重组人E1A激活基因阻遏子/myc-His融合糖蛋白的表达纯化及功能鉴定
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摘要
目的E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes, CREG)是1998年Gill博士利用酵母双杂交技术克隆的一个转录调控相关因子。CREG基因可与外源性腺病毒蛋白E1A及哺乳动物体内转录因子E2F家族蛋白竞争性地结合在靶基因的启动子区,从而阻遏E1A蛋白和E2F对靶基因的转录,而E1A、E2F均具有促进细胞增殖的作用,所以CREG可能通过与E1A、E2F竞争性抑制作用而起到促进细胞分化、抑制细胞增殖的作用。本实验室前期研究发现,去血清能够诱导体外培养的人血管平滑肌细胞(vascular smooth muscle cells,VSMCs)从合成表型逆转为分化表型,同时CREG蛋白表达明显增加。大鼠颈动脉拉伤实验证实,CREG基因表达与VSMCs增殖能力呈负相关,推测其可能参与了VSMCs表型调控。Veal博士等证实CREG是一种分泌型糖蛋白,可以与胰岛素样生长因子II竞争性结合细胞膜胰岛素样生长因子受体II,以自分泌和旁分泌两种途径对细胞功能进行调控。为进一步明确CREG糖蛋白的功能,本研究采用逆转录-聚合酶链反应(RT-PCR)、蛋白印迹(Western blot)及Ni-NTA亲和层析等方法表达、纯化重组hCREG糖蛋白,并用流式细胞周期分析、5-溴脱氧尿嘧啶(BrdU)掺入实验等方法验证重组人CREG(hCREG)糖蛋白具备抑制体外培养的人胸廓内动脉平滑肌细胞(Human internal thoracic artery-Shenyang, HITASY)增殖的生物学功能,为hCREG蛋白的工程化生产提供前期研究基础。
方法①用RT-PCR技术扩增终止密码突变的hCREG开放读码框并构建pcDNA3.1 myc-His/hCREG真核表达载体。②Lipofectamine 2000转染人293F细胞株并筛选稳定表达细胞克隆,Western blot方法鉴定hCREG/myc-His融合蛋白的表达,糖苷酶法及Western blotting行hCREG/myc-His融合蛋白的糖基化分析。③根据6×His亲和层析原理,应用Ni-NTA柱纯化重组hCREG蛋白,纯化后蛋白通过HiTrap脱盐柱脱盐。④用流式细胞周期分析研究添加0.5μg /mL、1μg /mL及2μg /mL重组hCREG/myc-His融合糖蛋白对体外培养HITASY细胞增殖的影响。用BrdU掺入方法研究添加重组蛋白对体外培养HITASY细胞增殖的影响。
结果RT-PCR扩增含终止密码突变的hCREG cDNA片段,经BamH I和EcoR I双酶切构建了pcDNA 3.1myc-His/hCREG真核表达载体,酶切及测序结果均证实构建的重组质粒正确。
稳定表达pcDNA 3.1 myc-His/hCREG的293F细胞及未转染的对照细胞裂解产物,分别以Anti-hCREG、Anti-myc及Anti-His行Western blot检测。hCREG抗体可检测到转染组较对照组多两条约30KD的融合蛋白表达条带,myc抗体及His抗体均检测到大小约为30KD的融合蛋白表达。将稳定表达pcDNA 3.1 myc-His/hCREG的293F细胞裂解产物及经PNGaseF处理过的样品分别行Anti-hCREG、Anti-myc及Anti-His的Western blot检测。结果显示经PNGaseF处理后,hCREG融合蛋白分子量由30KD降低至25KD,电泳条带下移,证明带有myc和His标签的重组hCREG蛋白为糖基化蛋白。
根据6×His与Ni亲合层析的原理用Ni-NTA纯化hCREG重组蛋白。收集的洗脱液经Centriprep离心超滤管(10000NMWL)浓缩后,经BCA法测定并与蛋白标准曲线比较,重组hCREG蛋白浓度为1.6 mg/mL。考马斯亮兰染色可见30KD大小的较单一条带,经image-J软件分析,纯度为92%。浓缩并脱盐后的纯化蛋白经PNGaseF处理后分子量由30KD降至25KD,条带下移,证实纯化后的蛋白仍保留了糖基化形式。
流式细胞仪细胞周期分析结果提示重组hCREG蛋白与对照组相比可抑制体外培养的HITASY细胞增殖,G0/G1期细胞比率(共计数2万个细胞)显著增加,且低浓度组的抑制效应要高于高浓度组。BrdU掺入实验结果提示,添加2μg /mL重组hCREG蛋白组与对照组相比可显著抑制体外培养的HITASY细胞增殖,BrdU阳性细胞的比率降低。组间比较有统计学差异(P<0.05)。
结论pcDNA 3.1 myc-His/hCREG真核表达载体的构建及具有生物学功能的重组hCREG/myc-His糖蛋白的表达纯化,为hCREG蛋白的功能研究及生物工程制备提供了实验平台。纯化的重组hCREG蛋白可能作为一种新的药物涂于支架表面,通过其促进分化、抑制增殖的生物学功能实现降低支架内再狭窄的作用。
Objective The cellular repressor of E1A- stimulated genes (CREG), as a transcriptional regulator, was cloned by the yeast two-hybrid method by Dr. Gill in 1998. The result that CREG could inhibit the transcription of target gene by E1A protein and E2F may contribute to the binding of CREG competing with the exogenous adenovirus E1A protein and transcription factor of E2F family proteins in mammals to the promoter region of target genes. Both E1A protein and E2F can promote cellular dedifferentiation and proliferation, CREG is therefore likely to play a role in promoting differentiation and/or inhibiting cell growth by competing with E1A and E2F.
