论文标题:突变型人β干扰素的表达、纯化及新型干扰素样细胞因子全长新基因的发现与功能研究
Experimental studies on new hepatitis-associated viruses(HGV and TTV)
论文作者 陈国友
论文导师 曹雪涛,论文学位 博士,论文专业 微生物学
论文单位 第二军医大学,点击次数 163,论文页数 104页File Size4527k
2001-05-01论文网 http://www.lw23.com/lunwen_915352/ β─干扰素,基因工程,纯化,树突状细胞,干扰素样蛋白,全长新基因,大规模测序,基因克隆,细胞因子
Beta-interferon;genetic engineering;purification;dendritic cells;interferon-like protein;Full length cDNA;large scale sequencing;gene cloning;cytokines
干扰素（Interferon）是一组具有广泛生物学活性，包括抗病毒、抗肿瘤以及免疫调节 活性的细胞因子，在临床上具有很广阔的应用前景。目前国内基因工程人IFN-β的研制 工程尚未达到产业化水平。本研究应用新型表达测序一体化载体pLCM182，构建了表 达重组人突变型IFN-β（~(17)Ser-IFN-β）的原核表达载体，导入大肠杆菌后经反复筛选到， 得到稳定高效表达~(17)Ser-IFN-β的工程菌；通过优化了~(17)Ser-IFN-β程菌的发酵表达条 件，使其可以满足中试生产的要求；另外经过反复实验，完成了基因工程~(17)Ser-IFN-β 的纯化工艺研究，连续三批制品检定结果表明其质量己达到生物制品规程的相关要求， 并已通过国家药品及生物制品检定所的质量鉴定。 另外，本研究还通过大规模测序从人树突状细胞cDNA文库中克隆到一种编码新型 干扰素样细胞因子（IFN-CK）的全长新基因。生物信息学分析结果表明，IFN-CK含有Ⅰ 型干扰素家族特征性标鉴序列，与IFN-α及IFN-β具有较高的同源性。Northern印迹分 析结果表明，IFN-CK在肾、心脏、骨骼肌、肝和胰腺、胎盘、睾丸、卵巢、外周血白 细胞、结肠和肺等组织中均未检测到明显的表达。RT-PCR结果表明，该基因相对特 异地表达于树突状细胞。 初步生物学功能研究结果表明，IFN-CK无明显的抗病毒活性，但可抑制亚适剂量 PHA刺激的PBMNC增殖反应及IL-2的分泌，抑制T细胞表面CD45RO及IL-2受体 的表达，表明该新型干扰素样细胞因子可能作为树突状细胞特异性分泌的活性分子对机 体的免疫功能起调节作用。 干扰素（Interferon，IFN）是一组具有广泛生物学活性，包括抗病毒、抗肿瘤以及免疫调节活性的细胞因子。IFN是最早发现、研究最多、也是第一个被用于临床治疗疾病的细胞因子。根据干扰素的蛋白质的氨基酸结构、抗原性和细胞来源，可将其分为三大类：IFN-α、IFN-β和IFN-γ，其中IFN-α主要由白细胞产生，IFN-β主要由成纤维细胞产生，两者均具有相似的生物学活性，结合同样的细胞表面受体，统称为Ⅰ型干扰素。IFN-γ主要由T淋巴细胞产生，其抗病毒活性较弱，但免疫调节作用强，因此也被称为免疫干扰素或Ⅱ型干扰素。 自从IFN-β被发现以来，研究人员最先是从成纤维细胞通过病毒诱导后制备天然的IFN-β，在对IFN-β进行了广泛的基础研究后，已将其试用于多发性硬化症、肿瘤、及多种病毒性感染的治疗。天然的人IFN-β是一种糖基化蛋白，但天然来源的IFN-β生产工艺比较复杂，来源比较困难而且成本较高，限制了IFN-β临床研究和应用。随着重组DNA技术的迅速发展，研究人员克隆了IFN-β的基因并成功地进行了表达。目前国外多家研究机构已经在成功地大肠杆菌、酵母菌、哺乳动物细胞中进行了IFN-β的稳定、高效表达，基因工程人 IFN-β已于 1993年通过 FDA批准用于多发性硬化症的治疗。 由于互型干扰素对临床上多种疾病具有显著的治疗，因此国外己广泛开展了该方面的研制及临床研究工作，而国内干扰素的研制和应用多集中于D’i－a，除了IFN－第二军医大学博士学位论文 中文摘要a fo为我国自行发现外，大多数的工作均属于跟踪性研究。最近我们通过大规模随机测序，从人树突状细胞。DNA文库中发现了一条编码一种千扰素样细胞因子 门nterferondike cytokine，fN－CK）的新基因，因此我们在完成了重组人突变型 fN－p工程菌的构建、表达及纯化工艺的研究基础上（第一部分），进一步探讨了该新型干扰素样细胞因子基因的组织分布及其生物学功能（第二部分）。 第一部分 基因工程人 IFN平工程菌的构建、表达纯化 虽然国内WN一p的研制工作虽然已开展多年，但至今仍未达到产业化，也未被国家药品监督管理局批准开展临床试验研究。究其原因，主要是因为在IFN一B的研制过程中，许多生产工艺限制了其研制的进程。其中包括WN一p的原核表达量，发酵条件、复性条件及随后的IFN一p的纯化工艺等。 人 UN个由 165个氨基酸残基组成，其中含 3个半耽氨酸，分别位于第 17、31和141位，第引位与第141位的半眯氨酸将形成二硫键，对其发挥生物学活性不可缺少。在原核表达过程中，第17位半眺氨酸的存在会影响随后复性过程中蛋白质内二硫键的正确形成，从而降低产品生物学活性。因此我们将其突变为丝氨酸，表达一种新型的 17位丝氨酸的 IFN乃（’飞er侧小卜在本部分研究中，我们采用 RT—PCR方法从骨髓基质细胞中克隆了了人 UN’＊ 6的 cDNA，并将第 17位编码半耽氨酸序列突变为编码丝氨酸的序列，构建了高效原核表达载体、通过优化工程菌的发酵条件、建立‘七er DNLDNL D的稳定高效的原核表达体系，并摸索了一种适合于后续中试生产应用的重组蛋白质纯化工艺。 取体外培养的人骨髓基质细?
Interferons (IFNs) represent an important class of cytokines having a number of immunomodulatory, antiviral, and antiproliferative effects. IFNs are generally classified into three classes: IFN-α. IFN-β and IFN-γ, each representing protein molecules of different structure. The IFN cytokine family consists of type ⅠIIFNs (IFN-α and IFN-β) and type Ⅱ IFN (IFN-γ). Type ⅠIFNs were primarily considered as viral inhibitors, whereas type Ⅱ IFN, also termed “immune IFN”, was generally considered to be