schilling F, et al. Progressive pseudorheumatoid arthritis of childhood (PPAC). A hereditary disorder simulating rheumatoid arthritis. Eur J Pediatr, 1983, 14: 34-40.
[3] Rezai-Delui H, Mamoori G, Sadri-Mahvelati E, et al. Progressive pseudorheumatoid chondrodysplasia: a report of nine cases in three families. Skeletal Radiol, 1994, 23: 411-419.
[4] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 169.
[5] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 174.
[6] Mampaey S, Vanhoenacker F, Boven K, et al. Progressive pseudorheumatoid dysplasia. Eur Radiol. 2000; 10: 1832-1835
[7] Kaibara N, Takagishi K, Katsuki I, et al. Spondyloepiphyseal dysplasia tarda with progressive arthropathy. Skeletal Radiol 1983; 10:13-6
[8] Byrne PA,Rajan KT.Spondyloepiphyseal dysplasia tarda with progressive arthropathy mimicking juvenile chronic arthritis.Br J Rheumatol ,1998,37:233-234.
[9] Kocyigit H,Arkun R,Ozkinay F,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.Clin Rheumatol,2000,19:238-241.
[10] Van Buggenhout G, de Smet L, Maroteaux P, et al.Progressive pseudorheumatoid dysplasia: report of a patient with symptoms present at birth.Genet Couns, 1998, 9:277-281.
[11] Teebi AS, Al Awadi SA.Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed amongArabs.J Med Genet,1986,23:189-191.
[12] Balci S,Aypar E,Kasapcopur O,et al.An eleven-year-old female Turkish patient with progressive pseudorheumatoid dysplasia mimicking juvenile idiopathic arthritis.Clin Exp Rheumatol,2001,19:759.
[13] El-Shanti HE, Omari HZ, Qubain HI.Progressive pseudorheumatoid dysplasia: report of a family and review.J Med Genet, 1997, 34:559-563.
[14] Al-Awadi SA,Farag TI,Naguib K,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.J Med Genet,1984,21:193-196.
[15] Perbal B,Brigstock DR,Lau LF.Report on the second international workshop on the CCN family of genes.Mol Pathol,2003,56:80-85.
[16] Perbal B.NOV (nephroblastoma overexpressed) and CCN family of genes:structural and functional issues.Mol Pathol, 2001, 54:57-79.
[17] Brigstock DR.The CCN family: a new stimulus package.J Endocrinol, 2003,178:169-175.
[18] Pennica D,Swanson TA, Welsh JW,et al.WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumor. Proct Nat Acad Sci 1998; 95: 14717-14722.
[19] Bork P. The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Letters 1993; 327: 125-130.
[20] Schutze N, Noth Ul, Schneidereit J, et al. Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation. Cell Communication and Signaling 2005, 3: 1-12.
[21] Hurvitz JR, Suwairi WM, Van Hul W, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nature Genet 1999; 23: 94-98.
[22] Valerie Delague, Eliane Chouery, Sandra Corbani, et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: Identification of two novel mutations, and description of a founder effect. Am J Med Genet A. 2005; 138(2): 118-126.
[23] Ehl S, Uhl M, Berner R, Bonafe L, et al. Clinical, radiographic, and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy. Rheumatol Int 2004; 24: 53-56.
[24] 彭依群，廖二元，顾慧敏等．复合杂合性CCN6基因突变致晚发型脊柱骨骺发育不良伴进行性骨关节病的关节软骨病理和分子病因研究．中华医学杂志，2004：84：1796-1803．
[25] Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selectior. Proc Natl Acad Sci, 1985, 82(2): 488-492.
[26] Laird AD, Morrison DK, Shalloway D. Characterization of Raf-1 activation in mitosis. J Biol Chem, 1999,274(7):4430-4439.
[27] Dai JL, Bansal RK, Kern SE.G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: phenotypes reversed by a tumorigenic mutation.Proc Natl Acad Sci, 1999, 96(4):1427-1432.
[28] Fletcher MC,Kuderova A,Cygler M,et al.Creation of a ribonuclease abzyme through site-directed mutagenesis.Nat Bio technol,1998,16(11):1065-1067.
[29] Kono M, Miyaza KIG, Nakamura H, et al. Site-directed mutagenesis in hemoglobin: at tempts to control the oxygen affinity with cooperativity preserved.Protein, 1998, 11 (3): 199-204.
[30] Urban A,Neukirehen S,Jaeger KE.A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.Nucleic Acids Res,1997,25(ll):2227-2228.
[31] Warrens AN, Jones MD,Lechler RI. Splicing by overlap extension by PCR using asymmetric amplification:An improved technique for the generation of hybrid proteins of immunological interest.Gene,1997,186(1):29-35.
[32] Horton RM, Cai ZL, Horton SN.Gene splicing by overlap extension: Tailor-made genes using the polymerase chain reaction.Biotechniques, 1990,8(5):528-535.
[33] Chang SW,Shieh CJ.Mutiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 gene expressed in pichia pastoris.Appl Microbiol Biotechnol,2004,186(1):159-264.
[34] Rheinallt M, Jones R.Mutational analysis of the critical bases involved in activation of the AreR-regulated 54-dependent promoter in acinetobacter strain ADP.Appl Envir Microbiol, 2003, 69:5627-5635.
[35] Huang CY, Chang AK. Site-directed mutagenesis of ionizable group in the active site of zymomona mobilis pyruvate decarboxylase: Effect on activity and PH dependence.Eur J Biochem, 2001,268(12):3558-3565.
[36] Mukhopadhyay P, Roy KB.Protein engineering of BamH I restriction endonuclease: replacement of Cys 54 by Ala enhances catalytic activity.Protein, 1998, 11(10):931-945.
[37] Sarkar G, Sommer SS.The megaprimer method of site-directed mutagenesis.Biotechniques, 1990, 8(4):404-407.
[38] Colosimo A,Xu Z,Novelli G,et al.Simple version of megaprimer PCR for site-directed mutagenesis.Biotechniques,1999,26:870-873.
[39] Ke SH, Madison EL.Rapid and efficient site-directed mutagenesis by single-tube megaprimer PCR method.Nucleic Acids Res, 1997,25:3371-3372.
[40] Li CH, Fang HY. Construction of middle fragment deletion mutant with improved gene splicing by overlap extension PCR.Zhonghua YiXue YiChuan Xue ZaZhi, 2004,21(1):52-55.
[41] Chen K, Arnold FH.Turning the activity of an enzyme for unususl environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci, 1993,90:5618-5622.
[1] Spranger J,Albert C,Schilling F,et al.Progressive pseudorheumatoid arthritis of childhood (PPAC).A hereditary disorder simulating rheumatoid arthritis.Eur J Pediatr,1983,14:34-40.
[2] Rezai-Delui H,Mamoori G,Sadri-Mahvelati E,et al.Progressive pseudorheumatoid chondrodysplasia:a report of nine cases in three families.Skeletal Radiol, 1994,23:411-419.