Our early research had found that the expression of CREG synchronously increased when the human vascular smooth muscle cells (VSMCs) cultured in vitro converted from synthetic into differentiated phenotype induced by serum withdrawal. The negative relationship between CREG mRNA expression and the proliferative activity of VSMCs was confirmed in rat injury carotid artery. CREG, as a secreted glycoprotein reported by Dr. Veal, could bind to the transmembrane insulin-like growth factor II receptor competing with the insulin-like growth factor II and regulate cell function through both autocrine and paracrine pathway. To study the bio-function of CREG glycoprotein, in this study, the reverse transcriptionpolymerase chain reaction(RT-PCR), Western blot and affinity chromatograph by Ni-NTA were used to express and purify the recombinant hCREG/myc-his glycoprotein. Flow cytometic analysis and BrdU incorporation method were used to confirm the biological function of hCREG/myc-his which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro. The work provided prophase research basis for engineering production of hCREG protein.
Methods①The ORF(open reading frame) fragment of human CREG(hCREG) with terminator codon mutation was amplificated by RT-PCR and the eukaryotic expression vector pcDNA3.1 myc-His/hCREG was constructed.②The human 293F cells were transfected with pcDNA3.1 myc-His/hCREG using Lipofectamine 2000, and stably transfected cell clones were screened by G418. The expression of recombinant secreted hCREG/myc-His protein was identified by Western blotting. The glycosylation of hCREG/myc-His protein was analyzed by PNGaseF digestion and Western blot.③The recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. The recombinant hCREG/ myc-His protein was concentrated and desalted. The concentration of recombinant protein was analyzed by SDS-PAGE and the purity was calculated by bio-informational software.④ - 8 -The effect of recombinant hCREG/myc-His glycoprotein of different concentration(0.5μg /mL、1μg /mL and 2μg /mL)on proliferation of HITASY cells was confirmed by Flow cytometic analysis and BrdU incorporation method.
Results The ORF fragment of hCREG was amplificated by RT-PCR and inserted into the pcDNA3.1 myc-His vector with EcoRΙ/BamHΙdigestion. The recombinant eukaryotic expression vector (pcDNA3.1 myc-His/ hCREG) was confirmed to be constructed successfully by restricted endonuclease digestion and DNA sequencing.
The lysates of 293F cells with stably transfected pcDNA3.1 myc-His/hCREG plasmid were detected by Western blot with Anti-hCREG, Anti-myc and Anti-His respectively. The recombinanted fusion protein about 30KD was identified in transfected cells by Western blot using Anti-myc and Anti-His. The recombinant fusion protein in cell lysates was identified as a glycoprotein because the molecular weight of fusion protein decreased from 30KD to 25KD after PNGaseF digestion. The recombinant hCREG protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. After the elution was concentrated with Centriprep centrifugal filter devices(10000NMWL), the concentration of recombinant protein was determined to be 1.6 mg/mL by BCA assay. The purity of recombinant protein reached 92% identified with image-J software analysis. Glycoprotein was identified using PNGaseF digestion.
The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro by Flow cytometic analysis and BrdU incorporation method. The inhibition effect of recombinant hCREG protein is stronger in low-dosage treatment than in high-dosage group. There was statistical difference among the groups(P<0.05).
Conclusion The construction of eukaryotic expression vector of pcDNA3.1 myc-His/hCREG and the expression and purification of recombinant hCREG/myc-His glycoprotein with biological activity provided an experiment platform for function study and engineering production of hCREG protein. The incidence of restenosis may be reduced if the stents were coated with recombinant hCREG/myc-His glycoprotein.
方法①用RT-PCR技术扩增终止密码突变的hCREG开放读码框并构建pcDNA3.1 myc-His/hCREG真核表达载体。②Lipofectamine 2000转染人293F细胞株并筛选稳定表达细胞克隆,Western blot方法鉴定hCREG/myc-His融合蛋白的表达,糖苷酶法及Western blotting行hCREG/myc-His融合蛋白的糖基化分析。③根据6×His亲和层析原理,应用Ni-NTA柱纯化重组hCREG蛋白,纯化后蛋白通过HiTrap脱盐柱脱盐。④用流式细胞周期分析研究添加0.5μg /mL、1μg /mL及2μg /mL重组hCREG/myc-His融合糖蛋白对体外培养HITASY细胞增殖的影响。用BrdU掺入方法研究添加重组蛋白对体外培养HITASY细胞增殖的影响。
结果RT-PCR扩增含终止密码突变的hCREG cDNA片段,经BamH I和EcoR I双酶切构建了pcDNA 3.1myc-His/hCREG真核表达载体,酶切及测序结果均证实构建的重组质粒正确。
稳定表达pcDNA 3.1 myc-His/hCREG的293F细胞及未转染的对照细胞裂解产物,分别以Anti-hCREG、Anti-myc及Anti-His行Western blot检测。hCREG抗体可检测到转染组较对照组多两条约30KD的融合蛋白表达条带,myc抗体及His抗体均检测到大小约为30KD的融合蛋白表达。将稳定表达pcDNA 3.1 myc-His/hCREG的293F细胞裂解产物及经PNGaseF处理过的样品分别行Anti-hCREG、Anti-myc及Anti-His的Western blot检测。结果显示经PNGaseF处理后,hCREG融合蛋白分子量由30KD降低至25KD,电泳条带下移,证明带有myc和His标签的重组hCREG蛋白为糖基化蛋白。
根据6×His与Ni亲合层析的原理用Ni-NTA纯化hCREG重组蛋白。收集的洗脱液经Centriprep离心超滤管(10000NMWL)浓缩后,经BCA法测定并与蛋白标准曲线比较,重组hCREG蛋白浓度为1.6 mg/mL。考马斯亮兰染色可见30KD大小的较单一条带,经image-J软件分析,纯度为92%。浓缩并脱盐后的纯化蛋白经PNGaseF处理后分子量由30KD降至25KD,条带下移,证实纯化后的蛋白仍保留了糖基化形式。
流式细胞仪细胞周期分析结果提示重组hCREG蛋白与对照组相比可抑制体外培养的HITASY细胞增殖,G0/G1期细胞比率(共计数2万个细胞)显著增加,且低浓度组的抑制效应要高于高浓度组。BrdU掺入实验结果提示,添加2μg /mL重组hCREG蛋白组与对照组相比可显著抑制体外培养的HITASY细胞增殖,BrdU阳性细胞的比率降低。组间比较有统计学差异(P<0.05)。
结论pcDNA 3.1 myc-His/hCREG真核表达载体的构建及具有生物学功能的重组hCREG/myc-His糖蛋白的表达纯化,为hCREG蛋白的功能研究及生物工程制备提供了实验平台。纯化的重组hCREG蛋白可能作为一种新的药物涂于支架表面,通过其促进分化、抑制增殖的生物学功能实现降低支架内再狭窄的作用。
Objective The cellular repressor of E1A- stimulated genes (CREG), as a transcriptional regulator, was cloned by the yeast two-hybrid method by Dr. Gill in 1998. The result that CREG could inhibit the transcription of target gene by E1A protein and E2F may contribute to the binding of CREG competing with the exogenous adenovirus E1A protein and transcription factor of E2F family proteins in mammals to the promoter region of target genes. Both E1A protein and E2F can promote cellular dedifferentiation and proliferation, CREG is therefore likely to play a role in promoting differentiation and/or inhibiting cell growth by competing with E1A and E2F.
