TIE2配体寡肽的设计、筛选及其在基因治疗中的靶向导入作用
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摘要
肿瘤基因治疗将成为除手术、放疗和化疗等方法外又一新的抗癌策略。尤其在抗肿瘤转移和复发方面将起重要作用。目前还缺乏将基因导入人体细胞的高效靶向性载体系统,这是基因治疗至今尚未成为临床常规治疗措施的关键因素之一。因此研发一种高效靶向性基因导入系统成为当务之急。本研究旨在建立一种靶向Tie2受体的非病毒基因导入系统并检验其有效性,为今后肿瘤的基因治疗提供可靠的理论依据与实践指导。
第一部分
Tie2配体寡肽的设计与筛选
【目的】掌握特定受体的配体寡肽的设计与筛选方法;获得Tie2受体的候选配体寡肽; 【方法】 ⑴ 应用基于Tie2受体天然配体Angiopoietin-2的同源序列比较/二级结构分析及疏水性分析方法设计Tie2受体的配体寡肽并化学合成; ⑵ RT-PCR 和Western Bloting筛选Tie2阴性表达细胞株;构建pCDNA3.0-ExTie2质粒并转染Tie2表达阴性细胞,G418筛选出Tie2稳定表达细胞系;分别以重组人Tie2融合蛋白(rh-Tie2/Fc)与稳定表达Tie2受体的细胞为筛选靶,用噬菌体展示随机12肽库进行筛选。经过5轮筛选的噬菌体,经测序、ELISA、免疫组化及噬菌体回收试验鉴定出高度富集的阳性噬菌体克隆;然后化学合成高亲和力阳性噬菌体克隆展示肽。【结果】基于Tie2受体的配体Angiopoietin-2 设计的22肽命名为GA3(WTIIQRREDGSVDFQRTWKEYK);筛选到Tie2阴性表达细胞株SMMC7721; 建立了Tie2稳定表达细胞系SMMC7721-ExTie2;以rh-Tie2/Fc与SMMC7721-ExTie2为筛选靶获得的高亲和力富集噬菌体展示肽序列羧基端各加一个酪氨酸后分别命名为GA4
(HATGTHGLSLSHY)和GA5(NSLSNASEFRAPY)。【结论】本研究使用的设计及筛选方法可能是获得受体的配体寡肽的一种可行途径;候选的配体寡肽需要进一步鉴定。
Gene therapy will be a novel anticancer strategy in addition to commonly used surgery、radiotherapy and chemotherapy. It could play an important role in preventing cancer recurrence and metastasis. Lack of an efficient targeting gene transfer system which can transfer gene into human cells was contributed to one of the key reasons for its having not been an efficacious anticancer therapy. So, It is very urgent for developing an cancer targeting gene transfer system.Our researches aim to construct Tie2 receptor-mediated nonvirus gene transfer system and to confirm its targetability and efficacy. It will provide evidences and practical guide for future cancer gene therapy.
Section I Design and Screening of Oligopeptide ligand of Tie2
【Objective】To design and screening oligopeptide ligand of defenited receptor and obtain candidate oligopeptide ligand of Tie2 receptor. 【Methods】(1) Angiopoietin-2-baesd hydropathic analysis and comparative sequence/structure analysis were used to design the peptide ligand of Tie2. (2) Cell line which had no apparent Tie2 expression was identified by RT-PCR and Western bloting; pCDNA3.0-ExTie2 plasmid was constructed and transferred into cells which had no apparent Tie2 expression; Cell line which stably expressed Tie2 was established via G418 selecting. Recombined human Tie2/Fc fusion and stable cell line were used as targets for screening 12-mer phage disply library. After 5-rounds screening , sequencing、 ELISA、immunohischemostry and phage recovery assay were used to find out enriched positive phage clones.Then, peptides inserted in positive phage were chemistry synthsised. 【Results】 A 22 peptide which was designed based on structure of Ang2 protein was denoted as GA3 (WTIIQRREDGSVDFQRTWKEYK). Two peptide isolated by phage display from screening rh-Tie2 and SMCC7721-ExTie2 cells which stably expressing Tie2 were respectively added
tyrosine(Y) to their C-terminit and named as GA4 (HATGTHGLSLSHY)and GA5 (NSLSNASEFRAPY). 【Conclusions】Approaches applied in designing and screening peptide ligand seem promising for developing oligopeptide ligand for receptors. The effectivity of candidate oligopeptide ligands waited for further validation.