[3] Perbal B.NOV (nephroblastoma overexpressed) and CCN family of genes: structural and functional issues.Mol Pathol, 2001, 54:57-79.
[4] Brigstock DR.The CCN family: a new stimulus package.J Endocrinol, 2003,178:169-175.
[5] Pennica D,Swanson TA, Welsh JW,et al.WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumor.Proct Nat Acad Sci 1998;95:14717-14722.
[6] Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression. Science, 1994, 263:802-805.
[7] Gerdes HH, Kaether C. Green fluorescent protein: applicationsin cell biology.FEBS Lett, 1996, 389:44-47.
[8] Kain SR, Adams M, Kondepudi A, et al. Green fluorescent protein as a reporter of gene expression and protein localization.Biotechniques, 1995 ,19 :650-655
[9] Gubin AN, et al. Long - term stable expression of green fluorescent protein in mammalian cells Biochem. Biophys Res Commun ,1997 ;236 :347-350
[10] Derek AP, James AA, Esther RA, et al. Retroviral - mediated tranfer of the green fluorescent protein gene into murine hematopoieticcells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo. Blood , 1997 ;90 (5) : 1777-1786
[11] John PL, Rebecca RM, Sergei Z, et al. Retroviral transfer and expression of a humanized, red-shifted green fluorescent protein gene into human tumor cells.Nature Biotechm ,1996 ;14 (5) :610-614
[12] Holt JR, Johns DC, Wang S, et al. Functional expression of exogenous proteins in mammalian sensoryhair cells infected eith adenoviral vectors. J Neurophysiol ,1999 ;81 (4) .1881-1888
[13] Weingart CL, Broitman - Maduro G, Dean G, et al. Fluorescent labels influence phagocytosis of Bordetella Pertussis by human neutophils. Infect Immun, 1999; 67(8): 4264-4267
[14] Liu HS, Jan MS, Chou CK, et al. Is green fluorescent protein toxic to the living cells? Biochen Biophys Res Commun, 1999; 260(3): 712-717
[15] Dreyer EB, Vorwerk CK, Zurakowski D, et al. Infection with adeno-associated virus may protect excitotoxicity. Neuroreport, 1999; 10(14): 2887-2890
[16] Takada T, et al. Selective production of transgenic nice using green fluorescent protein as a marker. Nat Biotechnol, 1997; 15: 458-461
[17] Stewart CN, Richards HA, Halfhill MD. Transgenic plants and biosafety: science, misconceptions and public perceptions. Biotechniques, 2000; 29 (4): 838-843
[18] Seethala R, Menxel R. A fluorescence polarization competition immunoassay for tyrosine kinases. J Annal Biochem, 1998; 255-257
[19] Bronstein I, Martin CS, Fortin JJ, et al. Chemliuminescince: sensitive detection technology for receptor gene assays. Clin Chem, 1996; 42(9): 1542
[20] Xu W. Structural organiztion of the human vesicular monoamine transporter type-2 gene and promoter analysis using the jelly fish green fluorescent protein as a reporter. Brain Res Mol Brain Res, 1997; 45: 41-49
[21] Casper SJ, et al. Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vactor. Gene, 1996; 173: 69～73
[22] 彭依群，廖二元，顾慧敏等．复合杂合性CCN6基因突变致晚发型脊柱骨骺发育不良伴进行性骨关节病的关节软骨病理和分子病因研究．中华医学杂志，2004；84：1796-1803．
[23] 宋今丹。医学细胞分子生物学，第一版。北京：人民卫生出版社，2003，141-161
[24] 卢圣栋。现代分子生物学实验技术，第一版。北京：高等教育出版社，1993， 3421-3432
[25] 瞿中和。细胞生物学，第一版。北京：高等教育出版社，1995，1021
[1] Spranger J, Albert C, Schilling F, et al. Progressive pseudorheumatoid arthritis of childhood (PPAC). A hereditary disorder simulating rheumatoid arthritis. Eur J Pediatr, 1983, 14: 34-40.
[2] Rezai-Delui H, Mamoori G; Sadri-Mahvelati E, et al. Progressive pseudorheumatoid chondrodysplasia: a report of nine cases in three families. Skeletal Radiol, 1994, 23: 411-419.
[3] Hurvitz JR, Suwairi WM, Van Hul W, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nature Genet 1999; 23: 94-98.
[4] Valerie Delague, Eliane Chouery, Sandra Corbani, et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: Identification of two novel mutations, and description of a founder effect. Am J Med Genet A. 2005; 138(2): 118-126.
[5] Ehl S, Uhl M, Berner R, Bonafe L, et al. Clinical, radiographic, and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy. Rheumatol Int 2004; 24: 53-56.
[6] Wynne-Davies R, Hall C, Ansell BM1. Spodyloepiphyseal dysplasia tarda with progressive arthropathy1 A"new"disorder of autosomal recessive inheritance1. J Bone Joint Surg Br, 1982, 64: 442-445.
[7] Byrne PA, Rajan KT. Spondyloepiphyseal dysplasia tarda with progressive arthropathy mimicking juvenile chronic arthritis. Br J Rheumatol, 1998 ,37:233-234.
[8] Kocyigit H,Arkun R,Ozkinay F,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.Clin Rheumatol,2000,19:23 8-241.
[9] Van Buggenhout G, de Smet L, Maroteaux P, et al.Progressive pseudorheumatoid dysplasia: report of a patient with symptoms present at birth.Genet Couns, 1998, 9:277-281.
[10] Teebi AS, Al Awadi SA.Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed amongArabs.J Med Genet,1986,23:189-191.
[11] Balci S,Aypar E,Kasapcopur O,et al.An eleven-year-old female Turkish patient with progressive pseudorheumatoid dysplasia mimicking juvenile idiopathic arthritis.Clin Exp Rheumatol,2001,19:759.
[12] El-Shanti HE, Omari HZ, Qubain HI.Progressive pseudorheumatoid dysplasia:report of a family and review.J Med Genet, 1997, 34:559-563.
[13] Al-Awadi SA,Farag TI,Naguib K,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.J Med Genet, 1984,21:193-196.
[14] Spranger J,Albert C,Schilling F,et al.Progressive pseudorheumatoid arthritis of childhood ( PPAC).A hereditary disorder simulating rheumatoid arthritis.Eur J Pediatr,1983,14:34-40.
[15] Rezai-Delui H,Mamoori G,Sadri-Mahvelati E,et al.Progressive pseudorheumatoid chondrodysplasia:a report of nine cases in three families.Skeletal Radiol, 1994,23:411 -419.
[16] Liao EY, Peng YQ, Zhou HD, et al.Gene symbol: WISP3. Disease:spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet.2004 Jul; 115(2): 169.
[17] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 174.
[18] 周后德，彭依群，谷卫等。SEDT-PA患者关节软骨细胞生物学特性及基因表达谱的改变。中华内科杂志，2005，44：16-21
[19] Goldring MB, Birkhead JR, Suen LF, et al. Interleukin 1 beta modulated gene expression in immortalized human chondrocytes. J Clin Invest 1994; 94: 2307-2316.
