靶向人FoxMlc的多肽先导药物筛选与分子模拟
|
摘要
恶性肿瘤是严重危害人类生命健康的重大疾病。在与肿瘤相关的各类转录因子中,Fox家族蛋白成员众多,其突变和表达异常与发育畸形、代谢性疾病以及肿瘤发生密切相关。FoxM1作为Forkhead家族的一个特殊成员在肿瘤等活跃的分裂细胞中大量表达,是控制包括增殖、成熟、死亡在内的整个细胞生命周期的重要基因之一,与肿瘤生长和转移密切相关。目前,国内外对FoxM1的研究尚处于起步阶段。本研究对于深入了解FoxM1c的生物信息基础和进一步的靶向FoxM1c药物研究具有重要的意义和价值。通过NCBI FoxMlc基因的蛋白质序列查询,选出8条来自不同物种具有代表性的序列,确定了不同物种中表达FoxM1c基因之间的同源关系,并发现FoxM1c基因或功能区域在其它物种中的表达。同时,依据ClustalX等生物信息学软件对人FoxMlc基因蛋白序列的理化性质、结构以及功能进行预测。
研究结果表明,人FoxM1c蛋白序列共有748个氨基酸组成,人FoxM1c蛋白序列的两大功能域分别为“叉头框”(Forkhead,236-314)和转录激活区(AD,688-748),“叉头框”是一个非常好的保守区域。p19ARF蛋白的26-46的氨基酸残基能与AD相结合,从而约束人FoxM1c和减少人FoxM1c对细胞核靶向作用的转录活性。
进一步地,通过对人FoxM1c全长片段、DNA结合区和转录激活区的PCR扩增和表达载体pQE30的重组,成功构建了各相应的重组表达系统。经SDS-PAGE凝胶电泳,确认DNA结合区成功表达。在此基础上,对DNA结合区表达系统pQD进行大量表达并通过Ni-NTA亲和层析进行蛋白纯化与定量,获得了mg级的DNA结合区蛋白。
以FoxM1c的DNA结合区蛋白为筛选靶标,利用噬菌体随机十二肽库筛选技术,通过“吸附—洗脱—扩增”的循环筛选获得富集噬菌体,经4轮筛选,将最后一轮筛选富集的噬菌体铺板,挑取单克隆噬菌体制备ssDNA并测序获得18条不同序列的十二肽。经分子对接软件MVD进行对接,靶向FoxM1的DNA结合区蛋白的富集噬菌体至少含有以下一种多肽结构序列：第一组：WHL/Q/D/N;第二组：DLY, DLLY, DHYN,DLNY；第三组：SSLWN/E;第四组：HLDY, HLYE, YHLE, LYHDD, YHLEE;第五组FYNL, NYFL;第六组：YPH, YPL, YPS。所述结合肽多肽结构序列分子模拟对接在FoxM1上的结合结构域预测主要是Arg-Arg(R-R, aa:254,256)和Glu-Thr-Ser-Ala-Asn-Gly-Lys(E-T-S-A-N-G-K, aa:298-304)。为进一步的靶向FoxM1c小分子先导肽的发现建立了基础。
Cancer is a major disease that is seriously harmful to human life and health. Among various types of tumor associated transcription factors, Fox Family is the most important with large members. Developmental malformation, metabolic disease and tumorigenesis are related to its mutation and abnormality expression. FoxMl is one of the gene family which controls the entire cell life cycle, tumor growth and metastasis as a special member of Forkhead family. It was closely related to the tumor. At present, Foxmlc transcription factor was less reported especially in bioinformatics analysis. The work is of great value and consequences for the further understanding of the bioinformatics bases of FoxM1c and the selection and finding of small molecular leading peptides targeting FoxMlc.
This work mainly includes three parts:1) the bioinformatics analysis of FoxM1c; 2) the cloning and exprsssion of human FoxM1c protein; 3) the selection and molecular simulation of lead peptide targeting human FoxM1c protein.
Homology among FoxM1c genes were found by searching eight representative sequences from different species by protein sequence query in NCBI, as well the finding of FoxMlc or its domain in other species, meanwhile, physico-chemical property, primary, secondary and tertiary structure and protein function of human FoxMlc were presented.