uniquely involved in immune reactions. Interferon beta is originally characterized from the supernatants of flbroblasts induced by virus infection. After extensive basic research , IFN-β is now found to be of great therapeutical value in clinic for virus infection, autoimmune disease, and malignant neoplasm. The native human IFN-β is a glycosylation protein, but it is difficult to get the native IFN-β for its complicated preparation and high cost, which restricts the use of IFN-β in clinical trial. With the development of genetic engineering technology, IFN-β has now been recombinantly expressed successfully in E. coil, yeast as well as CHO systems, and has been produced on large scale. Since then, recombinant IFN-β has been studied extensively on basic research and clinical trial. In 1993 IFN-β was proved by FDA for the treatment of multiple sclerosis. It is now used in daily clinical treatment of multiple sclerosis. And it is also used for treatment of chronic viral hepatitis, chronic granulomatous disease, and some malignant tumor. Interferons are also widely studied in our country, but most domestic studies were concentrated on IFN-α. Up to date, IFN-β have not been recombinantly produced yet because of its low expression level and complicated preparation. In the fist part of our present , we have successfully constructed a prokarocytic express vector for recombinant ~(17)Ser-IFN-β. The recombinant IFN- P was expressed at high level in E.coli and produced on large scale . Moreover, we have recently cloned a novel gene encoding interferon-like cytokine (WN-CK) from human dendritic cell cDNA library by large scale sequencing. Functional analysis of this novel gene is presented in part Ⅱ Part ⅠExpression and Purification of human recombinant IFN-β IFN beta is one of the first new therapeutic products resulting from recombinant DNA technology. Although it has been studied for many years, IFN- P are not proved by National Drug Administration to perform clinical trial in China just for its low amount of prokaryotic expression, the complicated preparation techniques including fermentation, refold and purification Human IFN-~3 comprises 165 amino acids and has three cysteine residues, located at amino acid positions 17, 31, and 141. The disu.lfide bond was formed between Cys-31 and Cys- 141, which was necessary for the activity of WN-~. The existence of Cys-17 could affect the formation of correct disulfide bond during protein renaturation. In this study, the codon for cysteine-17 was mutated to serine by the technique of site-specific mutagenesis with a synthetic oligonucleotide primer. A stable and high efficient expression system was established on the basis of the prokaryotic system. The total mRNA was extracted from the cultured human bone marrow stromal cells with polyAltract system. The eDNA was synthesized by reverse transcription with oligo-dT15 as the primer. IFN- P eDNA was amplified by PCR and 17-cysteine was mutated to serine. The
|
