CN102558362A - A fusion protein for treating diabetes and its preparation method - Google Patents

Abstract

The invention relates to the technical field of diabetes treatment medicines, in particular to a fusion protein for treating diabetes and a preparation method thereof, wherein the fusion protein is composed of 2-8 Exendin-4-linkers and human IgGFc mutant weights; linker is a flexible peptide segment; the human IgGFc mutant is an IgG1Fc, IgG2Fc or IgG4Fc mutant; the invention also discloses a preparation method thereof, which is a recombinant fusion protein efficiently expressed in CHO cells and obtained by affinity, ion exchange and molecular sieve chromatography, has the biological activity that Exendin-4 can stimulate the secretion of insulin and inhibit the release of glucagon after meals, also has the characteristic of prolonging the half-life period of the recombinant fusion protein in serum, is used for treating type I and type II diabetes, effectively reduces the psychological and physiological burden of patients, is safe to use and strong in practicability, and can be produced and sold in a large scale.

Description

Translated fromChinese

一种用于治疗糖尿病的融合蛋白及其制备方法A fusion protein for treating diabetes and its preparation method

技术领域technical field

本发明涉及糖尿病治疗药物技术领域，特别涉及一种用于治疗糖尿病的融合蛋白及其制备方法。The invention relates to the technical field of drugs for treating diabetes, in particular to a fusion protein for treating diabetes and a preparation method thereof.

背景技术Background technique

糖尿病是一种严重的自身代谢紊乱性疾病，可引起严重的心血管功能紊乱，具有高发病率和高死亡率等特点，严重危害人类健康和生存质量，给病人和社会造成了沉重的经济负担。据流行病学调查统计，目前全球糖尿病患者总数已逾1.2亿，其中90%左右为Ⅱ型糖尿病，而此数字还将不断上涨，估计到2025年，世界范围将有3亿人患上糖尿病。因此，特效糖尿病药物的开发具有很高的社会效益和市场前景。Diabetes is a serious metabolic disorder of its own, which can cause serious cardiovascular dysfunction and has the characteristics of high morbidity and mortality, which seriously endangers human health and quality of life, and causes a heavy economic burden to patients and society. . According to epidemiological survey statistics, the total number of diabetic patients in the world has exceeded 120 million, of which about 90% aretype 2 diabetes, and this number will continue to rise. It is estimated that by 2025, 300 million people worldwide will suffer from diabetes. Therefore, the development of specific diabetes drugs has high social benefits and market prospects.

目前Ⅰ型糖尿病主要靠注射胰岛素来进行治疗，Ⅱ型糖尿病的治疗则以口服降糖药为主，包括磺脲素药物、双胍类药物、α-葡萄糖苷酶抑制剂、噻唑烷二酮衍生物类、以及注射用胰岛素等，这些药物的主要缺点是不良反应较大，长期使用会产生药物耐受，如胰岛素会引起低血糖的危险，其它口服药物会引起肝、肾等主要脏器损伤，而且最终病人会对药物产生耐受导致治疗失败，并且现在多是两种或三种药物联合用药，增加了患者的身体和经济负担。因此，利用新靶点和新治疗方法开发出更加安全、有效的糖尿病药物, 如不引起低血糖且能改善病人β细胞功能等，是糖尿病药物研究的热点。At present,type 1 diabetes is mainly treated by injecting insulin, while the treatment oftype 2 diabetes is mainly oral hypoglycemic drugs, including sulfonylurea drugs, biguanide drugs, α-glucosidase inhibitors, thiazolidinedione derivatives The main disadvantage of these drugs is that they have relatively large adverse reactions, and long-term use will cause drug tolerance. For example, insulin will cause the risk of hypoglycemia, and other oral drugs will cause damage to major organs such as liver and kidney. And eventually the patient will develop tolerance to the drug, leading to treatment failure, and now mostly two or three drugs are used in combination, which increases the physical and economic burden of the patient. Therefore, using new targets and new treatment methods to develop safer and more effective diabetes drugs, such as not causing hypoglycemia and improving the patient's β-cell function, is a hot spot in diabetes drug research.