第一部分
Tie2配体寡肽的设计与筛选
【目的】掌握特定受体的配体寡肽的设计与筛选方法;获得Tie2受体的候选配体寡肽; 【方法】 ⑴ 应用基于Tie2受体天然配体Angiopoietin-2的同源序列比较/二级结构分析及疏水性分析方法设计Tie2受体的配体寡肽并化学合成; ⑵ RT-PCR 和Western Bloting筛选Tie2阴性表达细胞株;构建pCDNA3.0-ExTie2质粒并转染Tie2表达阴性细胞,G418筛选出Tie2稳定表达细胞系;分别以重组人Tie2融合蛋白(rh-Tie2/Fc)与稳定表达Tie2受体的细胞为筛选靶,用噬菌体展示随机12肽库进行筛选。经过5轮筛选的噬菌体,经测序、ELISA、免疫组化及噬菌体回收试验鉴定出高度富集的阳性噬菌体克隆;然后化学合成高亲和力阳性噬菌体克隆展示肽。【结果】基于Tie2受体的配体Angiopoietin-2 设计的22肽命名为GA3(WTIIQRREDGSVDFQRTWKEYK);筛选到Tie2阴性表达细胞株SMMC7721; 建立了Tie2稳定表达细胞系SMMC7721-ExTie2;以rh-Tie2/Fc与SMMC7721-ExTie2为筛选靶获得的高亲和力富集噬菌体展示肽序列羧基端各加一个酪氨酸后分别命名为GA4
(HATGTHGLSLSHY)和GA5(NSLSNASEFRAPY)。【结论】本研究使用的设计及筛选方法可能是获得受体的配体寡肽的一种可行途径;候选的配体寡肽需要进一步鉴定。
Gene therapy will be a novel anticancer strategy in addition to commonly used surgery、radiotherapy and chemotherapy. It could play an important role in preventing cancer recurrence and metastasis. Lack of an efficient targeting gene transfer system which can transfer gene into human cells was contributed to one of the key reasons for its having not been an efficacious anticancer therapy. So, It is very urgent for developing an cancer targeting gene transfer system.Our researches aim to construct Tie2 receptor-mediated nonvirus gene transfer system and to confirm its targetability and efficacy. It will provide evidences and practical guide for future cancer gene therapy.
Section I Design and Screening of Oligopeptide ligand of Tie2
【Objective】To design and screening oligopeptide ligand of defenited receptor and obtain candidate oligopeptide ligand of Tie2 receptor. 【Methods】(1) Angiopoietin-2-baesd hydropathic analysis and comparative sequence/structure analysis were used to design the peptide ligand of Tie2. (2) Cell line which had no apparent Tie2 expression was identified by RT-PCR and Western bloting; pCDNA3.0-ExTie2 plasmid was constructed and transferred into cells which had no apparent Tie2 expression; Cell line which stably expressed Tie2 was established via G418 selecting. Recombined human Tie2/Fc fusion and stable cell line were used as targets for screening 12-mer phage disply library. After 5-rounds screening , sequencing、 ELISA、immunohischemostry and phage recovery assay were used to find out enriched positive phage clones.Then, peptides inserted in positive phage were chemistry synthsised. 【Results】 A 22 peptide which was designed based on structure of Ang2 protein was denoted as GA3 (WTIIQRREDGSVDFQRTWKEYK). Two peptide isolated by phage display from screening rh-Tie2 and SMCC7721-ExTie2 cells which stably expressing Tie2 were respectively added
tyrosine(Y) to their C-terminit and named as GA4 (HATGTHGLSLSHY)and GA5 (NSLSNASEFRAPY). 【Conclusions】Approaches applied in designing and screening peptide ligand seem promising for developing oligopeptide ligand for receptors. The effectivity of candidate oligopeptide ligands waited for further validation.