[20] Chansky H, Robbins JR, Cha S, et al. Expression ofcartilage extracellular matrix and potential regulatory genes in a new human chondrosarcoma cell line. J Orthop Res 199816(5): 521-530.
[21] Lee D, Long SA, Adams JL, et al. Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality. J Biol Chem 2000; 275: 16007-16014.
[22] Browning JA, Wilkins RJ. The effect of intracellular alkalinisation on intracellular Ca2+homeostasis in a human chondrocyte cell line. Eur J Physiol 2002; 444: 744-751.
[23] Bork P. The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Letters 1993; 327: 125-130.
[24] Hurvitz JR, Suwairi WM, Van Hul W, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nature Genet 1999; 23: 94-98.
[25] Kleer CG, Zhang Y, Pan Q, et al. WISP3 is a novel tumor suppressor gene of inflammatory breast cancer. Oncogene, 2002, 21: 3172-3180.
[26] Kleer CG, Zhang Y, Pan Q, et al. WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer. Breast Cancer Res. 2004; 6(2): R110-115.
[27] Kleer CG, Zhang Y, Pan Q, et al.WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer.Neoplasia.2004; 6(2): 179-185.
[28] Zhang Y,Pan Q,Zhong H,et al.Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells.Breast Cancer Res.2005;7(6):R1080-1089.
[29] Schutze N,Noth Ul, Schneidereit J,et al.Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation.Cell Communication and Signaling 2005, 3:1-12.
[30] Lake AC, Castellot JJ J r.CCN5modulates the antiproliferativeeffect of heparin and regulates cell motility in vascular smooth muscle cells.Cell Commun Signal,2003,1:5.
[31] Perbal B.CCN proteins: multifunctional signalling regulators.Lancet, 2004, 363:62-64.
[32] Sen M,Cheng YH,Goldring MB,et al.WISP3-dependent regulation of type II collagen and aggrecan production in chondrocytes.Arthritis Rheum,2004,50:488-497.
[1] Perbal B, Brigstock DR, Lau LF. Report on the second international workshop on the CCN family of genes. Mol Pathol, 2003, 56: 80-85.
[2] Perbal B. NOV (nephroblastoma overexpressed) and CCN family of genes: structural and functional issues. Mol Pathol, 2001, 54: 57-79.
[3] Brigstock DR. The CCN family: a new stimulus package. J Endocrinol, 2003, 178:169-175.
[4] Pennica D,Swanson TA, Welsh JW,et al.WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumor.Proct Nat Acad Sci 1998;95:14717-14722.
[5] Bork P.The modular architecture of a new family of growth regulators related to connective tissue growth factor.FEBS Letters 1993; 327:125-130.
[6] Brigstock DR. The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocrine Reviews 1999;20:189-206.
[7] Lau LF & Lam SC.The CCN family of angiogenic regulators: the integrin connection.Experimental Cell Research 1999; 248:44-57.
[8] Mancuso DJ,Tuley EA,Westfield LA,et al.Structure of the gene for human von Willebrand factor.J Biol Chem. 1989; 264(33):19514-19527.
[9] Holt GD,Pangburn MK,Ginsburg V.Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates.J Biol Chem. 1990; 265(5):2852-2855.
[10] Voorberg J,Fontijn R,Calafat J,et al.Assembly and routing of von Willebrand factor variants: the requirements for disulfide-linked dimerization reside within the carboxy-terminal 151 amino acids.J Cell Biol 1991; 113(1):195-205.
[11] Schutze N,Noth Ul, Schneidereit J,et al.Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation.Cell Communication and Signaling 2005, 3:1-12.
[12] Lake AC, Castellot JJ J r.CCN5modulates the antiproliferativeeffect of heparin and regulates cell motility in vascular smooth muscle cells.Cell Commun Signal, 2003,1:5.
[13] Perbal B.CCN proteins: multifunctional signalling regulators.Lancet, 2004, 363: 62-64.
[14] Wynne-Davies R,Hall C,Ansell BM.Spodyloepiphyseal dysplasia tarda with progressive arthropathyl A"new"disorder of autosomal recessive inheritancel.J Bone Joint Surg Br, 1982,64:442-445.
[15] Byrne PA,Rajan KT.Spondyloepiphyseal dysplasia tarda with progressive arthropathy mimicking juvenile chronic arthritis.Br J Rheumatol ,1998 ,37:233-234.
[16] Kocyigit H,Arkun R,Ozkinay F,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.Clin Rheumatol,2000,19:23 8-241.
[17] Van Buggenhout G, de Smet L, Maroteaux P, et al.Progressive pseudorheumatoid dysplasia: report of a patient with symptoms present at birth.Genet Couns, 1998,9:277-281.
[18] Teebi AS, Al Awadi SA.Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed amongArabs.J Med Genet,1986,23:189-191.
[19] Balci S,Aypar E,Kasapcopur O,et al.An eleven-year-old female Turkish patient with progressive pseudorheumatoid dysplasia mimicking juvenile idiopathic arthritis.Clin Exp Rheumatol,2001,19:759.
[20] El-Shanti HE, Oman HZ, Qubain HI.Progressive pseudorheumatoid dysplasia:report of a family and review.J Med Genet, 1997, 34:559-563.
[21] Al-Awadi SA,Farag TI,Naguib K,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.J Med Genet, 1984,21:193 -196.
[22] Spranger J,Albert C,Schilling F,et al.Progressive pseudorheumatoid arthritis of childhood ( PPAC).A hereditary disorder simulating rheumatoid arthritis.Eur J Pediatr, 1983, 14: 34-40.
[23] Rezai-Delui H, Marnoori G, Sadri-Mahvelati E, et al. Progressive pseudorheumatoid chondrodysplasia: a report of nine cases in three families. Skeletal Radiol, 1994, 23: 411-419.
[24] Hurvitz JR, Suwairi WM, Van Hul W, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nature Genet 1999; 23: 94-98.
[25] Valerie Delague, Eliane Chouery, Sandra Corbani, et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: Identification of two novel mutations, and description of a founder effect. Am J Med Genet A. 2005; 138(2): 118-126.
[26] Ehl S, Uhl M, Berner R, Bonafe L, et al. Clinical, radiographie, and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy. Rheumatol Int 2004; 24: 53-56.
[27] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 169.
[28] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 174.
[29] 彭依群，廖二元，顾慧敏等．复合杂合性CCN6基因突变致晚发型脊柱骨骺发育不良伴进行性骨关节病的关节软骨病理和分子病因研究．中华医学杂志，2004：84：1796-1803．
[30] 周后德，彭依群，谷卫等。SEDT-PA患者关节软骨细胞生物学特性及基因表达谱的改变。中华内科杂志，2005，44：16-21
[31] Matsuno H, Yudoh K, Nakazawa F, et al. Antirheumatic effects of humanized anti-Fas monoclonal antibody in human rheumatoid arthritis/SCID mouse chimera. J Rheumatol, 2002,29:1609-1614.