Our early research had found that the expression of CREG synchronously increased when the human vascular smooth muscle cells (VSMCs) cultured in vitro converted from synthetic into differentiated phenotype induced by serum withdrawal. The negative relationship between CREG mRNA expression and the proliferative activity of VSMCs was confirmed in rat injury carotid artery. CREG, as a secreted glycoprotein reported by Dr. Veal, could bind to the transmembrane insulin-like growth factor II receptor competing with the insulin-like growth factor II and regulate cell function through both autocrine and paracrine pathway. To study the bio-function of CREG glycoprotein, in this study, the reverse transcriptionpolymerase chain reaction(RT-PCR), Western blot and affinity chromatograph by Ni-NTA were used to express and purify the recombinant hCREG/myc-his glycoprotein. Flow cytometic analysis and BrdU incorporation method were used to confirm the biological function of hCREG/myc-his which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro. The work provided prophase research basis for engineering production of hCREG protein.
Methods①The ORF(open reading frame) fragment of human CREG(hCREG) with terminator codon mutation was amplificated by RT-PCR and the eukaryotic expression vector pcDNA3.1 myc-His/hCREG was constructed.②The human 293F cells were transfected with pcDNA3.1 myc-His/hCREG using Lipofectamine 2000, and stably transfected cell clones were screened by G418. The expression of recombinant secreted hCREG/myc-His protein was identified by Western blotting. The glycosylation of hCREG/myc-His protein was analyzed by PNGaseF digestion and Western blot.③The recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. The recombinant hCREG/ myc-His protein was concentrated and desalted. The concentration of recombinant protein was analyzed by SDS-PAGE and the purity was calculated by bio-informational software.④ - 8 -The effect of recombinant hCREG/myc-His glycoprotein of different concentration(0.5μg /mL、1μg /mL and 2μg /mL)on proliferation of HITASY cells was confirmed by Flow cytometic analysis and BrdU incorporation method.
Results The ORF fragment of hCREG was amplificated by RT-PCR and inserted into the pcDNA3.1 myc-His vector with EcoRΙ/BamHΙdigestion. The recombinant eukaryotic expression vector (pcDNA3.1 myc-His/ hCREG) was confirmed to be constructed successfully by restricted endonuclease digestion and DNA sequencing.
The lysates of 293F cells with stably transfected pcDNA3.1 myc-His/hCREG plasmid were detected by Western blot with Anti-hCREG, Anti-myc and Anti-His respectively. The recombinanted fusion protein about 30KD was identified in transfected cells by Western blot using Anti-myc and Anti-His. The recombinant fusion protein in cell lysates was identified as a glycoprotein because the molecular weight of fusion protein decreased from 30KD to 25KD after PNGaseF digestion. The recombinant hCREG protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. After the elution was concentrated with Centriprep centrifugal filter devices(10000NMWL), the concentration of recombinant protein was determined to be 1.6 mg/mL by BCA assay. The purity of recombinant protein reached 92% identified with image-J software analysis. Glycoprotein was identified using PNGaseF digestion.
The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro by Flow cytometic analysis and BrdU incorporation method. The inhibition effect of recombinant hCREG protein is stronger in low-dosage treatment than in high-dosage group. There was statistical difference among the groups(P<0.05).
Conclusion The construction of eukaryotic expression vector of pcDNA3.1 myc-His/hCREG and the expression and purification of recombinant hCREG/myc-His glycoprotein with biological activity provided an experiment platform for function study and engineering production of hCREG protein. The incidence of restenosis may be reduced if the stents were coated with recombinant hCREG/myc-His glycoprotein.
引文
1 Kunita R, Otomo A, Ikeda JE. Identification and characterization of novel members of the CREG family, putative secreted glycoproteins expressed specifically in brain[J]. Genomics. 2002; 80(5):456~460.
2 Di Bacco A, Gill G. The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-like growth factor II receptor[J]. Oncogene. 2003; 22(35): 5436~5445.
3 Veal E, Groisman R, Eisenstein M, et al. The secreted glycoprotein CREG enhances differentiation of NTERA-2 human embryonal carcinoma cells[J]. Oncogene. 2000; 19(11): 2120~2128.
4 Veal E, Eisenstein M, Tseng A, et al. A cellular repressor of E1A- stimulated genes that inhibits activation by E2F[J]. Mol Cell Biol. 1998; 18(7): 5032~5041.
5韩雅玲,胡叶,刘海伟,等. E1A激活基因阻遏子促进人血管平滑肌细胞分化和迁移[J].生物化学和生物物理进展. 2005; 32(6): 517~522.
6 Mats Hellstr?m, Mattias Kalén, Per Lindahl, et al. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse[J]. Development. 1999; 126 (14) : 3047~3055.
7韩雅玲,康建,张剑,等.对去血清后HITASY细胞分子表达及表型分析[J].生物化学和生物物理进展. 2003; 30(6): 868~873.