The results showed that "fork head box" of FoxM1 protein was a conservative region. Homology between Fox family members such as C and S, J and K, H and Q, P and R, M and O, were respectively higher. Human FoxMlc was composed of a total of 748 amino acids. Human FoxM1c protein sequence had two domain:One is Fork head box (Forkhead, 236-314) and transcriptional activation domain (AD,688-748), to which p19ARF protein in the amino acid 26-46 binding and hence restraining human FoxM1c and reducing the transcription activity of human FoxM1c nuclear targeting. As we failed in obtaining the reliable tertiary structure of the AD, the precise mechanism of action requires further more study.
Also, the human full-length FoxMlc cDNA was identified and cloned in the lab. The work offered great basis for the cloning and expression of human full-length FoxM1c cDNA or its functional domains which would be the important steps in the selection and finding of leading peptides targeting FoxM1c.
Here, at first the full length, the DNA binding domain and the transcriptional activation domain of human FoxM1 cDNA were amplified by PCR, then they were genetically cloned into the prokaryotic expression vector pQE30 and confirmed by restriction analysis and sequencing, by IPTG induction and polyacrylamide gel electrophoresis the FoxMl proteins were successfully expressed and confirmed and later purified by Ni-NTA agarose gel packing and quantified up to 0.8mg protein/ml solution by BCA assay.
By four rounds of selection of phage random dodecapeptide library against this expressed FoxMlc protein, the phages were enriched and eighteen different sequences were obtained by sequencing of different phages clones, importantly several clones showed higher affinities for FoxM1c protein with the highest near to P/N of 200. In addition, important consensus sequence were also found. It was the first report of the finding of lead peptides targeting FoxMlc by phages display technology.
It was found that the binding peptides for FoxMl protein may contain at least one of the following sequence characteristics:the first group:WHL/Q/D/N; the second group:DLY, DLLY, DHYN, DLNY; the third group:SSLWN/E; the fourth group:HLDY, HLYE, YHLE, LYHDD, YHLEE; the fifth group:FYNL, NYFL; the sixth group:YPH, YPL, YPS. By molecular simulation of docking software MVD, it was predicted that the above described binding peptides may target FoxM1-binding domain mainly on the sites of Arg-Arg (RR, aa: 254,256), and Glu-Thr-Ser-Ala-Asn-Gly-Lys (E-T-S-A-N-G-K, aa:298-304). The further analysis and study of the peptides are still in progress.
研究结果表明,人FoxM1c蛋白序列共有748个氨基酸组成,人FoxM1c蛋白序列的两大功能域分别为“叉头框”(Forkhead,236-314)和转录激活区(AD,688-748),“叉头框”是一个非常好的保守区域。p19ARF蛋白的26-46的氨基酸残基能与AD相结合,从而约束人FoxM1c和减少人FoxM1c对细胞核靶向作用的转录活性。
进一步地,通过对人FoxM1c全长片段、DNA结合区和转录激活区的PCR扩增和表达载体pQE30的重组,成功构建了各相应的重组表达系统。经SDS-PAGE凝胶电泳,确认DNA结合区成功表达。在此基础上,对DNA结合区表达系统pQD进行大量表达并通过Ni-NTA亲和层析进行蛋白纯化与定量,获得了mg级的DNA结合区蛋白。
以FoxM1c的DNA结合区蛋白为筛选靶标,利用噬菌体随机十二肽库筛选技术,通过“吸附—洗脱—扩增”的循环筛选获得富集噬菌体,经4轮筛选,将最后一轮筛选富集的噬菌体铺板,挑取单克隆噬菌体制备ssDNA并测序获得18条不同序列的十二肽。经分子对接软件MVD进行对接,靶向FoxM1的DNA结合区蛋白的富集噬菌体至少含有以下一种多肽结构序列：第一组：WHL/Q/D/N;第二组：DLY, DLLY, DHYN,DLNY；第三组：SSLWN/E;第四组：HLDY, HLYE, YHLE, LYHDD, YHLEE;第五组FYNL, NYFL;第六组：YPH, YPL, YPS。所述结合肽多肽结构序列分子模拟对接在FoxM1上的结合结构域预测主要是Arg-Arg(R-R, aa:254,256)和Glu-Thr-Ser-Ala-Asn-Gly-Lys(E-T-S-A-N-G-K, aa:298-304)。为进一步的靶向FoxM1c小分子先导肽的发现建立了基础。
Cancer is a major disease that is seriously harmful to human life and health. Among various types of tumor associated transcription factors, Fox Family is the most important with large members. Developmental malformation, metabolic disease and tumorigenesis are related to its mutation and abnormality expression. FoxMl is one of the gene family which controls the entire cell life cycle, tumor growth and metastasis as a special member of Forkhead family. It was closely related to the tumor. At present, Foxmlc transcription factor was less reported especially in bioinformatics analysis. The work is of great value and consequences for the further understanding of the bioinformatics bases of FoxM1c and the selection and finding of small molecular leading peptides targeting FoxMlc.
