【摘要】：1995年P(guān)．B．Fisher用差減雜交技術(shù)發(fā)現(xiàn)一個(gè)新的與人黑色素瘤分化相關(guān)基因，當(dāng)時(shí)稱為mda-7(melanoma differentiation-associated gene 7)，并發(fā)現(xiàn)隨著黑色素瘤細(xì)胞生長(zhǎng)、發(fā)展和轉(zhuǎn)移，mda-7的表達(dá)逐漸減少至最終消失，，證實(shí)了其表達(dá)產(chǎn)物MDA-7具有抑制黑色素瘤細(xì)胞增生、促進(jìn)黑色素瘤細(xì)胞終末分化的能力。隨后，MDA-7一直作為腫瘤抑制物被研究。直到2002年，Caudell等研究證實(shí)了MDA-7的細(xì)胞因子屬性，重新命名為人白細(xì)胞介素24(Human interleukin-24，hIL-24)。迄今為止，已有大量實(shí)驗(yàn)證明hIL-24具有顯著的選擇性抑制多種腫瘤生長(zhǎng)、誘導(dǎo)腫瘤細(xì)胞凋亡的能力，這種抑制作用不依賴p53、Rb和p16等抑癌基因的狀態(tài)，且對(duì)正常細(xì)胞沒有影響。hIL-24作為細(xì)胞因子，不僅在激活免疫細(xì)胞、調(diào)節(jié)整個(gè)抗癌免疫反應(yīng)和造血系統(tǒng)中起重要作用，還具有選擇性誘導(dǎo)腫瘤細(xì)胞凋亡和直接抑制腫瘤細(xì)胞的增殖、轉(zhuǎn)移作用，其顯著的抗腫瘤特性使之成為腫瘤治療研究的新熱點(diǎn)。2004年，Introgen公司研制的基因治療制劑Ad．IL-24，即重組復(fù)制缺陷型腺病毒-IL-24，商品名為INGN 241，獲美國(guó)FDA批準(zhǔn)進(jìn)入Ⅱ期臨床試驗(yàn)，體內(nèi)外試驗(yàn)和臨床實(shí)驗(yàn)均證實(shí)了hIL-24顯著的抗腫瘤效果。隨著對(duì)hIL-24作用機(jī)制和基因治療方面深入的研究，一方面肯定了hIL-24生物學(xué)功能和治療效果，另一方面表現(xiàn)出對(duì)hIL-24的大量需求，所以，通過基因工程手段大規(guī)模生產(chǎn)高純度有活性的重組hIL-24成為必然。本研究分別采用大腸桿菌(原核)表達(dá)系統(tǒng)制備了融合蛋白Trx-IL-24，并用腸激酶酶切Trx-IL-24得到重組蛋白IL-24，以及采用畢赤酵母(真核)表達(dá)系統(tǒng)分泌表達(dá)了重組糖蛋白rIL-24，分析鑒定了三種重組蛋白誘導(dǎo)腫瘤細(xì)胞凋亡的生物學(xué)活性，為深入研究其誘導(dǎo)腫瘤細(xì)胞凋亡機(jī)制和腫瘤治療應(yīng)用奠定基礎(chǔ)。 研究目的：構(gòu)建hIL-24的大腸桿菌表達(dá)系統(tǒng)和畢赤酵母表達(dá)系統(tǒng)，獲得三種重組蛋白Trx-IL-24、IL-24和rIL-24。建立Trx-IL-24和IL-24的制備、純化及復(fù)性的中試生產(chǎn)工藝。篩選高效分泌表達(dá)rIL-24的重組畢赤酵母工程菌。鑒定三種重組蛋白誘導(dǎo)腫瘤細(xì)胞凋亡的生物學(xué)活性。 研究方法： 1) 人白細(xì)胞介素24基因的克隆 采用RT-PCR和nested-PCR方法，從ConA刺激培養(yǎng)的人外周血單個(gè)核細(xì)胞中，克隆帶信號(hào)肽的hIL-24編碼序列(hmIL-24)和不帶信號(hào)肽的hIL-24編碼序列(hIL-24)。 2) 人白細(xì)胞介素24基因在大腸桿菌中的表達(dá)與純化 為避免引入多余的氨基酸，利用載體pET32a(+)上的腸激酶識(shí)別位點(diǎn)編碼堿基之前的Kpn I位點(diǎn)，及多克
[Abstract]:In 1995, P. B. Fisher, using the subtractive hybridization technique, found a new gene associated with human melanoma differentiation, called mda-7 (melanoma differentiation-associated gene 7), and found that with the growth, development and transfer of melanoma cells, the mda-7 expression was gradually reduced to a final loss, It is confirmed that the expression product of MDA-7 has the ability to inhibit the proliferation of melanoma cells and promote the terminal differentiation of melanoma cells. Subsequently, MDA-7 has been studied as a tumor suppressor. Until 2002, Caudell et al. studied the cytokine profile of MDA-7 and renamed human interleukin-24 (hIL-24). So far, there have been a lot of experiments that have shown that hIL-24 has the ability to selectively inhibit the growth of various tumors and induce the apoptosis of tumor cells, which does not depend on the state of tumor suppressor genes such as p53, Rb and p16, and has no effect on normal cells. hIL-24 as a cytokine not only plays an important role in activating immune cells, regulating the whole anti-cancer immune response and the hematopoietic system, but also has the function of selectively inducing the apoptosis of the tumor cells and directly inhibiting the proliferation and the transfer of the tumor cells, In 2004, the gene therapy preparation Ad. IL-24 developed by Introgen Inc., the recombinant replication defective adenovirus-IL-24, the product named INGN 241, was approved by the US FDA to enter the Phase II clinical trial. The anti-tumor effect of hIL-24 was confirmed both in vivo and in clinical experiments. With the further study of the mechanism of hIL-24 and gene therapy, on the one hand, the biological function and therapeutic effect of hIL-24 are confirmed, and on the other hand, there is a great demand for hIL-24, so it is necessary to produce high-purity active recombinant hIL-24 by genetic engineering. The fusion protein Trx-IL-24 was prepared by using E. coli (Prokaryotic) expression system, and the recombinant protein IL-24 was obtained by using enterokinase and Trx-IL-24, and the recombinant glycoprotein rIL-24 was expressed by the expression system of Pichia pastoris (Eukaryotic). The biological activity of three recombinant proteins to induce the apoptosis of tumor cells was analyzed and the basis for further study on the mechanism of inducing tumor cell apoptosis and the application of tumor therapy was established. Objective: To construct the expression system of hIL-24 and the expression system of Pichia pastoris. Three recombinant proteins, Trx-IL-24 and IL, were obtained. Preparation and purification of Trx-IL-24 and IL-24 And refolding the trial-production process, and screening the high-efficiency secretion expression rIL-24. The recombinant Pichia pastoris engineering bacteria can be used for identifying three recombinant proteins to induce the tumor. cell-apoptotic Biological activity. Methods:1) The human interleukin-24 gene was cloned by RT-PCR and nested-PCR, and the hIL-24 coding sequence (h) with signal peptide was cloned from the peripheral blood mononuclear cells of human peripheral blood cultured by ConA. mIL-24 and hIL-24 coding sequence without signal peptide (hIL-24).2) Expression and purification of human interleukin-24 gene in E. coli
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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