8 Michael Sacher, Alessandra Di Bacco, Vladimir V. Lunin et al.The crystal structure of CREG, a secreted glycoprotein involved in cellular growth and differentiation[J]. Proc Natl Acad Sci U S A. 2005; 102(51):18326~18331.
9 Alessandra Di Bacco, Grace Gill, The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-like growth factor II receptor[J]. Oncogene. 2003; 22(35): 5436~5445.
10 Kobata A. Structures and functions of the sugar chains of glycoproteins[J]. Eur J Biochem. 1992; 209(2): 483~501.
11 Lloyd RC, BG. Davis, J B Jones. Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity[J]. Bioorg Med Chem. 2000; 8(7): 1537~1544.
12 Kwon KS, MH Yu. Effect of glycosylation on the stability of alpha1-antitrypsin toward urea denaturation and thermal deactivation[J]. Biochim Biophys Acta. 1997; 1335(3): 265~272.
13 Suzuki S, M Furuhashi, N Suganuma. Additional N-glycosylation at Asn(13) rescues the human LHbeta-subunit from disulfide-linked aggregation[J]. Mol Cell Endocrinol. 2000; 160(1~2): 157~163.
14 Sasaki K, Nishi T, Yasumura S, et a1. Modified granulocyte-colony stimulating factor polypeptide with added carbohydrate chains.United States Patent:52l8092.
15 Coloma M J, Trinh R K, Martinez A R, et al. Position effects of variable region carbohydrate on the affinity and in vivo behavior of an anti-(1-6) dextran antibody[J]. J Immunol. 1999; 162(4): 2162~2170.
16 Davies J, Jiang L Y, Pan L z, et al. Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII[J]. Biotechnol Bioeng. 2001; 74(4): 288~294.
17 Tams JW, J Vind, KG Welinder. Adapting protein solubility by glycosylation. N-glycosylation mutants of Coprinus cinereus peroxidase in salt and organicsolutions[J]. Biochim Biophys Acta. 1999; 1432(2): 214~221.
18 Linden HM, K Kaushansky. The glycan domain of thrombopoietin enhances its secretion[J]. Biochemistry. 2000; 39(11): 3044~3051.
19 Egrie JC, JK Browne. Development and characterization of novel erythropoiesis stimulating protein (NESP) [J]. Br J Cancer. 2001; 84 (Suppl 1):3~10.
20 Mistry PK, EP Wraight, TM Cox. Therapeutic delivery of proteins to macrophages: implications for treatment of Gaucher's disease[J]. Lancet. 1996; 348(9041): 1555~1559.
21 Dell A, HR Morris. Glycoprotein structure determination by mass spectrometry[J]. Science. 2001; 291(5512): 2351~2356.
22 Rudd PM, et al. A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands[J]. Proteomics. 2001; 1(2): 285~294.
23 Rudd PM, et al. Glycosylation and the immune system[J]. Science. 2001; 291(5512): 2370~2376.
24 Spellman MW. Carbohydrate characterization of recombinant glycoproteins of pharmaceutical interest[J]. Anal Chem. 1990; 62(17): 1714~1722.
25 Fryksdale BG., et al. Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis[J]. Electrophoresis. 2002; 23(14): 2184~2193.
26 Hardy MR, RR Townsend. Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection[J]. Proc Natl Acad SciU S A. 1988; 85(10):3289~3293.
27 Karty JA, et al. Artifacts and unassigned masses encountered in peptide mass mapping[J]. J Chromatogr B Analyt Technol Biomed Life Sci. 2002; 782(1~2): 363~383
28 Settineri CA, et al. Characterization of O-glycosylation sites in recombinant B-chain of platelet-derived growth factor expressed in yeast using liquid secondary ion mass spectrometry, tandem mass spectrometry and Edman sequence analysis[J]. Biomed Environ Mass Spectrom. 1990; 19(11): 665~676.
29 Greis KD, et al. Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry[J]. Anal Biochem. 1996; 234(1): 38~49.
30 Hanisch FG., M Jovanovic, J Peter-Katalinic. Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments[J]. Anal Biochem. 2001; 290(1): 47~59.
31 Apweiler R, H Hermjakob, N Sharon. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database[J]. Biochim Biophys Acta. 1999; 1473(1): 4~8.
32 Devenyi T , Gergely J .实用蛋白质化学技术.上海:上海科技出版社, 1982 , 52.
33 David R. Glycoprotein Detection in Polyacryamide Gel with Thymol and Sulfuric Acid[J]. Anal Biochem. 1979; 99(2): 474～476
34孙秀琴,中村健,伊滕洋一,等.曼氏裂头蚴蛋白分解酶的糖链研究[J].中国寄生虫病防治杂志. 2001;14 (4) :285～287.
35孙建中,王克夷.植物凝集素的超级家族[J].生物化学与生物物理进展.1994; 21 (2) :104～109.
36张维杰.复合多糖生化研究技术.上海:上海科技出版社, 1987 , 160.
37 Mechref Y, MV Novotny. Structural investigations of glycoconjugates at high sensitivity[J]. Chem Rev. 2002; 102(2): 321～337.
38 Dell A, HR Morris. Glycoprotein structure determination by mass spectrometry[J]. Science. 2001; 291(5512): 2351～2356.
39 Van Langenhove A, VN Reinhold. Determination of polysaccharide linkage and branching by reductive depolymerization. Gas-liquid chromatography and gas-liquid chromatography-mass spectrometry reference data[J]. Carbohydr Res. 1985; 143.
40 Barber M, Bordoli RS, Sedgwick RD, et al. Fast atom bombardment of solids as an ion source in mass spectrometry [J]. Nature. 1981; 293:270.
41 Aberth W, Straub KM, Burlingame AL. Secondary ion mass spectrometry with cesium ion ... trix for analysis of bioorganic compounds[J]. Anal Chem. 1982; 54: 20～29.