目前，作为一种新机制及靶点，以肠促胰岛素为基础的新型糖尿病药物被越来越多的生物研究机构及制药公司关注。胰高血糖素样肽（incretin-Glucagon-like-peptide-1, GLP-1）是体内稳定胰岛素水平和胰高血糖素水平的关键调控因子之一。Exendin-4是从南美大毒蜥（Gila monster Heoderma suspectrum）口腔分泌物中分离出的一种含有39个氨基酸的多肽，与内源GLP-1具有53%的同源性，生物活性与内源性GLP-1相似。而且，由于Exendin-4第2位为甘氨酸，而不是GLP-1的丙氨酸，Exendin-4进入体内后不受二肽基肽酶（DPP-IV）分解的影响， GLP-1在体内半衰期仅1～2分钟，即被DPP-IV分解，相比于GLP-1，Exendin-4的药效时间更长。在糖尿病动物模型上，无论口服、舌下含服、肺部、气道还是鼻腔给予Exendin-4均有效。At present, as a new mechanism and target, incretin-based new diabetes drugs are attracting more and more attention from biological research institutions and pharmaceutical companies. Glucagon-like peptide (incretin-Glucagon-like-peptide-1, GLP-1) is one of the key regulators for stabilizing insulin and glucagon levels in the body. Exendin-4 is a polypeptide containing 39 amino acids isolated from the oral secretion of Gila monster Heoderma suspectrum. It has 53% homology with endogenous GLP-1, and its biological activity is similar to that of endogenous Sex GLP-1 similar. Moreover, since the second position of Exendin-4 is glycine instead of alanine of GLP-1, Exendin-4 will not be affected by the decomposition of dipeptidyl peptidase (DPP-IV) after entering the body, and the half-life of GLP-1 in vivo It is decomposed by DPP-IV in only 1 to 2 minutes. Compared with GLP-1, Exendin-4 has a longer drug effect time. In animal models of diabetes, Exendin-4 is effective regardless of oral, sublingual, pulmonary, airway or nasal administration.

GLP-1受体激动剂Exendin-4在治疗糖尿病方面具有独特的作用机制：它是以糖依赖方式调控血糖水平的，即高血糖时刺激胰岛素的分泌，而低血糖时不刺激胰岛素的分泌。因此，与其他糖尿病类药物相比具有很多优势：①可以显著降低糖化血红蛋白水平和控制血糖水平；②由于其调控功能依赖血糖水平，因此不会导致低血糖的发生；③增强饱食感，降低食物过量摄入及减肥；④改善β细胞功能；⑤控制餐后高血糖的发生。从目前临床数据反馈来看，不良反应轻微，大大提高了用药安全。该靶点研究较为突出的如：礼来公司的Byetta（合成Exendin-4）于2005年经FDA批准上市，诺和诺德公司的利拉鲁肽(Liraglutide，其为GLP-1模拟肽)于2010年1月经FDA批准上市。大量实验资料及临床数据已经证实，GLP-1及其类似物Exendin-4作为新型糖尿病治疗药物是安全、有效的。GLP-1 receptor agonist Exendin-4 has a unique mechanism of action in the treatment of diabetes: it regulates blood sugar levels in a glucose-dependent manner, that is, it stimulates the secretion of insulin when the blood sugar is high, but does not stimulate the secretion of insulin when the blood sugar is low. Therefore, compared with other diabetes drugs, it has many advantages: ①It can significantly reduce the level of glycosylated hemoglobin and control the blood sugar level; ②Because its regulation function depends on the blood sugar level, it will not cause hypoglycemia; ③Enhance the feeling of satiety, reduce Excessive food intake and weight loss; ④ Improve β-cell function; ⑤ Control the occurrence of postprandial hyperglycemia. Judging from the current clinical data feedback, the adverse reactions are mild, which greatly improves the safety of medication. The research on this target is more prominent, such as: Eli Lilly's Byetta (synthetic Exendin-4) was approved by the FDA in 2005, and Novo Nordisk's Liraglutide (Liraglutide, which is a GLP-1 mimic peptide) was launched in 2005. In January 2010, it was approved for marketing by FDA. A large number of experimental data and clinical data have confirmed that GLP-1 and its analogue Exendin-4 are safe and effective as a new type of diabetes treatment drug.

