结缔组织生长因子特异性结合肽的表达及分离纯化-东南大学学报医学版

结缔组织生长因子特异性结合肽的表达及分离纯化

单位：1.东南大学,临床医学院,江苏,南京,210009； 2.东南大学附属中大医院,临床检验中心,江苏,南京,210009

分类号：Q78

出版年·卷·期（页码）：2008·27·第四期（259-263）

摘要：

目的:利用基因工程技术获得与结缔组织生长因子(CTGF)特异性结合的小肽.方法:设计并合成带有肠激酶切割位点的基因片段,插入pET-32(a)+质粒载体中硫氧化还原蛋白基因下游,构建重组质粒,转入E.coli BL21表达菌中,用终浓度1mmol·L-1的IPTG诱导表达融合蛋白;用热变性和硫酸铵分级沉淀对融合蛋白进行初步纯化,然后用亲和层析进一步纯化,经肠激酶切割,最后用凝胶过滤层析分离获得目的小肽;用反相色谱对所获小肽进行纯度鉴定.结果:所构建的重组质粒测序结果与预期一致;诱导表达后用SDS-PAGE鉴定,发现在相对分子质量20 000处有浓密的表达条带出现,与预期一致;融合蛋白用热变性和硫酸铵分级沉淀进行初步纯化,得率分别为78.6%和52.3%;用亲和层析纯化得率为50.5%;经肠激酶切割后用凝胶过滤层析分离获得目的小肽,用反相色谱鉴定其纯度大于95%;最终每升发酵液获得3.4mg目的肽.结论:成功制备CTGF特异性结合肽,为进一步研究该小肽的生物学活性打下基础.

Objective To obtain the peptide specifically binding to connective tissue growth factor by genetic engineering technique.Methods A gene fragment with enterokinase cleavage site was designed and synthesized by PCR method,and inserted into downstream of fusion partner thioredoxin gene of pET-32(a)+ plasmid vector.The recombinant plasmid was constructed and transfered into E.coli BL21(DE3).The fusion protein was induced by IPTG up to the final concentration of 1 mmol·L-1,and was then purified using methods of heat denaturation and fractional precipitation with ammonium sulfate.For further purification,an affinity chromatograph was applied.The interest peptide was finally obtained by gel filtration chromatography(GFC) after fusion protein was digested with enterokinase.The purity of the peptide was identified by reversal-phase chromatography.Results The sequencing result of the recombinant plasmid was consistent with the expectance.There was an expressive band of 20 000 which was consistent with the expected molecular weight by SDS-PAGE analysis.The yields of fusion protein purified by heat denaturation,fractional precipitation with ammonium sulfate and affinity chromatograph were 78.6%,52.3% and 50.5% respectively.The peptide was finally obtained by GFC after cleavage by enterokinase.The purity identified by reversal-phase chromatography was more then 95%.The peptide of 3.4 milligram per liter broth was detected.Conclusion The peptide specifically binding to connective tissue growth factor was obtained successfully.And it is valuable for the further study of the biologic activity of the peptide.

参考文献：

[1] BRADHAM D M, IGARASHI A, POTTER R L. Connective tissue growth factor:a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10, 1991(6)
[2] TAKEHERA K. Growth regulation of skin fibroblasts. 2000. doi:10.1016/S0923-1811(0)00144-4
[3] WAHAB N A, YEVDOKIMOVA N, WESTON B S. Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy. 2001. doi:10.1042/0264-6021:3590077
[4] HAYASHI N, KAKIMUMA T, SOMA Y. Connective tissue growth factor is directly related to liver fibrosis. 2002(43)
[5] KUCICH U, ROSENBLOOM J C, HERRICK D J. Signaling events required for transforming growth factor-beta stimulation of connective tissue growth factor expression by cultured human lung fibroblasts. 2001(1). doi:10.1006/abbi.2001.2571
[6] FAN W H, KARNOVSKY M J. Increased MMP-2 expression in connective tissue growth factor over expression vascular smooth muscle cells. 2002(12). doi:10.1074/jbc.M111213200
[7] JEDSADAYANMATA A, CHEN C C, KIREEVA M L. Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin alpha (IIb) beta (3). 1999. doi:10.1074/jbc.274.34.24321
[8] CHEN C C, CHEN N, LAU L F. The angiogenic factors Cyr61 and connective tissue growth factor induce adhesive signaling in primary human skin fibroblasts. 2001. doi:10.1074/jbc.M008087200
[9] HISHIKAWA K, OEMAR B S, NAKAKI T. Static pressure regulates connective tissue growth factor expression in human mesangial cells. 2001. doi:10.1074/jbc.M010722200
[10] HISHIKAWA K, NAKAKI T, FUJII T. Connective tissue growth factor induces apoptosis via caspase 3 in cultured human aortic smooth muscle cells. 2000(1/2). doi:10.1016/S0014-2999(0)00115-1
[11] HISHIKAWA K, OEMAR B S, TANNER F C. Connective tissue growth factor induces apoptosis in human breast cancer cell line MCF-7. 1999. doi:10.1074/jbc.274.52.37461
[12] SHIMO T, NAKANISHI T, NISHIDA T. Connective tissue growth factor induces the proliferation,migration,and tube formation of vascular endothelial cells in vitro,and angiogenesis in vivo, 1999
[13] 吴国球, 王明虹, 张臣. 十二肽库中与结缔组生长因子特异结合噬菌体的筛选. 中国药科大学学报2007(1)
[14] CHEN R, HUANG C, MORINELLI T A. Blockade of the effects of TGF-beta1 on mesangial cells by overexpression of Smad7. 2002(4)
[15] YOSIMICHI G, NAKANISHI T, NISHIDA T. CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK),and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK). 2001. doi:10.1046/j.0014-2956.2001.02553.x
[16] SCHAEFFER H J, WEBER M J. Mitogen-activated protein kinases:specific messages from ubiquitous messengers. 1999
[17] LALI F V, HUNT A E, TURNER S J. The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity,protein kinase B phosphorylation,and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. 2000. doi:10.1074/jbc.275.10.7395
[18] LI J H, ZHU H J, HUANG X R. Smad7 inhibits fibrotic effect of TGF-beta on renal tubular epithelial cells by blocking Smad2 activation. 2002(6). doi:10.1097/01.ASN.0000014252.37680.E4
[19] LAN H Y, MU W, TOMITA N. Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. 2003(6). doi:10.1097/01.ASN.0000067632.04658.B8
[20] RUPEREZ M, SANCHEZ-LOPEZ E, BLANCO-COLIO L M. The Rho-kinase pathway regulates angiotensin II-induced renal damage. 2005. doi:10.1111/j.1523-1755.2005.09908.x
[21] MATSUI Y, SADOSHIMA J. Rapid upregulation of CTGF in cardiac myocytes by hypertrophic stimuli:implication for cardiac fibrosis and hypertrophy. 2004. doi:10.1016/j.yjmcc.2004.05.012

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录