This work mainly includes three parts:1) the bioinformatics analysis of FoxM1c; 2) the cloning and exprsssion of human FoxM1c protein; 3) the selection and molecular simulation of lead peptide targeting human FoxM1c protein.
Homology among FoxM1c genes were found by searching eight representative sequences from different species by protein sequence query in NCBI, as well the finding of FoxMlc or its domain in other species, meanwhile, physico-chemical property, primary, secondary and tertiary structure and protein function of human FoxMlc were presented.
The results showed that "fork head box" of FoxM1 protein was a conservative region. Homology between Fox family members such as C and S, J and K, H and Q, P and R, M and O, were respectively higher. Human FoxMlc was composed of a total of 748 amino acids. Human FoxM1c protein sequence had two domain:One is Fork head box (Forkhead, 236-314) and transcriptional activation domain (AD,688-748), to which p19ARF protein in the amino acid 26-46 binding and hence restraining human FoxM1c and reducing the transcription activity of human FoxM1c nuclear targeting. As we failed in obtaining the reliable tertiary structure of the AD, the precise mechanism of action requires further more study.
Also, the human full-length FoxMlc cDNA was identified and cloned in the lab. The work offered great basis for the cloning and expression of human full-length FoxM1c cDNA or its functional domains which would be the important steps in the selection and finding of leading peptides targeting FoxM1c.
Here, at first the full length, the DNA binding domain and the transcriptional activation domain of human FoxM1 cDNA were amplified by PCR, then they were genetically cloned into the prokaryotic expression vector pQE30 and confirmed by restriction analysis and sequencing, by IPTG induction and polyacrylamide gel electrophoresis the FoxMl proteins were successfully expressed and confirmed and later purified by Ni-NTA agarose gel packing and quantified up to 0.8mg protein/ml solution by BCA assay.
By four rounds of selection of phage random dodecapeptide library against this expressed FoxMlc protein, the phages were enriched and eighteen different sequences were obtained by sequencing of different phages clones, importantly several clones showed higher affinities for FoxM1c protein with the highest near to P/N of 200. In addition, important consensus sequence were also found. It was the first report of the finding of lead peptides targeting FoxMlc by phages display technology.
It was found that the binding peptides for FoxMl protein may contain at least one of the following sequence characteristics:the first group:WHL/Q/D/N; the second group:DLY, DLLY, DHYN, DLNY; the third group:SSLWN/E; the fourth group:HLDY, HLYE, YHLE, LYHDD, YHLEE; the fifth group:FYNL, NYFL; the sixth group:YPH, YPL, YPS. By molecular simulation of docking software MVD, it was predicted that the above described binding peptides may target FoxM1-binding domain mainly on the sites of Arg-Arg (RR, aa: 254,256), and Glu-Thr-Ser-Ala-Asn-Gly-Lys (E-T-S-A-N-G-K, aa:298-304). The further analysis and study of the peptides are still in progress.
引文
[1]徐宁志,程书钧.恶性肿瘤癌变机理及早期诊治与预防的基础研究[J].生命科学,2005,17(6)：477-478.
[2]唐保东,刘思纯,徐雅,曾志荣,胡品津.人类Foxm1b基因的原核表达、抗体制备及其对肝癌细胞中Foxm1b蛋白的识别[J].中山大学学报(医学科学版),2007,28(6)：658-661.
[3]K.H. Kaestner, W. Knochel, D.E. Martinez, Unified nomenclature for the winged helix/forkhead transcription factors [J]. Genes Dev,2000,14:142-146.