虽然，Exendin-4具有和GLP-1相同的生物学活性和优点（如前所述），且具有较长于GLP-1的半衰期，但其半衰期仍较短，病人需要每天注射两次，在实际使用中给病人带来不便。因此，基于GLP-1及GLP-1受体激动剂Exendin-4开发出更为长效的糖尿病药物是目前该领域研究的热点。Although Exendin-4 has the same biological activity and advantages as GLP-1 (as mentioned above), and has a longer half-life than GLP-1, its half-life is still short, and patients need to be injected twice a day. Inconvenience to the patient in use. Therefore, the development of longer-acting diabetes drugs based on GLP-1 and GLP-1 receptor agonist Exendin-4 is currently a research hotspot in this field.

药物长效化研究近年来被生物技术领域科学家广泛关注。人们通过各种方式来延长多肽药物的体内半衰期，常用方法如：生物可降解材料缓释制剂、氨基酸突变/修饰抗蛋白酶突变体、多肽PEG化修饰、重组融合蛋白如与人血清白蛋白融合、与免疫球蛋白Fc片断融合等。Drug long-acting research has been widely concerned by scientists in the field of biotechnology in recent years. People use various methods to prolong the in vivo half-life of polypeptide drugs, such as: biodegradable material sustained-release preparations, amino acid mutation/modification anti-protease mutants, polypeptide PEGylation, recombinant fusion proteins such as fusion with human serum albumin, Fusion with Fc fragment of immunoglobulin, etc.

IgG类免疫球蛋白（immunoglobulin G，IgG）是人体血液中最丰富的蛋白之一，其半衰期可达21天。现已证实，IgG分子在体内半衰期长与其Fc段和新生Fc受体（FcRn）以pH依赖的方式结合后再循环有关。目前，以与IgG分子Fc段融合来延长蛋白质药物半衰期已经有许多成功例子上市，如Nplate, Orencia, Enbrel, Arcalyst, Amevive等。我国SFDA也批准了一种Enbrel的类似药品（益赛普）用来治疗类风湿性关节炎及其相关疾病。这些都进一步证实了蛋白质与IgG分子Fc段融合表达是提高药物半衰期行之有效的方法。IgG class immunoglobulin (immunoglobulin G, IgG) is one of the most abundant proteins in human blood, and its half-life can reach 21 days. It has been confirmed that the long half-life of IgG molecules in vivo is related to its Fc segment and neonatal Fc receptor (FcRn) binding and recirculation in a pH-dependent manner. At present, there are many successful examples of prolonging the half-life of protein drugs by fusing with the Fc segment of IgG molecules, such as Nplate, Orencia, Enbrel, Arcalyst, Amevive, etc. my country's SFDA has also approved a similar drug to Enbrel (Yisaipu) for the treatment of rheumatoid arthritis and related diseases. All of these further confirm that the fusion expression of protein and Fc segment of IgG molecule is an effective method to improve the half-life of the drug.

IgG类免疫球蛋白主要分为四个亚型，分别为IgG1、IgG2、IgG3和IgG4，其中尤以IgG1型在人体内含量最高，其分子结构图如说明书附图图1所示。IgG immunoglobulins are mainly divided into four subtypes, namely IgG1, IgG2, IgG3 and IgG4, among which the IgG1 type has the highest content in the human body, and its molecular structure is shown in Figure 1 of the accompanying drawing.