42 Olivares JA, Nhung T, Nguyen NT , et al .On-line mass spectrometric detection for capillary zone electrophoresis[J]. Anal Chem. 1987; 59:12～30.
43 Mechref YM, V Novotny. Mass spectrometric mapping and sequencing of N-linked oligosaccharides derived from submicrogram amounts of glycoproteins[J]. Anal Chem. 1998; 70(3): 455～463.
44 Colangelo JR, Orlando. On-target exoglycosidase digestions/MALDI-MS for determining the primary structures of carbohydrate chains[J]. Anal Chem. 1999; 71(7): 1479～1482.
45 Geyer H, Schmitt S, Wuhrer M, et al. Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS[J]. Anal Chem.1999; 71(2): 476～482.
46 Hirabayashi J, Hayama K, Kaji H, et a1. Afinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans[J]. J Biochem(Tokyo). 2002; 132(1):103~114.
47 Mawuenyega KG, Kaji H, Yamuchi Y, et a1. Large-scale identification of Caenorhabditis elegans proteins by multi-dimensional liquid chromatography-tandem mass spec-trometry[J]. J Proteome Res. 2003; 2(1): 23~35.
48 Yang Z, Hancock WS. Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin afinity column [J].J Chromatogr A. 2004; 1 053(1~2): 79~88.
49 Hirabayashi J. Lectin-based structural glycomics:glycoproteomics and glycan profiling[J]. Glycoconj J. 2004; 21(1~2): 35~40.
50 Dwek MV, Ross HA, Leathem AJ. Proteome and glycosylation mapping identifies post-translational modifications associated with aggressive breast cancer[J]. Proteomics. 200l; 1(6):756~762.
51 Schulenberg B, Beechem J M, Patton W F.Mapping glycosylation changes related to cancer using the Multiplexed Proteomics technology:a protein diferential display approach[J]. J Chromatogr B Analyt Technol Biomed Life Sci. 2003; 793(1): 127~139.
52 Sihlbom C, Davidsson P, Emmett MR, et a1. Glycoproteomics of Cerebrospinal Fluid in Neurodegenerative Disease[J]. Int J Mass Spectrom. 2004; 234(1~3): 145~152.
53 Wu J, Lenchik NJ, Pabst MJ, et a1. Functional characterization of two-dimensional gel-separated proteins using sequential staining[J]. Electrophoresis. 2005; 26(1): 225~237.
54 Ailor E, Takahashi N, Tsukamoto Y, et a1. N-glycan patterns of human transferrin produced in Trichoplusia ni insect cells: effects of mammalian galactosyltransferase[J]. Glycobiololgy. 2000; l0(8): 837~847.
55 Wada Y, Tajiri M, Yoshida S. Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics[J]. Anal Chem. 2004; 76(22): 6560~6565.
56 Wang D, Liu S, Trummer BJ, et a1. Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells[J]. Nat Biotechnol. 2002; 20(3): 272~281.
57 Wang D, Lu J. Glycan arrays lead to the discovery of autoimmunogenic activity of SARS-CoV [J].Physiol Genomics. 2004; 1 8(2): 245~248.
58 Disney MD, Seeberger PH. The use of carbohydrate microarrays to study carbohydrate-cell interactions and to detect pathogens[J]. Chem Biol. 2004; 11 (12): 1701~1707.
59 Adams EW, Ratner DM, Bokesch H R, et a1. Oligosaccharide and glycoprotein microarrays as tools in HIV glycobiology; glycan-dependent gp 120/protein interactions [J]. Chem Biol. 2004; 11(6): 875~881.
60 Khan I, Desai DV, Kumar A. Carbochips: a new energy for old biobuilders[J]. J Biosci Bioeng. 2004; 98(5): 331~337.
61 Hang HC, Yu C, Kato DL, et al. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation[J]. Proc Natl Acad Sci U S A. 2003. 100(25): 14846.
62 Vocadlo DJ, Hang HC, Kin EJ, et al. A chemical approach for identifying O-GlcNAc-modified proteins in cells[J]. Proc Natl Acad Sci U S A. 2003; 100(16): 9116.
63 Khidekel N, Arndt S, Lamarre-vincent N, et al. A chemoenzymaticapproach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications[J]. J Am Chem Soc. 2003; 125(52): 16162.
64 Khidekel N, Ficarro SB, Peters EC, et al. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain[J]. Proc Natl Acad Sci U S A. 2004; 101(36): 13132.
65 Yu W, Vath JE, Huberty MC, et al. Identification of the facile gas-phase cleavage of the Asp-Pro and Asp-Xxx peptide bonds in matrix-assisted laser desorption time-of-flight mass spectrometry[J]. Anal Chem. 1993; 65(21): 3015.
66 Wysocki VH, Tsaprailis G, Smith LL, et al. Mobile and localized protons: a framework for understanding peptide dissociation[J]. J Mass Spectrom. 2000; 35(12): 1399.
67 Biemann K. Mass spectrometric methods for protein sequencing. Anal Chem. 1986; 58(13): 1288A.
68 Biemann K, Martin SA. Mass spectrometric determination of the amino acid sequence of peptides and proteins[J]. Mass spectrum Rev. 1987; 6(1):1.
69 Hunt DF, Yates JR 3rd, Shabanowitz J. Protein sequencing by tandem mass spectrometry[J]. Proc Natl Acad Sci U S A. 1986; 83(17): 6233.
70 Keough T, Youngquist RS, Lacey MP. A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry[J]. Proc Natl Acad Sci U S A. 1999; 96(13): 7131.
71 Keough T, Youngquist RS, Lacey M P. Sulfonic acid derivatives for peptide sequencing by MALDI MS[J]. Anal Chem. 2003; 75(7): 156A.
72 Standing, KG.. Peptide and protein de novo sequencing by mass spectrometry[J]. Curr Opin Struct Biol. 2003; 13(5): 595.
73 Czeszak X, Morelle W, Ricart G.. Localization of the O-glycosylated sites in peptides by fixed-charge derivatization with a phosphonium group[J]. Anal Chem. 2004; 76(15): 4320.