[4]Kaestner K H, Knochel W, Martinez D E. Unified nomenclature for the winged helix/ forkhead transcription factors [J]. Genes Dev,2000,14(2):142-146.
[5]孙言伟,邹立君,金梅.生物信息学的研究进展[J].中华医学图书情报杂志,2002,4(11)：1-3.
[6]Silveira, N J, Freitas U, Hugo B, et al. Structural bioinformatics study of PNP from Schistosoma mansoni [J]. Biochemical and Biophysical Research Communications, 2004,(1):100-104.
[7]胡德华,方平.生物信息学研究进展[J].情报科学,2000,18(9)：784-786.
[8]Eric J. Current trends in bioinformatics [J]. Trends in Biotechnology,2002,20(8): 317-319.
[9]Zheng CJ, Han LY, Yap CW, et al. Trends in exploration of therapeutic targets [J]. Drug News Perspect,2005,18(2):109-127.
[10]Austen M, Dohrmann C. Phenotype-first screening for the identification of novel drug targets[J]. Drug Discov Today,2005,10(4):275-282.
[11]章波,向渝梅,白云,许雪青,王燕.小鼠peroxiredoxin基因家族的生物信息学分析[J].第三军医大学学报,2005,27(9)：847-849.
[12]马忠石,杨克,孟丽蕊等.青少年牙周炎相关基因SEC14L5的生物信息学分析[J].生物技术通报,2007,3：105-110.
[13]周林福,朱海红,陈离伟等.肺炎衣原体膜表面蛋白重组质粒的构建[J].科技通报,2004,20(2)：172-177.
[14]Bailon P, Ehr lich K G, Fung W J, et al. Affinity chromatography:methods and protocols [M]. New Jersey:Humana Press,2000.
[15]王莉,刘道杰,李连之.亲和层析在蛋白质研究中的应用进展[J].理化检验-化学分册, 2007,43(6)：515-517.
[16]贾翠娟,董兆麟.一种自制T-载体的构建[J].微生物学通报,2001,28(5)：56-60.
[17]吕勇刚,窦科峰,赵爱志等.T载体在噬菌体单链抗体库中的应用[J].中国新医药,2004,3(6)：4-5.
[18]李杨,韩梅.亲和层析技术在生物学中的应用及发展[J].生命科学研究,2006,10(1)：12-16.
[19]Robb V A, Li W, Gutmann D H. Disruption of binding does not impair Protein 4.1 B growth suppression [J]. Ontogeny,2004,23(20):3589-3596.
[20]Scott JK, Smith GP. Searching for peptide ligands with an epitode library [J]. Science, 1990,249:386-390.
[21]吴懿娜,杜欣军,张伟伟等.噬菌体抗体库技术及其应用[J].细胞与分子免疫学杂志,2010,26(4)：404-406.
[22]田波,李传昭,孙仑泉等[M].分子进化过程第1版,科学出版社,1999：7-8.
[23]D. R. Littler, M. Alvarez-Fernandez, A. Stein, et al. Structure of the FoxM1 DNA-recognition domain bound to a promoter sequence [J]. Nucleic Acids Research, 2010:1-12.
[24]朱志远,张燕,李征等.受体蛋白与药物分子对接的研究进展[J].中国临床药理学与治疗学,2009,14(11)：1308-1312.
[25]Comeau SR, Gatchell DW, Vajda S, et al. ClusPro:an automated docking and discrimination method for the prediction of protein complexes [J]. Bioinformatics,2004, 20(1):45-50.
[26]李春华,马晓慧,陈慰祖,王存新.蛋白质-蛋白质分子对接方法研究进展[J]Progress in Biochemistry and Biophysics,2006,33(7):616-621.
[27]许伟,蔡萍,严明,许琳Thermus thermophilus木糖异构酶与木糖醇的分子对接及模型分析[J].高等学校化学学报,2007,5：971-973.
[28]Fahmy A, Wagner G. J. [J].Am. Chem. Soc,2002,124(7):1241.
[29]Romkes M, Buch SC. Genotyping technologies:application to biotransformation enzyme genetic polymorphism screening [J]. Methods Mol Biol,2005,291(4):399-414.
[30]Smellie A, Stanton R, Henne R, et al. Conformational analysis by intersection [J]. Comput Chem,2003,24(3):10-20.
