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  本文選題：胰島素樣生長因子-1 + 人胰島素樣生長因子結(jié)合蛋白-3�。� 參考：《天津醫(yī)科大學(xué)》2008年碩士論文

【摘要】：胰島素樣生長因子結(jié)合蛋白3(IGFBP-3)是一種多功能的內(nèi)分泌因子,是該家族的重要成員,是人類血液中數(shù)量最多、作用最強(qiáng)的IGFBP,可結(jié)合血液中95%-99%的胰島素樣生長因子-1(IGF-1)。重組人IGF-1 (rhIGF-1)早已被應(yīng)用于治療侏儒癥,并發(fā)現(xiàn)IGF-1對于糖尿病、神經(jīng)損傷、和骨質(zhì)疏松癥也有治療作用。但已有報道證明單獨(dú)應(yīng)用rhIGF1治療時,可引發(fā)一些輕度和中度不良反應(yīng),包括四肢浮腫、頭疼、關(guān)節(jié)疼、下顎觸痛、體位性低血壓、視神經(jīng)水腫等。IGFBP-3可使IGF-1的半衰期延長,控制IGF-1的生物利用度而使不良反應(yīng)顯著減少。rhIGF-1和rhIGFBP-3合并成復(fù)合物使用,當(dāng)可成為提高藥效降低副作用之最佳方案。而后者也有望被開發(fā)為一種新藥,并具有廣泛的應(yīng)用前景。與此同時多年研究確認(rèn)IGFBP-3是細(xì)胞凋亡的誘導(dǎo)物,這一作用是獨(dú)立于IGFs之外的。IGFBP-3具有促細(xì)胞凋亡、抑制細(xì)胞生長、增強(qiáng)癌細(xì)胞的放射敏感性等作用,IGFBP-3對生長失控細(xì)胞的作用機(jī)制,已成為抗腫瘤治療研究的一個新熱點(diǎn)。而國內(nèi)對于IGFBP-3的研究,僅限于將其作為糖尿病、腫瘤等的標(biāo)志物,而對于表達(dá)純化及功能方面的報道甚少。 我實(shí)驗(yàn)室已成功構(gòu)建了IGF-1的高效表達(dá)系統(tǒng)。本文目的旨在建立IGFBP-3的原核表達(dá)系統(tǒng),為進(jìn)一步深入研究IGFBP-3提供條件。我們提取人肝組織總RNA,逆轉(zhuǎn)錄聚合酶鏈反應(yīng)擴(kuò)增IGFBP-3的cDNA,經(jīng)巢式PCR擴(kuò)增得到目的片段。將其克隆入pGEM-T Easy載體。測序證實(shí)序列正確后,將目的基因插入融合型原核表達(dá)載體pQE-30Xa,將己構(gòu)建好的表達(dá)載體pQE-30Xa/ IGFBP-3重組質(zhì)粒轉(zhuǎn)化大腸桿菌M15,異丙基硫代-8-D-半乳糖苷(IPTG)誘導(dǎo)表達(dá)。采用親和層析法純化目的蛋白。ELISA法測定rhIGFBP-3與IGF-1的結(jié)合活性。MTT法檢測rhIGFBP-3對Hela細(xì)胞增殖的影響。結(jié)果SDS-PAGE可見與預(yù)期大小相符的蛋白條帶,Western Blot證實(shí)該條帶即為rhIGFBP-3。經(jīng)純化后,用Chemi Genius凝膠成像分析系統(tǒng)分析,重組蛋白的純度超過95%。生物活性研究顯示它有抑制Hela細(xì)胞增殖的作用,且在體外具有與IGF-1結(jié)合的活性。我們成功建立了rhIGFBP-3的高效表達(dá)系統(tǒng),rhIGFBP-3的表達(dá)量可達(dá)到菌體總蛋白量的50.2%,而且具有很好的生物學(xué)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R346

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