重组人白细胞介素24的表达及其生物学活性鉴定
发布时间：2019-03-07 15:55
【摘要】：1995年P．B．Fisher用差减杂交技术发现一个新的与人黑色素瘤分化相关基因，当时称为mda-7(melanoma differentiation-associated gene 7)，并发现随着黑色素瘤细胞生长、发展和转移，mda-7的表达逐渐减少至最终消失，，证实了其表达产物MDA-7具有抑制黑色素瘤细胞增生、促进黑色素瘤细胞终末分化的能力。随后，MDA-7一直作为肿瘤抑制物被研究。直到2002年，Caudell等研究证实了MDA-7的细胞因子属性，重新命名为人白细胞介素24(Human interleukin-24，hIL-24)。迄今为止，已有大量实验证明hIL-24具有显著的选择性抑制多种肿瘤生长、诱导肿瘤细胞凋亡的能力，这种抑制作用不依赖p53、Rb和p16等抑癌基因的状态，且对正常细胞没有影响。hIL-24作为细胞因子，不仅在激活免疫细胞、调节整个抗癌免疫反应和造血系统中起重要作用，还具有选择性诱导肿瘤细胞凋亡和直接抑制肿瘤细胞的增殖、转移作用，其显著的抗肿瘤特性使之成为肿瘤治疗研究的新热点。2004年，Introgen公司研制的基因治疗制剂Ad．IL-24，即重组复制缺陷型腺病毒-IL-24，商品名为INGN 241，获美国FDA批准进入Ⅱ期临床试验，体内外试验和临床实验均证实了hIL-24显著的抗肿瘤效果。随着对hIL-24作用机制和基因治疗方面深入的研究，一方面肯定了hIL-24生物学功能和治疗效果，另一方面表现出对hIL-24的大量需求，所以，通过基因工程手段大规模生产高纯度有活性的重组hIL-24成为必然。本研究分别采用大肠杆菌(原核)表达系统制备了融合蛋白Trx-IL-24，并用肠激酶酶切Trx-IL-24得到重组蛋白IL-24，以及采用毕赤酵母(真核)表达系统分泌表达了重组糖蛋白rIL-24，分析鉴定了三种重组蛋白诱导肿瘤细胞凋亡的生物学活性，为深入研究其诱导肿瘤细胞凋亡机制和肿瘤治疗应用奠定基础。 研究目的：构建hIL-24的大肠杆菌表达系统和毕赤酵母表达系统，获得三种重组蛋白Trx-IL-24、IL-24和rIL-24。建立Trx-IL-24和IL-24的制备、纯化及复性的中试生产工艺。筛选高效分泌表达rIL-24的重组毕赤酵母工程菌。鉴定三种重组蛋白诱导肿瘤细胞凋亡的生物学活性。 研究方法： 1) 人白细胞介素24基因的克隆 采用RT-PCR和nested-PCR方法，从ConA刺激培养的人外周血单个核细胞中，克隆带信号肽的hIL-24编码序列(hmIL-24)和不带信号肽的hIL-24编码序列(hIL-24)。 2) 人白细胞介素24基因在大肠杆菌中的表达与纯化 为避免引入多余的氨基酸，利用载体pET32a(+)上的肠激酶识别位点编码碱基之前的Kpn I位点，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
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392
本文编号：2436245
[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
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 姚斌,张春义,王建华,范云六;高效表达具有生物学活性的植酸酶的毕赤酵母[J];中国科学C辑:生命科学;1998年03期
2 欧阳立明,张惠展,张嗣良,刘志敏;巴斯德毕赤酵母的基因表达系统研究进展[J];生物化学与生物物理进展;2000年02期
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