[32] Cho TJ, Lehmann W, Edgar C, et al.Tumor necrosis factor alpha activation of the apoptotic cascade in murine articular chondrocytes is associated with the induction of metalloproteinases and specific pro-resorptive factors.Arthritis Rheum, 2003, 48:2845-2854.
[33] Sen M,Cheng YH,Goldring MB,et al.WISP3-dependent regulation of type II collagen and aggrecan production in chondrocytes.Arthritis Rheum,2004,50:488-497.
[34] Kutz WE, Gong Y, Warman ML.WISP3, the gene responsible for the human skeletal disease progressive pseudorheumatoid dysplasia, is not essential for skeletal function in mice. Mol Cell Biol 2005; 25(1):414-421.
[35] Kleer CG Zhang Y, Pan Q, et al.WISP3 is a novel tumor suppressor gene of inflammatory breast cancer.Oncogene, 2002, 21:3172-3180.
[36] Kleer CG,Zhang Y,Pan Q,et al.WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer.Breast Cancer Res.2004;6(2):R110-115.
[37] Kleer CG, Zhang Y, Pan Q, et al.WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer.Neoplasia.2004r; 6(2): 179-185.
[38] Zhang Y,Pan Q,Zhong H,et al.Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells.Breast Cancer Res.2005;7(6):R1080-1089.
[1] Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selectior. Proc Natl Acad Sci, 1985, 82(2): 488-492.
[2] Laird AD, Morrison DK, Shalloway D. Characterization of Raf-1 activation in mitosis. J Biol Chem, 1999, 274(7): 4430-4439.
[3] Dai JL, Bansal RK, Kern SE. G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: phenotypes reversed by a tumorigenic mutation. Proc Natl Acad Sci, 1999, 96(4): 1427-1432.
[4] Fletcher MC, Kuderova A, Cygler M, et al. Creation of a ribonuclease abzyme through site-directed mutagenesis. Nat Bio technol, 1998, 16(11): 1065-1067.
[5] Kono M, Miyaza KIG, Nakamura H, et al. Site-directed mutagenesis in hemoglobin: at tempts to control the oxygen affinity with cooperativity preserved. Protein, 1998, 11(3): 199-204.
[6] Wane A, Pakl KA. Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability. Biochemistry, 1998, 37(38): 13120-13127.
[7] Horton SN, Hunt HD. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 1989, 77(1): 51-59.
[8] Senanayake SD, Brain DA. Precise large deletions by the PCR-based overlap extension.Mol Biotechnol, 1995,4(1):13-15.
[9] Urban A,Neukirehen S,Jaeger KE.A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.Nucleic Acids Res,1997,25(ll):2227-2228.
[10] Warrens AN, Jones MD, Lechler RI. Splicing by overlap extension by PCR using asymmetric amplification: An improved technique for the generation of hybrid proteins of immunological interest.Gene, 1997, 186(l):29-35.
[11] Horton RM, Cai ZL, Horton SN.Gene splicing by overlap extension:Tailor-made genes using the polymerase chain reaction.Biotechniques, 1990,8(5):528-535.
[12] Chang SW,Shieh CJ.Mutiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 gene expressed in pichia pastoris.Appl Microbiol Biotechnol,2004,186(1): 159-264.
[13] Rheinallt M, Jones R.Mutational analysis of the critical bases involved in activation of the AreR-regulated 54-dependent promoter in acinetobacter strain ADP l.Appl Envir Microbiol, 2003,69:5627-5635.
[14] Huang CY, Chang AK. Site-directed mutagenesis of ionizable group in the active site of zymomona mobilis pyruvate decarboxylase: Effect on activity and PH dependence.Eur J Biochem, 2001, 268(12):3558-3565.
[15] Mukhopadhyay P, Roy KB.Protein engineering of BamH I restriction endonuclease: replacement of Cys 54 by Ala enhances catalytic activity.Protein,1998, 11(10):931-945.
[16] Sarkar G, Sommer SS.The megaprimer method of site-directed mutagenesis.Biotechniques, 1990, 8(4):404-407.
[17] Colosimo A,Xu Z,Novelli G,et al.Simple version of megaprimer PCR for site-directed mutagenesis.Biotechniques, 1999,26:870-873.
[18] Ke SH, Madison EL. Rapid and efficient site-directed mutagenesis by single-tube megaprimer PCR method. Nucleic Acids Res, 1997, 25: 3371-3372.
[19] Li CH, Fang HY. Construction of middle fragment deletion mutant with improved gene splicing by overlap extension PCR. Zhonghua YiXue YiChuan Xue ZaZhi, 2004, 21(1): 52-55.
[20] Chen K, Arnold FH. Turning the activity of an enzyme for unususl environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci, 1993, 90: 5618-5622.
[21] 彭贵洪，马忠，薛宇鸣等。尿激酶变体Glul54-m tcu-PA的构建及其性质研究．生物化学与生物物理学报，1997，29(6)：547-552．
[2] Byrne PA,Rajan KT.Spondyloepiphyseal dysplasia tarda with progressive arthropathy mimicking juvenile chronic arthritis.Br J Rheumatol ,1998 ,37:233-234.
[3] Kocyigit H,Arkun R,Ozkinay F,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.Clin Rheumatol,2000,19:238-241.
[4] Van Buggenhout G, de Smet L, Maroteaux P, et al.Progressive pseudorheumatoid dysplasia: report of a patient with symptoms present at birth.Genet Couns, 1998, 9:277-281.
[5] Teebi AS, Al Awadi SA.Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed among Arabs.J Med Genet,1986,23:189-191.
[6] Balci S,Aypar E,Kasapcopur O,et al.An eleven-year-old female Turkish patient with progressive pseudorheumatoid dysplasia mimicking juvenile idiopathic arthritis.Clin Exp Rheumatol,2001,19:759.
[7] El-Shanti HE, Omari HZ, Qubain HI.Progressive pseudorheumatoid dysplasia: report of a family and review.J Med Genet, 1997, 34:559-563.
[8] Al-Awadi SA,Farag TI,Naguib K,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.J Med Genet,1984,21:193-196.
[9] Spranger J,Albert C,Schilling F,et al.Progressive pseudorheumatoid arthritis of childhood (PPAC).A hereditary disorder simulating rheumatoid arthritis.Eur J Pediatr,1983,14:34-40.
[10] Rezai-Delui H,Mamoori G,Sadri-Mahvelati E,et al.Progressive pseudorheumatoid chondrodysplasia:a report of nine cases in three families.Skeletal Radiol, 1994,23:411 -419.
[11] Hurvitz JR,Suwairi WM,Van Hul W,et al.Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia.Nature Genet 1999;23:94-98.
[12] Valerie Delague,Eliane Chouery,Sandra Corbani,et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia:Identification of two novel mutations,and description of a founder effect.Am J Med Genet A.2005;138(2):118-126.
