肥胖疾病一直以来验证威胁着人类的生命健康。研究证实过重的体重与多种疾病的发病机制有关，包括高血压、高血脂、糖尿病以及某些类型的癌症。化学药物治疗肥胖往往存在着副作用强，导致患者难以承受以及导致营养缺乏等不良后果。近年来生物技术药物的迅猛发展，为使用生物技术药物治疗肥胖疾病带来契机。睫状神经营养因子（CNTF）是一种分子量约20kDa，由四个反相alpha-螺旋结构组成的蛋白质。其在体内的生理功能主要参与神经元细胞的生长以及受损神经元的修复等。近年来临床研究发现CNTF在治疗神经损伤修复过程中，其还具有抑制个体食物摄取的生理活性。CNTF抑制个人食欲的机制主要是通过作用于下丘脑部位的受体来实现。相比于另一种抑制食欲的分子瘦蛋白（leptin），CNTF对食物诱导型肥胖同样有抑制食欲的效果，而瘦蛋白却没有。而食物诱导型肥胖往往与人类的肥胖发病机理更为贴近。因此，CNTF蛋白在肥胖疾病治疗方面显示出极大的应用前景。随后研究发现，CNTF在体内血液中的代谢半衰期及其短暂。大鼠药代动力学研究显示CNTF的血液清除半衰期小于10分钟，极大的限制了CNTF在临床上的应用。因此开发长效型的CNTF是当前对CNTF用于肥胖疾病治疗研究的主要方向。聚乙二醇修饰是目前蛋白类药物最主要的长效策略，将高亲水性的聚乙二醇修饰与蛋白质的表面活泼官能团偶联后，偶合物相比于原蛋白具有更大的分子体积、更强的抗细胞、蛋白酶降解的能力，继而延长在血液中的保留半衰期。聚乙二醇修饰的方式目前已经开发出了多种修饰偶联方式。而蛋白质的聚乙二醇修饰的关键在于修饰方式的选择优化和修饰后的分离纯化。此外，转铁蛋白在体内具有较长的代谢半衰期，约10天。同时得益于血脑屏障上高表达的转铁蛋白受体，因此转铁蛋白能够参与脑部与血液中的物质交换过程。因此基于转铁蛋白的脑部药物递送研究是当前脑部靶向递药方面研究的主要热点。考虑到CNTF及其短暂的代谢半衰期，同时其发挥作用的区域存在于与脑部下丘脑，因此本研究的研究目的在于制备、表征和对比聚乙二醇修饰CNTF和转铁蛋白偶联CNTF对CNTF在体外活性、理化性质以及体内生物活性等影响。在本文中通过对CNTF进行聚乙二醇20k的修饰和通过PEG5k与转铁蛋白的偶联，偶联产物相对于CNTF原蛋白，表观分子体积以及水相中的分子粒径都显著增大，在生物活性和抗体亲和力方面都有不同程度的下降（细胞活性和抗体亲和力PEG20k-CNTF分别为 50.6%和3.8%， Tf-PEG5k-CNTF 为65.8%和89.9%）。药代动力学结果显示CNTF，PEG20k-CNTF与Tf-PEG5k-CNTF在SD大鼠体内的代谢半衰期分别为（0.25 ± 0.12，5.34 ± 0.26和8.65 ± 0.60小时）。小鼠实验显示耦合物相比于CNTF，抑制小鼠食物摄取的能力都显著提升。;Obesity has long been a threat to human health. Studies have shown that overweight weight is linked to the pathogenesis of many diseases, including high blood pressure, hyperlipidemia, diabetes and certain types of cancer. The treatment of obesity by chemical drugs is often associated with strong side effects, which can be difficult to bear and result in poor nutrition. In recent years, the rapid development of biotechnology drugs has brought opportunities for the use of biotechnology drugs to treat obesity.Ciliary neurotrophic factor (CNTF) is a molecular weight of about 20kDa, consisting of four anti-phase alpha-helical structures. Its physiological function in vivo is mainly involved in the growth of neurons and repair of damaged neurons. In recent years, clinical studies have found that CNTF can inhibit the physiological activity of individual food intake during the treatment of nerve injury. The mechanism by which CNTF inhibits individual appetite is mainly through the receptor that acts on the hypothalamus. Compared to leptin, another appetite-suppressing molecule, CNTF also has an appetite suppressant effect on food-induced obesity, while leptin does not. And food induced obesity is more closely related to the obesity pathogenesis of human. Therefore, CNTF protein shows great application prospect in the treatment of obesity disease. Subsequently, the study found that CNTF had a brief period of metabolic half-life in the blood in the body. The study of pharmacokinetics of rats showed that the half-life of the blood removal of CNTF was less than 10 minutes, which greatly restricted the clinical application of CNTF. Therefore, the development of long-term CNTF is the main direction for the research on the treatment of obesity.Polyethylene glycol (PEG) modification is one of the main protein drugs currently long-term strategy, to high hydrophilic polyethylene glycol modification and surface active functional groups of protein coupling, coupling material compared to the original protein has a larger molecular volume and stronger resistance to cells, the protease degradation ability, then extend the reserved half-life in blood. The method of polyethylene glycol modification has been developed in many ways. The key to the modification of polyethylene glycol in protein is the selection optimization of the modified mode and the purification after modification. In addition, transferrin has a longer metabolic half-life in the body, about 10 days. It also benefits from the high expression of transferritin receptors on the blood-brain barrier, so transferrin can participate in the exchange of substances in the brain and blood. Therefore, the research on the brain drug delivery based on transferrin is the main research topic in the current brain targeting drug delivery.Considering the metabolism of CNTF and its short half-life, at the same time, its work area exists in the hypothalamus and brain, so the preparation, characterization and the research purpose of this study is to compare the polyethylene glycol modified CNTF and transferrin coupling CNTF activity, physical and chemical properties of CNTF in vitro and in vivo biological activity, etc. In this article, through the study of the modification of PEG20 k of CNTF and through PEG5k and transferrin coupling, coupling product relative to the CNTF protein, apparent molecular volume and molecular size was significantly increased in water phase, in the aspect of biological activity and antibody affinity have varying degrees of decline (cell activity and antibody affinity PEG20k - CNTF were 50.6% and 3.8% respectively, Tf - PEG5k - CNTF is 65.8% and 89.9%). The pharmacokinetic results showed that CNTF, PEG20k - CNTF and tf-peg5k-cntf had a metabolic half-life in SD rats (0.25 + + 0.12, 5.34 + 0.26 and 8.65 + 0.60 hours) respectively. Mice showed significant improvement in their ability to suppress food intake compared to CNTF.