人胰岛素样生长因子结合蛋白-3基因编码序列的克隆及其在大肠杆菌中的表达

发布时间：2018-04-29 12:36

本文选题：胰岛素样生长因子-1 + 人胰岛素样生长因子结合蛋白-3　；参考：《天津医科大学》2008年硕士论文

【摘要】：胰岛素样生长因子结合蛋白3(IGFBP-3)是一种多功能的内分泌因子,是该家族的重要成员,是人类血液中数量最多、作用最强的IGFBP,可结合血液中95%-99%的胰岛素样生长因子-1(IGF-1)。重组人IGF-1 (rhIGF-1)早已被应用于治疗侏儒症,并发现IGF-1对于糖尿病、神经损伤、和骨质疏松症也有治疗作用。但已有报道证明单独应用rhIGF1治疗时,可引发一些轻度和中度不良反应,包括四肢浮肿、头疼、关节疼、下颚触痛、体位性低血压、视神经水肿等。IGFBP-3可使IGF-1的半衰期延长,控制IGF-1的生物利用度而使不良反应显著减少。rhIGF-1和rhIGFBP-3合并成复合物使用,当可成为提高药效降低副作用之最佳方案。而后者也有望被开发为一种新药,并具有广泛的应用前景。与此同时多年研究确认IGFBP-3是细胞凋亡的诱导物,这一作用是独立于IGFs之外的。IGFBP-3具有促细胞凋亡、抑制细胞生长、增强癌细胞的放射敏感性等作用,IGFBP-3对生长失控细胞的作用机制,已成为抗肿瘤治疗研究的一个新热点。而国内对于IGFBP-3的研究,仅限于将其作为糖尿病、肿瘤等的标志物,而对于表达纯化及功能方面的报道甚少。我实验室已成功构建了IGF-1的高效表达系统。本文目的旨在建立IGFBP-3的原核表达系统,为进一步深入研究IGFBP-3提供条件。我们提取人肝组织总RNA,逆转录聚合酶链反应扩增IGFBP-3的cDNA,经巢式PCR扩增得到目的片段。将其克隆入pGEM-T Easy载体。测序证实序列正确后,将目的基因插入融合型原核表达载体pQE-30Xa,将己构建好的表达载体pQE-30Xa/ IGFBP-3重组质粒转化大肠杆菌M15,异丙基硫代-8-D-半乳糖苷(IPTG)诱导表达。采用亲和层析法纯化目的蛋白。ELISA法测定rhIGFBP-3与IGF-1的结合活性。MTT法检测rhIGFBP-3对Hela细胞增殖的影响。结果SDS-PAGE可见与预期大小相符的蛋白条带,Western Blot证实该条带即为rhIGFBP-3。经纯化后,用Chemi Genius凝胶成像分析系统分析,重组蛋白的纯度超过95%。生物活性研究显示它有抑制Hela细胞增殖的作用,且在体外具有与IGF-1结合的活性。我们成功建立了rhIGFBP-3的高效表达系统,rhIGFBP-3的表达量可达到菌体总蛋白量的50.2%,而且具有很好的生物学活性。
[Abstract]:Insulin-like growth factor-binding protein (IGFBP-3) is a multifunctional endocrine factor and an important member of the family. IGFBP-3 is the most abundant and most effective IGFBPin human blood. IGFBP-3 can bind 95 to 99% of IGFBP-1 in blood. Recombinant human IGF-1 rhIGF-1 has long been used in the treatment of dwarfism, and it has been found that IGF-1 also has therapeutic effects on diabetes, nerve damage, and osteoporosis. However, it has been reported that rhIGF1 alone can lead to mild and moderate adverse reactions, including limb edema, headache, joint pain, jaw tenderness, postural hypotension, optic nerve edema and so on. IGFBP-3 can prolong the half-life of IGF-1. Controlling the bioavailability of IGF-1 significantly reduced the adverse reactions. RhIGF-1 and rhIGFBP-3 were combined to form a complex, which could be the best way to improve the efficacy and reduce the side effects. The latter is also expected to be developed as a new drug and has broad application prospects. At the same time, years of studies have confirmed that IGFBP-3 is an inducer of cell apoptosis, which is independent of IGFs. IGFBP-3 can promote cell apoptosis, inhibit cell growth and enhance the radiosensitivity of cancer cells. It has become a new hot spot in the research of anti-tumor therapy. However, the study of IGFBP-3 in China is limited to being used as a marker of diabetes, tumor and so on, but there are few reports on expression purification and function. Our laboratory has successfully constructed an efficient expression system for IGF-1. The purpose of this paper is to establish prokaryotic expression system of IGFBP-3 and to provide conditions for further study of IGFBP-3. We extracted total RNAs from human liver tissues and amplified IGFBP-3 cDNAs by reverse transcriptase polymerase chain reaction (RT-PCR). The target fragments were obtained by nested PCR amplification. It was cloned into pGEM-T Easy vector. After the sequence was confirmed, the target gene was inserted into the fusion prokaryotic expression vector pQE-30Xa. the constructed recombinant plasmid pQE-30Xa/ IGFBP-3 was transformed into E. coli M15 and induced by isopropylthio-8-D-galactoside IPTG. The binding activity of rhIGFBP-3 and IGF-1 was determined by affinity chromatography. The effect of rhIGFBP-3 on the proliferation of Hela cells was detected by Elisa. Results SDS-PAGE showed that the protein band was rhIGFBP-3, which was confirmed by Western Blot. After purification, the purity of recombinant protein was more than 95% by Chemi Genius gel imaging analysis system. The bioactivity study showed that it could inhibit the proliferation of Hela cells and bind to IGF-1 in vitro. We have successfully established a highly efficient expression system of rhIGFBP-3, rhIGFBP-3, which can reach 50.2% of the total bacterial protein, and has good biological activity.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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