[13] Ehl S,Uhl M,Berner R,Bonafe L,et al.Clinical,radiographic,and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy.Rheumatol Int 2004;24: 53-56.
[14] Perbal B,Brigstock DR,Lau LF.Report on the second international workshop on the CCN family of genes.Mol Pathol,2003,56:80-85.
[15] Perbal B.NOV (nephroblastoma overexpressed) and CCN family of genes:structural and functional issues.Mol Pathol, 2001, 54:57-79.
[16] Brigstock DR.The CCN family: a new stimulus package.J Endocrinol, 2003,178:169-175.
[17] Liao EY, Peng YQ, Zhou HD, et al.Gene symbol: WISP3. Disease:spondyloepihyseal dysplasia tarda with progressive arthropathy.Hum Genet.2004 Jul;115(2):169.
[18] Liao EY,Peng YQ,Zhou HD,et al.Gene symbol: WISP3. Disease:spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul；115(2)：174．
[19] 彭依群，廖二元，顾慧敏等．复合杂合性CCN6基因突变致晚发型脊柱骨骺发育不良伴进行性骨关节病的关节软骨病理和分子病因研究．中华医学杂志，2004；84：1796-1803．
[20] 周后德，彭依群，谷卫等。SEDT-PA患者关节软骨细胞生物学特性及基因表达谱的改变。中华内科杂志，2005，44：16-21．
[1] Wynne-Davies R, Hall C, Ansell BM. Spodyloepiphyseal dysplasia tarda with progressive arthropathy1 A"new"disorder of autosomal recessive inheritance1. J Bone Joint Surg Br, 1982, 64: 442-445.
[2] Spranger J, Albert C, Schilling F, et al. Progressive pseudorheumatoid arthritis of childhood (PPAC). A hereditary disorder simulating rheumatoid arthritis. Eur J Pediatr, 1983, 14: 34-40.
[3] Rezai-Delui H, Mamoori G, Sadri-Mahvelati E, et al. Progressive pseudorheumatoid chondrodysplasia: a report of nine cases in three families. Skeletal Radiol, 1994, 23: 411-419.
[4] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 169.
[5] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 174.
[6] Mampaey S, Vanhoenacker F, Boven K, et al. Progressive pseudorheumatoid dysplasia. Eur Radiol. 2000; 10: 1832-1835
[7] Kaibara N, Takagishi K, Katsuki I, et al. Spondyloepiphyseal dysplasia tarda with progressive arthropathy. Skeletal Radiol 1983; 10:13-6
[8] Byrne PA,Rajan KT.Spondyloepiphyseal dysplasia tarda with progressive arthropathy mimicking juvenile chronic arthritis.Br J Rheumatol ,1998,37:233-234.
[9] Kocyigit H,Arkun R,Ozkinay F,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.Clin Rheumatol,2000,19:238-241.
[10] Van Buggenhout G, de Smet L, Maroteaux P, et al.Progressive pseudorheumatoid dysplasia: report of a patient with symptoms present at birth.Genet Couns, 1998, 9:277-281.
[11] Teebi AS, Al Awadi SA.Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed amongArabs.J Med Genet,1986,23:189-191.
[12] Balci S,Aypar E,Kasapcopur O,et al.An eleven-year-old female Turkish patient with progressive pseudorheumatoid dysplasia mimicking juvenile idiopathic arthritis.Clin Exp Rheumatol,2001,19:759.
[13] El-Shanti HE, Omari HZ, Qubain HI.Progressive pseudorheumatoid dysplasia: report of a family and review.J Med Genet, 1997, 34:559-563.
[14] Al-Awadi SA,Farag TI,Naguib K,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.J Med Genet,1984,21:193-196.
[15] Perbal B,Brigstock DR,Lau LF.Report on the second international workshop on the CCN family of genes.Mol Pathol,2003,56:80-85.
[16] Perbal B.NOV (nephroblastoma overexpressed) and CCN family of genes:structural and functional issues.Mol Pathol, 2001, 54:57-79.
[17] Brigstock DR.The CCN family: a new stimulus package.J Endocrinol, 2003,178:169-175.
[18] Pennica D,Swanson TA, Welsh JW,et al.WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumor. Proct Nat Acad Sci 1998; 95: 14717-14722.
[19] Bork P. The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Letters 1993; 327: 125-130.
[20] Schutze N, Noth Ul, Schneidereit J, et al. Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation. Cell Communication and Signaling 2005, 3: 1-12.
[21] Hurvitz JR, Suwairi WM, Van Hul W, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nature Genet 1999; 23: 94-98.
[22] Valerie Delague, Eliane Chouery, Sandra Corbani, et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: Identification of two novel mutations, and description of a founder effect. Am J Med Genet A. 2005; 138(2): 118-126.
[23] Ehl S, Uhl M, Berner R, Bonafe L, et al. Clinical, radiographic, and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy. Rheumatol Int 2004; 24: 53-56.
[24] 彭依群，廖二元，顾慧敏等．复合杂合性CCN6基因突变致晚发型脊柱骨骺发育不良伴进行性骨关节病的关节软骨病理和分子病因研究．中华医学杂志，2004：84：1796-1803．
[25] Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selectior. Proc Natl Acad Sci, 1985, 82(2): 488-492.
[26] Laird AD, Morrison DK, Shalloway D. Characterization of Raf-1 activation in mitosis. J Biol Chem, 1999,274(7):4430-4439.
[27] Dai JL, Bansal RK, Kern SE.G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: phenotypes reversed by a tumorigenic mutation.Proc Natl Acad Sci, 1999, 96(4):1427-1432.
[28] Fletcher MC,Kuderova A,Cygler M,et al.Creation of a ribonuclease abzyme through site-directed mutagenesis.Nat Bio technol,1998,16(11):1065-1067.
[29] Kono M, Miyaza KIG, Nakamura H, et al. Site-directed mutagenesis in hemoglobin: at tempts to control the oxygen affinity with cooperativity preserved.Protein, 1998, 11 (3): 199-204.
[30] Urban A,Neukirehen S,Jaeger KE.A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.Nucleic Acids Res,1997,25(ll):2227-2228.
[31] Warrens AN, Jones MD,Lechler RI. Splicing by overlap extension by PCR using asymmetric amplification:An improved technique for the generation of hybrid proteins of immunological interest.Gene,1997,186(1):29-35.
[32] Horton RM, Cai ZL, Horton SN.Gene splicing by overlap extension: Tailor-made genes using the polymerase chain reaction.Biotechniques, 1990,8(5):528-535.
[33] Chang SW,Shieh CJ.Mutiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 gene expressed in pichia pastoris.Appl Microbiol Biotechnol,2004,186(1):159-264.
[34] Rheinallt M, Jones R.Mutational analysis of the critical bases involved in activation of the AreR-regulated 54-dependent promoter in acinetobacter strain ADP.Appl Envir Microbiol, 2003, 69:5627-5635.
