人GRIM19的cDNA克

发布时间：2018-12-12 18:14

【摘要】：Signal Transducer and Activator of Transcription 3(STAT3)受细胞因子、生长因子等的激活,在细胞生长、抗凋亡和细胞转化上起着重要的作用。人类多数肿瘤细胞中均有高于正常活性的 STAT3。GRIM19(Genes associated with Retinoid-IFN-induced Mortality)是维甲酸干扰素诱导的死亡相关基因,它能直接特异性作用于 STAT3,抑制 STAT3 激活靶基因转录的活性、抑制靶基因表达,从而抑制肿瘤的生长。肿瘤的靶向治疗需要特异性载体,将药物导向肿瘤。编码含人类高迁移组蛋白 2(HMGN2)氮端 31 个氨基酸的 F3 肽具有特异性与肿瘤细胞和肿瘤血管内皮细胞结合的功能,能将药物带入胞质和胞核,可以用作肿瘤靶向给药的载体。本课题运用基因克隆技术,从人胎盘组织中克隆了 GRIM19 的cDNA,并构建了 F3-GRIM19 表达载体。利用大肠杆菌表达体系表达 F3-GRIM19,亲和层析纯化获得 F3-GRIM19 融合蛋白,并鉴定了融合蛋白体外抗肿瘤细胞增殖的生物学活性,为进一步研究其作用机理和临床应用奠定基础。第一部分:以中国人胎盘组织为材料,提取组织总 RNA,用 RT-PCR方法扩增出 GRIM19 的 cDNA,克隆入 pET32a(+)载体中并测序鉴定,亚克隆构建原核表达载体 pQE30-GRIM19 及 pQE30-F3-GRIM19,IPTG 诱导表达。用 Ni2+-NTA 琼脂糖树脂亲和层析对 GRIM19 和 F3-GRIM19 进行纯化,并透析复性。第二部分:利用 MTT 比色法分析目的蛋白 GRIM19 和 F3-GRIM19 分别对正常 HUVEC 细胞和人乳腺癌 MCF7、MDA-MB231 细胞增殖活性的影
[Abstract]:Signal Transducer and Activator of Transcription _ 3 (STAT3), activated by cytokines and growth factors, plays an important role in cell growth, anti-apoptosis and cell transformation. STAT3.GRIM19 (Genes associated with Retinoid-IFN-induced Mortality, which is higher than normal activity in most human tumor cells, is a death related gene induced by retinoic acid. It can directly and specifically inhibit the activity of STAT3 activated target gene transcription by STAT3,. Inhibition of target gene expression, thereby inhibiting tumor growth. The targeted treatment of tumor requires a specific carrier to direct the drug to the tumor. F3 peptide, which encodes 31 amino acids in the nitrogen terminal of human high migration histone 2 (HMGN2), has the function of binding specifically to tumor cells and tumor vascular endothelial cells. F3 peptide can bring drugs into cytoplasm and nucleus and can be used as a drug carrier for tumor targeting delivery. In this study, cDNA, of GRIM19 was cloned from human placenta tissue by gene cloning technique and F3-GRIM19 expression vector was constructed. The fusion protein F3-GRIM19 was expressed in Escherichia coli and purified by affinity chromatography. The biological activity of the fusion protein against tumor cell proliferation in vitro was identified, which laid a foundation for further study on its mechanism and clinical application. Part one: using Chinese placental tissue as material, total RNA, of GRIM19 was amplified by RT-PCR and cloned into pET32a () vector and sequenced. Prokaryotic expression vectors pQE30-GRIM19 and pQE30-F3-GRIM19, were constructed by subclone. IPTG induced expression. GRIM19 and F3-GRIM19 were purified by Ni2-NTA agarose resin affinity chromatography. Part two: MTT colorimetric assay was used to analyze the proliferative activity of target protein GRIM19 and F3-GRIM19 on normal HUVEC cells and human breast cancer MCF7,MDA-MB231 cells, respectively.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78

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