74 Mirgorodskaya E, P Roepstorff, RA Zubarev. Localization of O-glycosylation sites in peptides by electron capture dissociation in a Fourier transform mass spectrometer[J]. Anal Chem. 1999; 71(20): 4431.
75 Zubarev RA, Kelleher NL, McLafferty FW. Electron capture dissociation of multiply chargedproteincations. A nonergodic process[J]. J Am Chem Soc. 1998; 120: 3265.
76 Zubarev RA, Electron-capture dissociation tandem mass spectrometry[J]. Curr Opin Biotechnol. 2004; 15(1): 12.
77 Mann M, ON Jensen. Proteomic analysis of post-translational modifications[J]. Nat Biotechnol. 2003; 21(3): 255.
78韩雅玲,刘海伟,闫承慧,等.人E1A激活基因阻遏子的表达、抗体制备及生物学活性分析[J].细胞与分子免疫学杂志. 2005; 21(5):570~574.
79韩雅玲,闫承慧,刘海伟,等. E1A激活基因阻遏子过表达诱导体外培养大鼠平滑肌细胞分化[J].生物化学和生物物理进展. 2004; 31(12):1099~1105.
80韩雅玲,邓捷,梁明,等. E1A激活基因阻遏子(CREG)抑制大鼠颈动脉球囊损伤后新生内膜形成[J].中国病理生理杂志(已接收,待发表).
81 Ryota Kunita, Asako Otomo, Joh-E Ikeda. Identification and characterization of novel members of the CREG family, putative secreted glycoproteins expressed specifically in brain[J]. Genomics. 2002;80(5):456~460.
82 Dilley RJ. A novel inhibitory role for CREG-mediated signalling in cardiac hypertrophy[J]?.J Hypertens. 2004; 22(8):1469~1471.
83 Xu Li, Liu Jun-Ming, Chen Lan-Ying. CREG, a new regulator of ERK1/2 in cardiac hypertrophy[J]. Journal of Hypertension. 2004; 22(8):1579~1587.
84 Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease[J] . Physiol Rev. 2004; 84:767~ 801.
85韩雅玲,刘海伟,康建,等. E1A激活基因阻遏子在不同表型血管平滑肌细胞中的表达[J].中华医学杂志. 2005; 85(1):49~53.
86韩雅玲,王效增,康建,等.大鼠颈动脉球囊损伤后血管平滑肌细胞E1A激活基因阻遏子的表达变化[J].中华心血管病杂志. 2004; 32(1): 53~58.
2 Di Bacco A, Gill G. The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-like growth factor II receptor[J]. Oncogene. 2003; 22(35): 5436~5445.
3 Veal E, Groisman R, Eisenstein M, et al. The secreted glycoprotein CREG enhances differentiation of NTERA-2 human embryonal carcinoma cells[J]. Oncogene. 2000; 19(11): 2120~2128.
4 Veal E, Eisenstein M, Tseng A, et al. A cellular repressor of E1A- stimulated genes that inhibits activation by E2F[J]. Mol Cell Biol. 1998; 18(7): 5032~5041.
5韩雅玲,胡叶,刘海伟,等. E1A激活基因阻遏子促进人血管平滑肌细胞分化和迁移[J].生物化学和生物物理进展. 2005; 32(6): 517~522.
6 Mats Hellstr?m, Mattias Kalén, Per Lindahl, et al. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse[J]. Development. 1999; 126 (14) : 3047~3055.
7韩雅玲,康建,张剑,等.对去血清后HITASY细胞分子表达及表型分析[J].生物化学和生物物理进展. 2003; 30(6): 868~873.
8 Michael Sacher, Alessandra Di Bacco, Vladimir V. Lunin et al.The crystal structure of CREG, a secreted glycoprotein involved in cellular growth and differentiation[J]. Proc Natl Acad Sci U S A. 2005; 102(51):18326~18331.
9 Alessandra Di Bacco, Grace Gill, The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-like growth factor II receptor[J]. Oncogene. 2003; 22(35): 5436~5445.
10 Kobata A. Structures and functions of the sugar chains of glycoproteins[J]. Eur J Biochem. 1992; 209(2): 483~501.
11 Lloyd RC, BG. Davis, J B Jones. Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity[J]. Bioorg Med Chem. 2000; 8(7): 1537~1544.
12 Kwon KS, MH Yu. Effect of glycosylation on the stability of alpha1-antitrypsin toward urea denaturation and thermal deactivation[J]. Biochim Biophys Acta. 1997; 1335(3): 265~272.
13 Suzuki S, M Furuhashi, N Suganuma. Additional N-glycosylation at Asn(13) rescues the human LHbeta-subunit from disulfide-linked aggregation[J]. Mol Cell Endocrinol. 2000; 160(1~2): 157~163.
14 Sasaki K, Nishi T, Yasumura S, et a1. Modified granulocyte-colony stimulating factor polypeptide with added carbohydrate chains.United States Patent:52l8092.
15 Coloma M J, Trinh R K, Martinez A R, et al. Position effects of variable region carbohydrate on the affinity and in vivo behavior of an anti-(1-6) dextran antibody[J]. J Immunol. 1999; 162(4): 2162~2170.
16 Davies J, Jiang L Y, Pan L z, et al. Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII[J]. Biotechnol Bioeng. 2001; 74(4): 288~294.
17 Tams JW, J Vind, KG Welinder. Adapting protein solubility by glycosylation. N-glycosylation mutants of Coprinus cinereus peroxidase in salt and organicsolutions[J]. Biochim Biophys Acta. 1999; 1432(2): 214~221.
18 Linden HM, K Kaushansky. The glycan domain of thrombopoietin enhances its secretion[J]. Biochemistry. 2000; 39(11): 3044~3051.
19 Egrie JC, JK Browne. Development and characterization of novel erythropoiesis stimulating protein (NESP) [J]. Br J Cancer. 2001; 84 (Suppl 1):3~10.