[35] Huang CY, Chang AK. Site-directed mutagenesis of ionizable group in the active site of zymomona mobilis pyruvate decarboxylase: Effect on activity and PH dependence.Eur J Biochem, 2001,268(12):3558-3565.
[36] Mukhopadhyay P, Roy KB.Protein engineering of BamH I restriction endonuclease: replacement of Cys 54 by Ala enhances catalytic activity.Protein, 1998, 11(10):931-945.
[37] Sarkar G, Sommer SS.The megaprimer method of site-directed mutagenesis.Biotechniques, 1990, 8(4):404-407.
[38] Colosimo A,Xu Z,Novelli G,et al.Simple version of megaprimer PCR for site-directed mutagenesis.Biotechniques,1999,26:870-873.
[39] Ke SH, Madison EL.Rapid and efficient site-directed mutagenesis by single-tube megaprimer PCR method.Nucleic Acids Res, 1997,25:3371-3372.
[40] Li CH, Fang HY. Construction of middle fragment deletion mutant with improved gene splicing by overlap extension PCR.Zhonghua YiXue YiChuan Xue ZaZhi, 2004,21(1):52-55.
[41] Chen K, Arnold FH.Turning the activity of an enzyme for unususl environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci, 1993,90:5618-5622.
[1] Spranger J,Albert C,Schilling F,et al.Progressive pseudorheumatoid arthritis of childhood (PPAC).A hereditary disorder simulating rheumatoid arthritis.Eur J Pediatr,1983,14:34-40.
[2] Rezai-Delui H,Mamoori G,Sadri-Mahvelati E,et al.Progressive pseudorheumatoid chondrodysplasia:a report of nine cases in three families.Skeletal Radiol, 1994,23:411-419.
[3] Perbal B.NOV (nephroblastoma overexpressed) and CCN family of genes: structural and functional issues.Mol Pathol, 2001, 54:57-79.
[4] Brigstock DR.The CCN family: a new stimulus package.J Endocrinol, 2003,178:169-175.
[5] Pennica D,Swanson TA, Welsh JW,et al.WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumor.Proct Nat Acad Sci 1998;95:14717-14722.
[6] Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression. Science, 1994, 263:802-805.
[7] Gerdes HH, Kaether C. Green fluorescent protein: applicationsin cell biology.FEBS Lett, 1996, 389:44-47.
[8] Kain SR, Adams M, Kondepudi A, et al. Green fluorescent protein as a reporter of gene expression and protein localization.Biotechniques, 1995 ,19 :650-655
[9] Gubin AN, et al. Long - term stable expression of green fluorescent protein in mammalian cells Biochem. Biophys Res Commun ,1997 ;236 :347-350
[10] Derek AP, James AA, Esther RA, et al. Retroviral - mediated tranfer of the green fluorescent protein gene into murine hematopoieticcells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo. Blood , 1997 ;90 (5) : 1777-1786
[11] John PL, Rebecca RM, Sergei Z, et al. Retroviral transfer and expression of a humanized, red-shifted green fluorescent protein gene into human tumor cells.Nature Biotechm ,1996 ;14 (5) :610-614
[12] Holt JR, Johns DC, Wang S, et al. Functional expression of exogenous proteins in mammalian sensoryhair cells infected eith adenoviral vectors. J Neurophysiol ,1999 ;81 (4) .1881-1888
[13] Weingart CL, Broitman - Maduro G, Dean G, et al. Fluorescent labels influence phagocytosis of Bordetella Pertussis by human neutophils. Infect Immun, 1999; 67(8): 4264-4267
[14] Liu HS, Jan MS, Chou CK, et al. Is green fluorescent protein toxic to the living cells? Biochen Biophys Res Commun, 1999; 260(3): 712-717
[15] Dreyer EB, Vorwerk CK, Zurakowski D, et al. Infection with adeno-associated virus may protect excitotoxicity. Neuroreport, 1999; 10(14): 2887-2890
[16] Takada T, et al. Selective production of transgenic nice using green fluorescent protein as a marker. Nat Biotechnol, 1997; 15: 458-461
[17] Stewart CN, Richards HA, Halfhill MD. Transgenic plants and biosafety: science, misconceptions and public perceptions. Biotechniques, 2000; 29 (4): 838-843
[18] Seethala R, Menxel R. A fluorescence polarization competition immunoassay for tyrosine kinases. J Annal Biochem, 1998; 255-257
[19] Bronstein I, Martin CS, Fortin JJ, et al. Chemliuminescince: sensitive detection technology for receptor gene assays. Clin Chem, 1996; 42(9): 1542
[20] Xu W. Structural organiztion of the human vesicular monoamine transporter type-2 gene and promoter analysis using the jelly fish green fluorescent protein as a reporter. Brain Res Mol Brain Res, 1997; 45: 41-49
[21] Casper SJ, et al. Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vactor. Gene, 1996; 173: 69～73
[22] 彭依群，廖二元，顾慧敏等．复合杂合性CCN6基因突变致晚发型脊柱骨骺发育不良伴进行性骨关节病的关节软骨病理和分子病因研究．中华医学杂志，2004；84：1796-1803．
[23] 宋今丹。医学细胞分子生物学，第一版。北京：人民卫生出版社，2003，141-161
[24] 卢圣栋。现代分子生物学实验技术，第一版。北京：高等教育出版社，1993， 3421-3432
[25] 瞿中和。细胞生物学，第一版。北京：高等教育出版社，1995，1021
[1] Spranger J, Albert C, Schilling F, et al. Progressive pseudorheumatoid arthritis of childhood (PPAC). A hereditary disorder simulating rheumatoid arthritis. Eur J Pediatr, 1983, 14: 34-40.
[2] Rezai-Delui H, Mamoori G; Sadri-Mahvelati E, et al. Progressive pseudorheumatoid chondrodysplasia: a report of nine cases in three families. Skeletal Radiol, 1994, 23: 411-419.
[3] Hurvitz JR, Suwairi WM, Van Hul W, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nature Genet 1999; 23: 94-98.
[4] Valerie Delague, Eliane Chouery, Sandra Corbani, et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: Identification of two novel mutations, and description of a founder effect. Am J Med Genet A. 2005; 138(2): 118-126.
[5] Ehl S, Uhl M, Berner R, Bonafe L, et al. Clinical, radiographic, and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy. Rheumatol Int 2004; 24: 53-56.
[6] Wynne-Davies R, Hall C, Ansell BM1. Spodyloepiphyseal dysplasia tarda with progressive arthropathy1 A"new"disorder of autosomal recessive inheritance1. J Bone Joint Surg Br, 1982, 64: 442-445.
[7] Byrne PA, Rajan KT. Spondyloepiphyseal dysplasia tarda with progressive arthropathy mimicking juvenile chronic arthritis. Br J Rheumatol, 1998 ,37:233-234.
[8] Kocyigit H,Arkun R,Ozkinay F,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.Clin Rheumatol,2000,19:23 8-241.