[31]Ma XH, Li CH, Shen LZ, et al. Biologically enhanced sampling geometric docking and backbone flexibility treatment with multi conformational superposition [J]. Proteins, 2005,60(2):319-323.
[32]曹冬梅,卢建.叉头框(Fox)转录因子家族的结构与功能[J].生命科学,2006,18(5)：491-496.
[33]J.M. Luscher-Firzlaff, R. Lilischkis, B. Luscher. Regulation of the transcription factor FOXM1c by Cyclin E/CDK2 [J]. FEBS Lett,2006,580:1716-1722.
[34]J. van den Boom, M. Wolter, R. Kuick, D.E. Misek, A.S. Youkilis, D.S.Wechsler, C. Sommer, G. Reifenberger, S.M. Hanash. Characterization of gene expression profiles associated with glioma progression using oligonucleotide-based microarray analysis and real-time reverse transcription-polymerase chain reaction, Am [J]. Pathol,2003, 163:1033-1043.
[35]Laoukili, J., Stahl, M., and Medema, R.H. FoxM1:at the crossroads of ageing and Cancer[J]. Biochim. Biophys. Acta.2007,1775:92-102.
[36]M.T. Teh, S.T. Wong, G.W. Neill, L.R. Ghali, M.P. Philpott, A.G. Quinn,FoxM1 isAdownstream target of Glil in basal cell carcinomas [J]. CancerRes,2002,62: 4773-4780.
[37]Li, X., Hussain, M., Sarkar, S.H., Eliason, J., Li, R., and Sarkar, F.H. Gene expression profiling revealed novel mechanism of action of Taxotere and Furtulon in prostate cancer cells [J]. BMC Cancer Ther,2005,5(7):689-672.
[38]M.L. Major, R. Lepe, R.H. Costa, Forkhead box M1B transcriptional activity requires binding of Cdk-Cyclin complexes for phosphorylation dependent recruitment of p300/CBP co-activators [J]. Mol. Cell. Biol,2004,24:2649-2661.
[39]Wierstra, I. and Alves, J. Despite its strong transactivation domain, transcription factor FOXMlc is kept almost inactive by two different inhibitory domains [J]. Biol. Chem. 2006,387:963-976.
[40]Wierstra, I. and Alves, J. FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2 and cyclin A/Cdkl, but repressed by GSK-3a [J]. Biochem.Biophys. Res. Commun,2006, 348:99-108.
[41]Wierstra, I. and Alves, J. The central domain of transcription factor FOXM1c directly interacts with itself in vivo and switches from an essential to an inhibitory domain depending on the FOXM1c binding site [J]. Biol. Chem,2007,388:805-818.
[42]Parvin, J.D., McCormick, R.J., Sharp, P.A., and Fisher, D.E. Pre-bending of a promote sequence enhances affinity for the TATA-binding factor [J]. Nature,1995,373: 724-727.
[43]D.R. Wonsey, M.T. Follettie, Loss of the forkhead transcription factor FoxMl causes centrosome amplification and mitotic catastrophe, Cancer Res,2005,65:5181-5189.
[44]Zhiwei Wang, Aamir Ahmad, Yiwei Li,et al. Forkhead box M1 transcription factor:A novel target for cancer therapy [J]. Cancer Treatment Reviews,2010,36:151-156.
[45]Heike Marxfeld, Frank Staedtler, Johannes H. Harleman. Characterisation of two rat mammary tumour models for breast cancer research by gene expression profiling [J]. Experimental and Toxicologic Pathology,2006,58:133-143.
[46]Liu, M., Dai, Rahman, K.M.W., Li, Y., Wang, Z., Sarkar, S.H., and Sarkar, F.H. Gene expression profiling revealed survivin asAtarget of 3,3'-diindolylmethane-induced cell growth inhibition and apoptosis in breast cancer cells [J]. Cancer Res,2006,66: 4952-4960.
[47]Kalin, T.V., Wang, I.C., Ackerson, T.J.,et al. Increased levels of the FoxMl transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice[J]. Cancer Res,2006,66:1712-1720.