引文
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Seth, P., Fitzgerald, D., Ginsberg, H., et al. Evidence that the penton base of adenovirus is involved in potentiation of toxicity of pseudomonas exotoxin conjugated to epidermal growth factor. Mol Cell Biol 1984, 4(8):1528-1536.
田培坤,任圣俊,任常春等 一种新的细胞表面受体为靶向的基因导入系统。 中国科学(C辑), 1998,28(6):554-560.
Li, J.M., Han, J.S., Huang,Y. et al. A novel gene delivery system targeting cells expression VEGF receptors. Cell Res, 1999, 9(1):11-25.
Partanen J ,Armstrong E, Makela TP, et al .A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains . Mol Cell Biol, 1992, 4 (8) 1698-1707.
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receptor , during embryonic angiogenesis. Cell, 1996, 87 (7):1171-1180.
Patan S,. Tie1 and Tie2 receptor tyrosine kinase inversely regulate embryonic angiogenesis by the mechanism of intussusceptive microvascular growth. Microvasc Res, 1998,56(1)1-21
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Wu, G.Y. and W, C.H. Receptor-mediated in vivo gene transformation by a soluble DNA carrier system. J Biol Chem, 1987, 262(10):4429-4432.
Wu, C.H., Wilson J.M. and Wu, G.Y. Targeting genes:delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J Biol Chem, 1989, 264(29):16985-16987.
Cotton M., Wgner E., Zatloukal. Et al. High-efficiency receptor-mediated delivery of small and large(48kb) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particales. Proc Natl Acad Sci USA,1992,89(13):6094-6098
Seth, P., Fitzgerald, D.J.P., Wilingham, M.C. et al. Role of a low-pH environment in adenovirus enhancement of the toxicityof a pseudomonas exotoxin-epidermal growth factor conjugate J. Virol ,1984, 51(3):650-655.
Seth, P., Fitzgerald, D., Ginsberg, H., et al. Evidence that the penton base of adenovirus is involved in potentiation of toxicity of pseudomonas exotoxin conjugated to epidermal growth factor. Mol Cell Biol 1984, 4(8):1528-1536.
田培坤,任圣俊,任常春等 一种新的细胞表面受体为靶向的基因导入系统。 中国科学(C辑), 1998,28(6):554-560.
Li, J.M., Han, J.S., Huang,Y. et al. A novel gene delivery system targeting cells expression VEGF receptors. Cell Res, 1999, 9(1):11-25.
Partanen J ,Armstrong E, Makela TP, et al .A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains . Mol Cell Biol, 1992, 4 (8) 1698-1707.
Suri C, Jones PF, Patan S, et al. Requisite role of angiopoietin-1, a ligand for the Tie2
receptor , during embryonic angiogenesis. Cell, 1996, 87 (7):1171-1180.
Patan S,. Tie1 and Tie2 receptor tyrosine kinase inversely regulate embryonic angiogenesis by the mechanism of intussusceptive microvascular growth. Microvasc Res, 1998,56(1)1-21
Sato TN, Tozawa Y, Deutsch, et al. Distinct roles of the receptor tyrosine kinase Tie1 and Tie2 in blood vessel formation. Nature, 1995, 376(6535):70-74.
Dumont DJ, Gradwohl G,Fong GH, et al. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, Tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev, 1994,8(16):1897-1909.
Currie MJ ,Gunningham SP, Turner K, et al . Expression of the angiopoietins and ther receptor Tie 2 in human renal clear cell carcinomas regulation by the von Hippel-Lindau gene and hypoxia . J Pathol ,2002, 198(4):502-510
Peters KG, Coogan A, Berry D, et al . Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumor angiogenesis. Br J Cancer, 1998,77(1):51-56.
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