[9] Van Buggenhout G, de Smet L, Maroteaux P, et al.Progressive pseudorheumatoid dysplasia: report of a patient with symptoms present at birth.Genet Couns, 1998, 9:277-281.
[10] Teebi AS, Al Awadi SA.Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed amongArabs.J Med Genet,1986,23:189-191.
[11] Balci S,Aypar E,Kasapcopur O,et al.An eleven-year-old female Turkish patient with progressive pseudorheumatoid dysplasia mimicking juvenile idiopathic arthritis.Clin Exp Rheumatol,2001,19:759.
[12] El-Shanti HE, Omari HZ, Qubain HI.Progressive pseudorheumatoid dysplasia:report of a family and review.J Med Genet, 1997, 34:559-563.
[13] Al-Awadi SA,Farag TI,Naguib K,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.J Med Genet, 1984,21:193-196.
[14] Spranger J,Albert C,Schilling F,et al.Progressive pseudorheumatoid arthritis of childhood ( PPAC).A hereditary disorder simulating rheumatoid arthritis.Eur J Pediatr,1983,14:34-40.
[15] Rezai-Delui H,Mamoori G,Sadri-Mahvelati E,et al.Progressive pseudorheumatoid chondrodysplasia:a report of nine cases in three families.Skeletal Radiol, 1994,23:411 -419.
[16] Liao EY, Peng YQ, Zhou HD, et al.Gene symbol: WISP3. Disease:spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet.2004 Jul; 115(2): 169.
[17] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 174.
[18] 周后德，彭依群，谷卫等。SEDT-PA患者关节软骨细胞生物学特性及基因表达谱的改变。中华内科杂志，2005，44：16-21
[19] Goldring MB, Birkhead JR, Suen LF, et al. Interleukin 1 beta modulated gene expression in immortalized human chondrocytes. J Clin Invest 1994; 94: 2307-2316.
[20] Chansky H, Robbins JR, Cha S, et al. Expression ofcartilage extracellular matrix and potential regulatory genes in a new human chondrosarcoma cell line. J Orthop Res 199816(5): 521-530.
[21] Lee D, Long SA, Adams JL, et al. Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality. J Biol Chem 2000; 275: 16007-16014.
[22] Browning JA, Wilkins RJ. The effect of intracellular alkalinisation on intracellular Ca2+homeostasis in a human chondrocyte cell line. Eur J Physiol 2002; 444: 744-751.
[23] Bork P. The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Letters 1993; 327: 125-130.
[24] Hurvitz JR, Suwairi WM, Van Hul W, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nature Genet 1999; 23: 94-98.
[25] Kleer CG, Zhang Y, Pan Q, et al. WISP3 is a novel tumor suppressor gene of inflammatory breast cancer. Oncogene, 2002, 21: 3172-3180.
[26] Kleer CG, Zhang Y, Pan Q, et al. WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer. Breast Cancer Res. 2004; 6(2): R110-115.
[27] Kleer CG, Zhang Y, Pan Q, et al.WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer.Neoplasia.2004; 6(2): 179-185.
[28] Zhang Y,Pan Q,Zhong H,et al.Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells.Breast Cancer Res.2005;7(6):R1080-1089.
[29] Schutze N,Noth Ul, Schneidereit J,et al.Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation.Cell Communication and Signaling 2005, 3:1-12.
[30] Lake AC, Castellot JJ J r.CCN5modulates the antiproliferativeeffect of heparin and regulates cell motility in vascular smooth muscle cells.Cell Commun Signal,2003,1:5.
[31] Perbal B.CCN proteins: multifunctional signalling regulators.Lancet, 2004, 363:62-64.
[32] Sen M,Cheng YH,Goldring MB,et al.WISP3-dependent regulation of type II collagen and aggrecan production in chondrocytes.Arthritis Rheum,2004,50:488-497.
[1] Perbal B, Brigstock DR, Lau LF. Report on the second international workshop on the CCN family of genes. Mol Pathol, 2003, 56: 80-85.
[2] Perbal B. NOV (nephroblastoma overexpressed) and CCN family of genes: structural and functional issues. Mol Pathol, 2001, 54: 57-79.
[3] Brigstock DR. The CCN family: a new stimulus package. J Endocrinol, 2003, 178:169-175.
[4] Pennica D,Swanson TA, Welsh JW,et al.WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumor.Proct Nat Acad Sci 1998;95:14717-14722.
[5] Bork P.The modular architecture of a new family of growth regulators related to connective tissue growth factor.FEBS Letters 1993; 327:125-130.
[6] Brigstock DR. The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocrine Reviews 1999;20:189-206.
[7] Lau LF & Lam SC.The CCN family of angiogenic regulators: the integrin connection.Experimental Cell Research 1999; 248:44-57.
[8] Mancuso DJ,Tuley EA,Westfield LA,et al.Structure of the gene for human von Willebrand factor.J Biol Chem. 1989; 264(33):19514-19527.
[9] Holt GD,Pangburn MK,Ginsburg V.Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates.J Biol Chem. 1990; 265(5):2852-2855.
[10] Voorberg J,Fontijn R,Calafat J,et al.Assembly and routing of von Willebrand factor variants: the requirements for disulfide-linked dimerization reside within the carboxy-terminal 151 amino acids.J Cell Biol 1991; 113(1):195-205.
[11] Schutze N,Noth Ul, Schneidereit J,et al.Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation.Cell Communication and Signaling 2005, 3:1-12.
[12] Lake AC, Castellot JJ J r.CCN5modulates the antiproliferativeeffect of heparin and regulates cell motility in vascular smooth muscle cells.Cell Commun Signal, 2003,1:5.
[13] Perbal B.CCN proteins: multifunctional signalling regulators.Lancet, 2004, 363: 62-64.
[14] Wynne-Davies R,Hall C,Ansell BM.Spodyloepiphyseal dysplasia tarda with progressive arthropathyl A"new"disorder of autosomal recessive inheritancel.J Bone Joint Surg Br, 1982,64:442-445.
[15] Byrne PA,Rajan KT.Spondyloepiphyseal dysplasia tarda with progressive arthropathy mimicking juvenile chronic arthritis.Br J Rheumatol ,1998 ,37:233-234.
[16] Kocyigit H,Arkun R,Ozkinay F,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.Clin Rheumatol,2000,19:23 8-241.
[17] Van Buggenhout G, de Smet L, Maroteaux P, et al.Progressive pseudorheumatoid dysplasia: report of a patient with symptoms present at birth.Genet Couns, 1998,9:277-281.
[18] Teebi AS, Al Awadi SA.Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed amongArabs.J Med Genet,1986,23:189-191.
[19] Balci S,Aypar E,Kasapcopur O,et al.An eleven-year-old female Turkish patient with progressive pseudorheumatoid dysplasia mimicking juvenile idiopathic arthritis.Clin Exp Rheumatol,2001,19:759.
[20] El-Shanti HE, Oman HZ, Qubain HI.Progressive pseudorheumatoid dysplasia:report of a family and review.J Med Genet, 1997, 34:559-563.