[48]I.M. Kim, T. Ackerson, S. Ramakrishna, M. Tretiakova, I.C. Wang, T.V.Kalin, M.L. Major, G.A. Gusarova, H.M. Yoder, R.H. Costa, V.V.Kalinichenko, The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer [J].Cancer Res,2006,66:2153-2161.
[49]Dai, B.D., Kang, S.H., Gong, W., et al. Aberrant FoxM1B expression increases matrix metal loproteinase-2 transcription andenhances the invasion of glioma cells [J]. Oncogene,2007,26:6212-6219.
[50]H. Okabe, S. Satoh, T. Kato, O. Kitahara, R. Yanagawa, Y. Yamaoka, T. Tsunoda, Y. Furukawa, Y. Nakamura, Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray:identification of genes involved in viral carcinogenesis and tumor progression [J].Cancer Res,2001,61:2129-2137.
[51]Yoshida, Y., Wang, I.C., Yoder, H.M.,et al. The Forkhead Box M1 transcription factor contributes to the development and growth of mouse colorectal cancer [J]. Gastroenterology,2007,132:1420-1431.
[52]A. Ruiz i Altaba, P. Sanchez, N. Dahmane, Gli and hedgehog in cancer:tumours, embryos and stem cells [J]. Nat. Rev., Cancer,2002,2:361-372.
[53]Li Y, Hong X, Hussain M, et al. Gene expression profiling revealed novel molecular targets of docetaxel and estramustine combination treatment in prostate cancer cells [J]. Mol Cancer Ther,2005,4:389-398.
[54]Gartel AL. FoxMl inhibitors as potential anticancer drugs [J]. Expert Opin Ther Targets, 2008,12(6):663-665.
[55]Costa, R.H. FoxM1 dances with mitosis [J]. Nat. Cell Biol,2005,7:108-110.
[56]Bhat UG, Halasi M, Gartel AL. Thiazole antibiotics target FoxM1 and induce apoptosis in human cancer cells [J]. PLoS One,2009,4:e5592.
[2]唐保东,刘思纯,徐雅,曾志荣,胡品津.人类Foxm1b基因的原核表达、抗体制备及其对肝癌细胞中Foxm1b蛋白的识别[J].中山大学学报(医学科学版),2007,28(6)：658-661.
[3]K.H. Kaestner, W. Knochel, D.E. Martinez, Unified nomenclature for the winged helix/forkhead transcription factors [J]. Genes Dev,2000,14:142-146.
[4]Kaestner K H, Knochel W, Martinez D E. Unified nomenclature for the winged helix/ forkhead transcription factors [J]. Genes Dev,2000,14(2):142-146.
[5]孙言伟,邹立君,金梅.生物信息学的研究进展[J].中华医学图书情报杂志,2002,4(11)：1-3.
[6]Silveira, N J, Freitas U, Hugo B, et al. Structural bioinformatics study of PNP from Schistosoma mansoni [J]. Biochemical and Biophysical Research Communications, 2004,(1):100-104.
[7]胡德华,方平.生物信息学研究进展[J].情报科学,2000,18(9)：784-786.
[8]Eric J. Current trends in bioinformatics [J]. Trends in Biotechnology,2002,20(8): 317-319.
[9]Zheng CJ, Han LY, Yap CW, et al. Trends in exploration of therapeutic targets [J]. Drug News Perspect,2005,18(2):109-127.
[10]Austen M, Dohrmann C. Phenotype-first screening for the identification of novel drug targets[J]. Drug Discov Today,2005,10(4):275-282.
[11]章波,向渝梅,白云,许雪青,王燕.小鼠peroxiredoxin基因家族的生物信息学分析[J].第三军医大学学报,2005,27(9)：847-849.
[12]马忠石,杨克,孟丽蕊等.青少年牙周炎相关基因SEC14L5的生物信息学分析[J].生物技术通报,2007,3：105-110.
[13]周林福,朱海红,陈离伟等.肺炎衣原体膜表面蛋白重组质粒的构建[J].科技通报,2004,20(2)：172-177.
[14]Bailon P, Ehr lich K G, Fung W J, et al. Affinity chromatography:methods and protocols [M]. New Jersey:Humana Press,2000.
[15]王莉,刘道杰,李连之.亲和层析在蛋白质研究中的应用进展[J].理化检验-化学分册, 2007,43(6)：515-517.