20 Mistry PK, EP Wraight, TM Cox. Therapeutic delivery of proteins to macrophages: implications for treatment of Gaucher's disease[J]. Lancet. 1996; 348(9041): 1555~1559.
21 Dell A, HR Morris. Glycoprotein structure determination by mass spectrometry[J]. Science. 2001; 291(5512): 2351~2356.
22 Rudd PM, et al. A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands[J]. Proteomics. 2001; 1(2): 285~294.
23 Rudd PM, et al. Glycosylation and the immune system[J]. Science. 2001; 291(5512): 2370~2376.
24 Spellman MW. Carbohydrate characterization of recombinant glycoproteins of pharmaceutical interest[J]. Anal Chem. 1990; 62(17): 1714~1722.
25 Fryksdale BG., et al. Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis[J]. Electrophoresis. 2002; 23(14): 2184~2193.
26 Hardy MR, RR Townsend. Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection[J]. Proc Natl Acad SciU S A. 1988; 85(10):3289~3293.
27 Karty JA, et al. Artifacts and unassigned masses encountered in peptide mass mapping[J]. J Chromatogr B Analyt Technol Biomed Life Sci. 2002; 782(1~2): 363~383
28 Settineri CA, et al. Characterization of O-glycosylation sites in recombinant B-chain of platelet-derived growth factor expressed in yeast using liquid secondary ion mass spectrometry, tandem mass spectrometry and Edman sequence analysis[J]. Biomed Environ Mass Spectrom. 1990; 19(11): 665~676.
29 Greis KD, et al. Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry[J]. Anal Biochem. 1996; 234(1): 38~49.
30 Hanisch FG., M Jovanovic, J Peter-Katalinic. Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments[J]. Anal Biochem. 2001; 290(1): 47~59.
31 Apweiler R, H Hermjakob, N Sharon. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database[J]. Biochim Biophys Acta. 1999; 1473(1): 4~8.
32 Devenyi T , Gergely J .实用蛋白质化学技术.上海:上海科技出版社, 1982 , 52.
33 David R. Glycoprotein Detection in Polyacryamide Gel with Thymol and Sulfuric Acid[J]. Anal Biochem. 1979; 99(2): 474～476
34孙秀琴,中村健,伊滕洋一,等.曼氏裂头蚴蛋白分解酶的糖链研究[J].中国寄生虫病防治杂志. 2001;14 (4) :285～287.
35孙建中,王克夷.植物凝集素的超级家族[J].生物化学与生物物理进展.1994; 21 (2) :104～109.
36张维杰.复合多糖生化研究技术.上海:上海科技出版社, 1987 , 160.
37 Mechref Y, MV Novotny. Structural investigations of glycoconjugates at high sensitivity[J]. Chem Rev. 2002; 102(2): 321～337.
38 Dell A, HR Morris. Glycoprotein structure determination by mass spectrometry[J]. Science. 2001; 291(5512): 2351～2356.
39 Van Langenhove A, VN Reinhold. Determination of polysaccharide linkage and branching by reductive depolymerization. Gas-liquid chromatography and gas-liquid chromatography-mass spectrometry reference data[J]. Carbohydr Res. 1985; 143.
40 Barber M, Bordoli RS, Sedgwick RD, et al. Fast atom bombardment of solids as an ion source in mass spectrometry [J]. Nature. 1981; 293:270.
41 Aberth W, Straub KM, Burlingame AL. Secondary ion mass spectrometry with cesium ion ... trix for analysis of bioorganic compounds[J]. Anal Chem. 1982; 54: 20～29.
42 Olivares JA, Nhung T, Nguyen NT , et al .On-line mass spectrometric detection for capillary zone electrophoresis[J]. Anal Chem. 1987; 59:12～30.
43 Mechref YM, V Novotny. Mass spectrometric mapping and sequencing of N-linked oligosaccharides derived from submicrogram amounts of glycoproteins[J]. Anal Chem. 1998; 70(3): 455～463.
44 Colangelo JR, Orlando. On-target exoglycosidase digestions/MALDI-MS for determining the primary structures of carbohydrate chains[J]. Anal Chem. 1999; 71(7): 1479～1482.
45 Geyer H, Schmitt S, Wuhrer M, et al. Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS[J]. Anal Chem.1999; 71(2): 476～482.
46 Hirabayashi J, Hayama K, Kaji H, et a1. Afinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans[J]. J Biochem(Tokyo). 2002; 132(1):103~114.
47 Mawuenyega KG, Kaji H, Yamuchi Y, et a1. Large-scale identification of Caenorhabditis elegans proteins by multi-dimensional liquid chromatography-tandem mass spec-trometry[J]. J Proteome Res. 2003; 2(1): 23~35.
48 Yang Z, Hancock WS. Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin afinity column [J].J Chromatogr A. 2004; 1 053(1~2): 79~88.
49 Hirabayashi J. Lectin-based structural glycomics:glycoproteomics and glycan profiling[J]. Glycoconj J. 2004; 21(1~2): 35~40.
50 Dwek MV, Ross HA, Leathem AJ. Proteome and glycosylation mapping identifies post-translational modifications associated with aggressive breast cancer[J]. Proteomics. 200l; 1(6):756~762.
51 Schulenberg B, Beechem J M, Patton W F.Mapping glycosylation changes related to cancer using the Multiplexed Proteomics technology:a protein diferential display approach[J]. J Chromatogr B Analyt Technol Biomed Life Sci. 2003; 793(1): 127~139.
52 Sihlbom C, Davidsson P, Emmett MR, et a1. Glycoproteomics of Cerebrospinal Fluid in Neurodegenerative Disease[J]. Int J Mass Spectrom. 2004; 234(1~3): 145~152.
53 Wu J, Lenchik NJ, Pabst MJ, et a1. Functional characterization of two-dimensional gel-separated proteins using sequential staining[J]. Electrophoresis. 2005; 26(1): 225~237.
54 Ailor E, Takahashi N, Tsukamoto Y, et a1. N-glycan patterns of human transferrin produced in Trichoplusia ni insect cells: effects of mammalian galactosyltransferase[J]. Glycobiololgy. 2000; l0(8): 837~847.