[21] Al-Awadi SA,Farag TI,Naguib K,et al.Spondyloepiphyseal dysplasia tarda with progressive arthropathy.J Med Genet, 1984,21:193 -196.
[22] Spranger J,Albert C,Schilling F,et al.Progressive pseudorheumatoid arthritis of childhood ( PPAC).A hereditary disorder simulating rheumatoid arthritis.Eur J Pediatr, 1983, 14: 34-40.
[23] Rezai-Delui H, Marnoori G, Sadri-Mahvelati E, et al. Progressive pseudorheumatoid chondrodysplasia: a report of nine cases in three families. Skeletal Radiol, 1994, 23: 411-419.
[24] Hurvitz JR, Suwairi WM, Van Hul W, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nature Genet 1999; 23: 94-98.
[25] Valerie Delague, Eliane Chouery, Sandra Corbani, et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: Identification of two novel mutations, and description of a founder effect. Am J Med Genet A. 2005; 138(2): 118-126.
[26] Ehl S, Uhl M, Berner R, Bonafe L, et al. Clinical, radiographie, and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy. Rheumatol Int 2004; 24: 53-56.
[27] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 169.
[28] Liao EY, Peng YQ, Zhou HD, et al. Gene symbol: WISP3. Disease: spondyloepihyseal dysplasia tarda with progressive arthropathy. Hum Genet. 2004 Jul; 115(2): 174.
[29] 彭依群，廖二元，顾慧敏等．复合杂合性CCN6基因突变致晚发型脊柱骨骺发育不良伴进行性骨关节病的关节软骨病理和分子病因研究．中华医学杂志，2004：84：1796-1803．
[30] 周后德，彭依群，谷卫等。SEDT-PA患者关节软骨细胞生物学特性及基因表达谱的改变。中华内科杂志，2005，44：16-21
[31] Matsuno H, Yudoh K, Nakazawa F, et al. Antirheumatic effects of humanized anti-Fas monoclonal antibody in human rheumatoid arthritis/SCID mouse chimera. J Rheumatol, 2002,29:1609-1614.
[32] Cho TJ, Lehmann W, Edgar C, et al.Tumor necrosis factor alpha activation of the apoptotic cascade in murine articular chondrocytes is associated with the induction of metalloproteinases and specific pro-resorptive factors.Arthritis Rheum, 2003, 48:2845-2854.
[33] Sen M,Cheng YH,Goldring MB,et al.WISP3-dependent regulation of type II collagen and aggrecan production in chondrocytes.Arthritis Rheum,2004,50:488-497.
[34] Kutz WE, Gong Y, Warman ML.WISP3, the gene responsible for the human skeletal disease progressive pseudorheumatoid dysplasia, is not essential for skeletal function in mice. Mol Cell Biol 2005; 25(1):414-421.
[35] Kleer CG Zhang Y, Pan Q, et al.WISP3 is a novel tumor suppressor gene of inflammatory breast cancer.Oncogene, 2002, 21:3172-3180.
[36] Kleer CG,Zhang Y,Pan Q,et al.WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer.Breast Cancer Res.2004;6(2):R110-115.
[37] Kleer CG, Zhang Y, Pan Q, et al.WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer.Neoplasia.2004r; 6(2): 179-185.
[38] Zhang Y,Pan Q,Zhong H,et al.Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells.Breast Cancer Res.2005;7(6):R1080-1089.
[1] Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selectior. Proc Natl Acad Sci, 1985, 82(2): 488-492.
[2] Laird AD, Morrison DK, Shalloway D. Characterization of Raf-1 activation in mitosis. J Biol Chem, 1999, 274(7): 4430-4439.
[3] Dai JL, Bansal RK, Kern SE. G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: phenotypes reversed by a tumorigenic mutation. Proc Natl Acad Sci, 1999, 96(4): 1427-1432.
[4] Fletcher MC, Kuderova A, Cygler M, et al. Creation of a ribonuclease abzyme through site-directed mutagenesis. Nat Bio technol, 1998, 16(11): 1065-1067.
[5] Kono M, Miyaza KIG, Nakamura H, et al. Site-directed mutagenesis in hemoglobin: at tempts to control the oxygen affinity with cooperativity preserved. Protein, 1998, 11(3): 199-204.
[6] Wane A, Pakl KA. Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability. Biochemistry, 1998, 37(38): 13120-13127.
[7] Horton SN, Hunt HD. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 1989, 77(1): 51-59.
[8] Senanayake SD, Brain DA. Precise large deletions by the PCR-based overlap extension.Mol Biotechnol, 1995,4(1):13-15.
[9] Urban A,Neukirehen S,Jaeger KE.A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.Nucleic Acids Res,1997,25(ll):2227-2228.
[10] Warrens AN, Jones MD, Lechler RI. Splicing by overlap extension by PCR using asymmetric amplification: An improved technique for the generation of hybrid proteins of immunological interest.Gene, 1997, 186(l):29-35.
[11] Horton RM, Cai ZL, Horton SN.Gene splicing by overlap extension:Tailor-made genes using the polymerase chain reaction.Biotechniques, 1990,8(5):528-535.
[12] Chang SW,Shieh CJ.Mutiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 gene expressed in pichia pastoris.Appl Microbiol Biotechnol,2004,186(1): 159-264.
[13] Rheinallt M, Jones R.Mutational analysis of the critical bases involved in activation of the AreR-regulated 54-dependent promoter in acinetobacter strain ADP l.Appl Envir Microbiol, 2003,69:5627-5635.
[14] Huang CY, Chang AK. Site-directed mutagenesis of ionizable group in the active site of zymomona mobilis pyruvate decarboxylase: Effect on activity and PH dependence.Eur J Biochem, 2001, 268(12):3558-3565.
[15] Mukhopadhyay P, Roy KB.Protein engineering of BamH I restriction endonuclease: replacement of Cys 54 by Ala enhances catalytic activity.Protein,1998, 11(10):931-945.
[16] Sarkar G, Sommer SS.The megaprimer method of site-directed mutagenesis.Biotechniques, 1990, 8(4):404-407.
[17] Colosimo A,Xu Z,Novelli G,et al.Simple version of megaprimer PCR for site-directed mutagenesis.Biotechniques, 1999,26:870-873.
[18] Ke SH, Madison EL. Rapid and efficient site-directed mutagenesis by single-tube megaprimer PCR method. Nucleic Acids Res, 1997, 25: 3371-3372.
[19] Li CH, Fang HY. Construction of middle fragment deletion mutant with improved gene splicing by overlap extension PCR. Zhonghua YiXue YiChuan Xue ZaZhi, 2004, 21(1): 52-55.
[20] Chen K, Arnold FH. Turning the activity of an enzyme for unususl environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci, 1993, 90: 5618-5622.
[21] 彭贵洪，马忠，薛宇鸣等。尿激酶变体Glul54-m tcu-PA的构建及其性质研究．生物化学与生物物理学报，1997，29(6)：547-552．