[16]贾翠娟,董兆麟.一种自制T-载体的构建[J].微生物学通报,2001,28(5)：56-60.
[17]吕勇刚,窦科峰,赵爱志等.T载体在噬菌体单链抗体库中的应用[J].中国新医药,2004,3(6)：4-5.
[18]李杨,韩梅.亲和层析技术在生物学中的应用及发展[J].生命科学研究,2006,10(1)：12-16.
[19]Robb V A, Li W, Gutmann D H. Disruption of binding does not impair Protein 4.1 B growth suppression [J]. Ontogeny,2004,23(20):3589-3596.
[20]Scott JK, Smith GP. Searching for peptide ligands with an epitode library [J]. Science, 1990,249:386-390.
[21]吴懿娜,杜欣军,张伟伟等.噬菌体抗体库技术及其应用[J].细胞与分子免疫学杂志,2010,26(4)：404-406.
[22]田波,李传昭,孙仑泉等[M].分子进化过程第1版,科学出版社,1999：7-8.
[23]D. R. Littler, M. Alvarez-Fernandez, A. Stein, et al. Structure of the FoxM1 DNA-recognition domain bound to a promoter sequence [J]. Nucleic Acids Research, 2010:1-12.
[24]朱志远,张燕,李征等.受体蛋白与药物分子对接的研究进展[J].中国临床药理学与治疗学,2009,14(11)：1308-1312.
[25]Comeau SR, Gatchell DW, Vajda S, et al. ClusPro:an automated docking and discrimination method for the prediction of protein complexes [J]. Bioinformatics,2004, 20(1):45-50.
[26]李春华,马晓慧,陈慰祖,王存新.蛋白质-蛋白质分子对接方法研究进展[J]Progress in Biochemistry and Biophysics,2006,33(7):616-621.
[27]许伟,蔡萍,严明,许琳Thermus thermophilus木糖异构酶与木糖醇的分子对接及模型分析[J].高等学校化学学报,2007,5：971-973.
[28]Fahmy A, Wagner G. J. [J].Am. Chem. Soc,2002,124(7):1241.
[29]Romkes M, Buch SC. Genotyping technologies:application to biotransformation enzyme genetic polymorphism screening [J]. Methods Mol Biol,2005,291(4):399-414.
[30]Smellie A, Stanton R, Henne R, et al. Conformational analysis by intersection [J]. Comput Chem,2003,24(3):10-20.
[31]Ma XH, Li CH, Shen LZ, et al. Biologically enhanced sampling geometric docking and backbone flexibility treatment with multi conformational superposition [J]. Proteins, 2005,60(2):319-323.
[32]曹冬梅,卢建.叉头框(Fox)转录因子家族的结构与功能[J].生命科学,2006,18(5)：491-496.
[33]J.M. Luscher-Firzlaff, R. Lilischkis, B. Luscher. Regulation of the transcription factor FOXM1c by Cyclin E/CDK2 [J]. FEBS Lett,2006,580:1716-1722.
[34]J. van den Boom, M. Wolter, R. Kuick, D.E. Misek, A.S. Youkilis, D.S.Wechsler, C. Sommer, G. Reifenberger, S.M. Hanash. Characterization of gene expression profiles associated with glioma progression using oligonucleotide-based microarray analysis and real-time reverse transcription-polymerase chain reaction, Am [J]. Pathol,2003, 163:1033-1043.
[35]Laoukili, J., Stahl, M., and Medema, R.H. FoxM1:at the crossroads of ageing and Cancer[J]. Biochim. Biophys. Acta.2007,1775:92-102.
[36]M.T. Teh, S.T. Wong, G.W. Neill, L.R. Ghali, M.P. Philpott, A.G. Quinn,FoxM1 isAdownstream target of Glil in basal cell carcinomas [J]. CancerRes,2002,62: 4773-4780.
[37]Li, X., Hussain, M., Sarkar, S.H., Eliason, J., Li, R., and Sarkar, F.H. Gene expression profiling revealed novel mechanism of action of Taxotere and Furtulon in prostate cancer cells [J]. BMC Cancer Ther,2005,5(7):689-672.