55 Wada Y, Tajiri M, Yoshida S. Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics[J]. Anal Chem. 2004; 76(22): 6560~6565.
56 Wang D, Liu S, Trummer BJ, et a1. Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells[J]. Nat Biotechnol. 2002; 20(3): 272~281.
57 Wang D, Lu J. Glycan arrays lead to the discovery of autoimmunogenic activity of SARS-CoV [J].Physiol Genomics. 2004; 1 8(2): 245~248.
58 Disney MD, Seeberger PH. The use of carbohydrate microarrays to study carbohydrate-cell interactions and to detect pathogens[J]. Chem Biol. 2004; 11 (12): 1701~1707.
59 Adams EW, Ratner DM, Bokesch H R, et a1. Oligosaccharide and glycoprotein microarrays as tools in HIV glycobiology; glycan-dependent gp 120/protein interactions [J]. Chem Biol. 2004; 11(6): 875~881.
60 Khan I, Desai DV, Kumar A. Carbochips: a new energy for old biobuilders[J]. J Biosci Bioeng. 2004; 98(5): 331~337.
61 Hang HC, Yu C, Kato DL, et al. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation[J]. Proc Natl Acad Sci U S A. 2003. 100(25): 14846.
62 Vocadlo DJ, Hang HC, Kin EJ, et al. A chemical approach for identifying O-GlcNAc-modified proteins in cells[J]. Proc Natl Acad Sci U S A. 2003; 100(16): 9116.
63 Khidekel N, Arndt S, Lamarre-vincent N, et al. A chemoenzymaticapproach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications[J]. J Am Chem Soc. 2003; 125(52): 16162.
64 Khidekel N, Ficarro SB, Peters EC, et al. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain[J]. Proc Natl Acad Sci U S A. 2004; 101(36): 13132.
65 Yu W, Vath JE, Huberty MC, et al. Identification of the facile gas-phase cleavage of the Asp-Pro and Asp-Xxx peptide bonds in matrix-assisted laser desorption time-of-flight mass spectrometry[J]. Anal Chem. 1993; 65(21): 3015.
66 Wysocki VH, Tsaprailis G, Smith LL, et al. Mobile and localized protons: a framework for understanding peptide dissociation[J]. J Mass Spectrom. 2000; 35(12): 1399.
67 Biemann K. Mass spectrometric methods for protein sequencing. Anal Chem. 1986; 58(13): 1288A.
68 Biemann K, Martin SA. Mass spectrometric determination of the amino acid sequence of peptides and proteins[J]. Mass spectrum Rev. 1987; 6(1):1.
69 Hunt DF, Yates JR 3rd, Shabanowitz J. Protein sequencing by tandem mass spectrometry[J]. Proc Natl Acad Sci U S A. 1986; 83(17): 6233.
70 Keough T, Youngquist RS, Lacey MP. A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry[J]. Proc Natl Acad Sci U S A. 1999; 96(13): 7131.
71 Keough T, Youngquist RS, Lacey M P. Sulfonic acid derivatives for peptide sequencing by MALDI MS[J]. Anal Chem. 2003; 75(7): 156A.
72 Standing, KG.. Peptide and protein de novo sequencing by mass spectrometry[J]. Curr Opin Struct Biol. 2003; 13(5): 595.
73 Czeszak X, Morelle W, Ricart G.. Localization of the O-glycosylated sites in peptides by fixed-charge derivatization with a phosphonium group[J]. Anal Chem. 2004; 76(15): 4320.
74 Mirgorodskaya E, P Roepstorff, RA Zubarev. Localization of O-glycosylation sites in peptides by electron capture dissociation in a Fourier transform mass spectrometer[J]. Anal Chem. 1999; 71(20): 4431.
75 Zubarev RA, Kelleher NL, McLafferty FW. Electron capture dissociation of multiply chargedproteincations. A nonergodic process[J]. J Am Chem Soc. 1998; 120: 3265.
76 Zubarev RA, Electron-capture dissociation tandem mass spectrometry[J]. Curr Opin Biotechnol. 2004; 15(1): 12.
77 Mann M, ON Jensen. Proteomic analysis of post-translational modifications[J]. Nat Biotechnol. 2003; 21(3): 255.
78韩雅玲,刘海伟,闫承慧,等.人E1A激活基因阻遏子的表达、抗体制备及生物学活性分析[J].细胞与分子免疫学杂志. 2005; 21(5):570~574.
79韩雅玲,闫承慧,刘海伟,等. E1A激活基因阻遏子过表达诱导体外培养大鼠平滑肌细胞分化[J].生物化学和生物物理进展. 2004; 31(12):1099~1105.
80韩雅玲,邓捷,梁明,等. E1A激活基因阻遏子(CREG)抑制大鼠颈动脉球囊损伤后新生内膜形成[J].中国病理生理杂志(已接收,待发表).
81 Ryota Kunita, Asako Otomo, Joh-E Ikeda. Identification and characterization of novel members of the CREG family, putative secreted glycoproteins expressed specifically in brain[J]. Genomics. 2002;80(5):456~460.
82 Dilley RJ. A novel inhibitory role for CREG-mediated signalling in cardiac hypertrophy[J]?.J Hypertens. 2004; 22(8):1469~1471.
83 Xu Li, Liu Jun-Ming, Chen Lan-Ying. CREG, a new regulator of ERK1/2 in cardiac hypertrophy[J]. Journal of Hypertension. 2004; 22(8):1579~1587.
84 Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease[J] . Physiol Rev. 2004; 84:767~ 801.
85韩雅玲,刘海伟,康建,等. E1A激活基因阻遏子在不同表型血管平滑肌细胞中的表达[J].中华医学杂志. 2005; 85(1):49~53.
86韩雅玲,王效增,康建,等.大鼠颈动脉球囊损伤后血管平滑肌细胞E1A激活基因阻遏子的表达变化[J].中华心血管病杂志. 2004; 32(1): 53~58.