>
网站首页期刊介绍通知公告编 委 会投稿须知电子期刊广告合作联系我们
最新消息:
RKIP的克隆、表达及其影响肿瘤细胞迁移性质的初步研究
作者:皮亚洲  华子春 
单位:南京大学 医药生物技术国家重点实验室, 江苏 南京 210093
关键词:Raf激酶抑制蛋白 表达 纯化 细胞迁移 
分类号:Q786
出版年·卷·期(页码):2014·33·第六期(752-755)
摘要:

目的: 研究Raf激酶抑制蛋白(RKIP)在细胞外对肿瘤细胞迁移的影响.方法: 通过PCR扩增得到RKIP基因片段,使用BamHⅠ和HindⅢ双酶切并回收,然后插入双酶切过的原核表达载体pET28a(+),得到pET28a-RKIP重组质粒.将重组质粒转化表达菌株BL21(DE3),经IPTG诱导表达,表达产物经Ni+金属螯合层析纯化得到His-RKIP重组蛋白.通过伤口愈合实验,观察不同浓度的重组RKIP在细胞外对肿瘤细胞迁移的影响.结果: 重组RKIP获得了高效表达和分离纯化,伤口愈合实验表明,在低浓度重组RKIP作用下A549肿瘤细胞迁移速度增加,在高浓度下肿瘤细胞迁移速度降低.结论: 重组RKIP在细胞外具有促进和抑制A549肿瘤细胞迁移的双重作用,具体表现为低浓度时促进A549肿瘤细胞迁移,高浓度时抑制A549肿瘤细胞迁移.

Objective: To investigate the Raf kinase inhibitor protein(RKIP)'s extracellular effect on tumor metastasis. Methods: The RKIP encoding gene fragment was amplified by PCR,digested with BamHⅠ and HindⅢ,and inserted into the same digested expression vector pET28a(+),thus becoming recombinant plasmid pET28a-RKIP.The expression plasmid pET28a-RKIP was transformed into E.coli BL21(DE3),then induced by IPTG.The recombinant protein His-RKIP was purified through Ni+ affinity chromatography. Through wound healing experiment, we could understand the extracellular effect of the concentration of RKIP protein on tumor metastasis. Results: Recombinant RKIP was expressed and purified.The wound healing experiment demonstrated that the rate of tumor cell migration at first grew as the concentration grew and then dropped as the concentration continued to grow. Conclusion: The RKIP protein could either promote or inhibit A549 cell migration depending on the concentration, low concentration of RKIP can promote the migration of A549 cells while high concentration inhibits it.

参考文献:

[1] BERNIER I,JOLLES P.Purification and characterization of a basic 23 kDa cytosolic protein from bovine brain[J].Biochim Biophys Acta,1984,790(2): 174-181.
[2] BERNIER I,TRESCA J P,JOLLES P.Ligand-binding studies with a 23 kDa protein purified from bovine brain cytosol[J].Biochim Biophys Acta,1986,871(1): 19-23.
[3] 林小伟,华子春.TDG变体的构建、表达、纯化及其DNA修复功能研究[J].东南大学学报: 医学版,2012,31(4): 389-392.
[4] YEUNG K,SEITZ T,LI S,et al.Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP[J].Nature,1999,401(6749): 173-177.
[5] TRAKUL N,ROSNER M R.Modulation of the MAP kinase signaling cascade by Raf kinase inhibitory protein[J].Cell Res,2005,15(1): 19-23.
[6] GEMS D,MAIZELS R M.An abundant trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins[J].J Biol Chem,1995,270(31): 18517-18522.
[7] PIKIELNY C W,HASAN G,ROUYER F,et al. Members of a family of Drosophila putative odorant-binding proteins are expressed in different subsets of olfactory hairs[J].Neuron,1994,12(1): 35-49.
[8] LORENZ K,LOHSE M J,QUITTERER U.Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2[J].Nature,2003,426(6966): 574-579.
[9] YEUNG K C,ROSE D W,DHILLON A S,et al.Raf kinase inhibitor protein interacts with NF-kB-inducing kinase and TAK1and inhibits NF-kB activation[J].Mol Cell Biol,2001(21): 7207-7217.
[10] FU Z,SMITH P C,ZHANG L,et al.Effects of raf kinase inhibitor protein expression on suppression of prostate cancer metastasis[J].J Natl Cancer Inst,2003,95(12): 878-889.
[11] KELLER E T,FU Z,BRENNAN M.The biology of a prostate cancer metastasis suppressor protein: Raf kinase inhibitor protein[J].J Cell Bichem,2005,94(2): 273-278.
[12] HAGAN S,AL-MULLA F,MALLON E,et al.Reduction of Raf-1 kinase inhibitor protein expression correlates with breast cancer metastasis[J].Clin Cancer Res,2005,11(20): 7392-7397.
[13] SCHUIERER M M,BATAILLE F,HAGAN S,et al.Reduction in Raf kinase inhibitor protein expression is associated with increased Ras-extracellular signal regulated kinase signaling in melanoma cell lines[J].Cancer Res,2004,64(15): 5186-5192.
[14] CORBIT K C,TRAKUL N,EVES E M,et al.Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein[J].J Biol Chem,2003,278(15): 13061-13068.
[15] KROSLAK T,KOCH T,KAHL E,et al.Human phosphatidylethanolamine binding protein facilitates heterotrimeric G protein dependent signaling[J].J Biol Chem,2001,276(43): 39772-39778.
[16] ZHANG L,FU Z,SIMEONE D M,et al.Raf kinase inhibitory protein inhibits b-cell proliferation[J].Surgery,2004,136(3): 708-715.
[17] SCHUIERER M M,HEILMEIER U,LANGMANN T,et al.Induction of Raf kinase inhibitor protein contributes to macrophage differentiation[J].Biochem Biophys Res Commun,2006,342(4): 1083-1087.
[18] CHATTERJEE D,BAI Y,YEUNG K C,et al.RKIP sensitizes prostate and breast cancer cells to drug induced apoptosis[J].J Biol Chem,2004,279(17): 17515-17523.

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
您是第 233105 位访问者


copyright ©《东南大学学报(医学版)》编辑部
联系电话:025-87232481 83272483
电子邮件:
bjb@pub.seu.edu.cn

苏ICP备09058364