[38]M.L. Major, R. Lepe, R.H. Costa, Forkhead box M1B transcriptional activity requires binding of Cdk-Cyclin complexes for phosphorylation dependent recruitment of p300/CBP co-activators [J]. Mol. Cell. Biol,2004,24:2649-2661.
[39]Wierstra, I. and Alves, J. Despite its strong transactivation domain, transcription factor FOXMlc is kept almost inactive by two different inhibitory domains [J]. Biol. Chem. 2006,387:963-976.
[40]Wierstra, I. and Alves, J. FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2 and cyclin A/Cdkl, but repressed by GSK-3a [J]. Biochem.Biophys. Res. Commun,2006, 348:99-108.
[41]Wierstra, I. and Alves, J. The central domain of transcription factor FOXM1c directly interacts with itself in vivo and switches from an essential to an inhibitory domain depending on the FOXM1c binding site [J]. Biol. Chem,2007,388:805-818.
[42]Parvin, J.D., McCormick, R.J., Sharp, P.A., and Fisher, D.E. Pre-bending of a promote sequence enhances affinity for the TATA-binding factor [J]. Nature,1995,373: 724-727.
[43]D.R. Wonsey, M.T. Follettie, Loss of the forkhead transcription factor FoxMl causes centrosome amplification and mitotic catastrophe, Cancer Res,2005,65:5181-5189.
[44]Zhiwei Wang, Aamir Ahmad, Yiwei Li,et al. Forkhead box M1 transcription factor:A novel target for cancer therapy [J]. Cancer Treatment Reviews,2010,36:151-156.
[45]Heike Marxfeld, Frank Staedtler, Johannes H. Harleman. Characterisation of two rat mammary tumour models for breast cancer research by gene expression profiling [J]. Experimental and Toxicologic Pathology,2006,58:133-143.
[46]Liu, M., Dai, Rahman, K.M.W., Li, Y., Wang, Z., Sarkar, S.H., and Sarkar, F.H. Gene expression profiling revealed survivin asAtarget of 3,3'-diindolylmethane-induced cell growth inhibition and apoptosis in breast cancer cells [J]. Cancer Res,2006,66: 4952-4960.
[47]Kalin, T.V., Wang, I.C., Ackerson, T.J.,et al. Increased levels of the FoxMl transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice[J]. Cancer Res,2006,66:1712-1720.
[48]I.M. Kim, T. Ackerson, S. Ramakrishna, M. Tretiakova, I.C. Wang, T.V.Kalin, M.L. Major, G.A. Gusarova, H.M. Yoder, R.H. Costa, V.V.Kalinichenko, The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer [J].Cancer Res,2006,66:2153-2161.
[49]Dai, B.D., Kang, S.H., Gong, W., et al. Aberrant FoxM1B expression increases matrix metal loproteinase-2 transcription andenhances the invasion of glioma cells [J]. Oncogene,2007,26:6212-6219.
[50]H. Okabe, S. Satoh, T. Kato, O. Kitahara, R. Yanagawa, Y. Yamaoka, T. Tsunoda, Y. Furukawa, Y. Nakamura, Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray:identification of genes involved in viral carcinogenesis and tumor progression [J].Cancer Res,2001,61:2129-2137.
[51]Yoshida, Y., Wang, I.C., Yoder, H.M.,et al. The Forkhead Box M1 transcription factor contributes to the development and growth of mouse colorectal cancer [J]. Gastroenterology,2007,132:1420-1431.
[52]A. Ruiz i Altaba, P. Sanchez, N. Dahmane, Gli and hedgehog in cancer:tumours, embryos and stem cells [J]. Nat. Rev., Cancer,2002,2:361-372.
[53]Li Y, Hong X, Hussain M, et al. Gene expression profiling revealed novel molecular targets of docetaxel and estramustine combination treatment in prostate cancer cells [J]. Mol Cancer Ther,2005,4:389-398.
[54]Gartel AL. FoxMl inhibitors as potential anticancer drugs [J]. Expert Opin Ther Targets, 2008,12(6):663-665.
[55]Costa, R.H. FoxM1 dances with mitosis [J]. Nat. Cell Biol,2005,7:108-110.
[56]Bhat UG, Halasi M, Gartel AL. Thiazole antibiotics target FoxM1 and induce apoptosis in human cancer cells [J]. PLoS One,2009,4:e5592.