现有技术中也有采用胰高血糖素类似肽GLP-1、GLP-1突变体或与其至少50%同源性的同功因子（Exendin-4）与IgG Fc的融合蛋白用于预防和治疗I型和Ⅱ型糖尿病。然而，人免疫球蛋白的Fc区域在体内具有重要的生物学活性，如（1）与细胞表面的Fc受体（FcγRs）结合，导致通过抗体依赖性细胞毒性作用（ADCC效应），来有效的杀伤病原体或者恶性细胞；（2）与第一补体成分C1q部分结合，引发补体依赖的细胞毒性作用（CDC效应）来有效杀伤病原体；（3）通过与新生Fc受体（FcRn）以pH依赖的方式结合后再循环从而获得长半衰期。作为药物融合模式来提高半衰期时，IgG Fc片段的生物学效应中ADCC效应和CDC效应是无用的，甚至是有害的，往往会造成不良反应的发生。另外，现有技术中GLP-1受体激动剂GLP-1或者Exendin-4与IgG的融合采用的是一个目的肽段与IgG融合，其比活性相对较低，有效剂量偏大，易造成不良反应风险提高，同时生产成本也较高。In the prior art, the fusion protein of glucagon-like peptide GLP-1, GLP-1 mutant or its isofunctional factor (Exendin-4) with at least 50% homology and IgG Fc is used for the prevention and treatment of I type andtype 2 diabetes. However, the Fc region of human immunoglobulins has important biological activities in vivo, such as (1) binding to Fc receptors (FcγRs) on the cell surface, resulting in effective cellular activity through antibody-dependent cellular cytotoxicity (ADCC effect). Kill pathogens or malignant cells; (2) combine with the first complement component C1q to trigger complement-dependent cytotoxicity (CDC effect) to effectively kill pathogens; (3) interact with newborn Fc receptors (FcRn) in a pH-dependent manner Recycling after binding in a manner to obtain a long half-life. When used as a drug fusion mode to increase the half-life, the ADCC effect and CDC effect in the biological effects of the IgG Fc fragment are useless, even harmful, and often cause adverse reactions. In addition, in the prior art, the fusion of GLP-1 receptor agonist GLP-1 or Exendin-4 with IgG uses a target peptide segment to be fused with IgG, its specific activity is relatively low, the effective dose is too large, and it is easy to cause adverse reactions. The reaction risk is increased, and the production cost is also higher.

发明内容Contents of the invention

本发明的目的是针对现有技术的不足而提供一种具有长效化特征的2-8个Exendin-4-Linker柔性肽段与人IgG Fc突变体重组成的用于治疗糖尿病的融合蛋白，其半衰期明显延长，依从性好，有效减少病人的生理和心理负担，使用安全且实用性强。The object of the present invention is to provide a fusion protein for treating diabetes composed of 2-8 Exendin-4-Linker flexible peptides with long-acting characteristics and human IgG Fc mutants for the deficiencies of the prior art. The half-life is significantly prolonged, the compliance is good, the physical and psychological burden of the patient is effectively reduced, and the use is safe and practical.

本发明的另一目的是针对现有技术的不足而提供一种具有长效化特征的2个Exendin-4-Linker柔性肽段与人IgG Fc突变体重组成的用于治疗糖尿病的融合蛋白的制备方法，其工艺成熟，有利于实现产业化生产和销售，制得的融合蛋白半衰期明显延长，依从性好，有效减少病人的生理和心理负担，使用安全且实用性强。Another object of the present invention is to provide a preparation of a fusion protein composed of two Exendin-4-Linker flexible peptides with long-acting characteristics and human IgG Fc mutants for the treatment of diabetes in view of the deficiencies in the prior art The method has a mature process, is conducive to realizing industrialized production and sales, and the half-life of the prepared fusion protein is obviously extended, the compliance is good, the physiological and psychological burden of patients can be effectively reduced, and the use is safe and practical.

为实现上述目的，本发明采用如下技术方案。In order to achieve the above object, the present invention adopts the following technical solutions.

提供一种用于治疗糖尿病的融合蛋白，所述融合蛋白为N个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白N(Exendin-4-Linker)- 人IgG Fc突变体；A fusion protein for treating diabetes is provided, the fusion protein is a fusion protein N (Exendin-4-Linker)-human IgG Fc mutant composed of N Exendin-4-Linker and human IgG Fc mutant;

其中，Linker为一组由疏水氨基酸组成的柔性肽段，其氨基酸的个数少于25个；Among them, Linker is a group of flexible peptides composed of hydrophobic amino acids, and the number of amino acids is less than 25;

其中，人IgG Fc突变体为IgG1 Fc、IgG2 Fc或IgG4 Fc突变体；Wherein, the human IgG Fc mutant is IgG1 Fc, IgG2 Fc or IgG4 Fc mutant;

其中，N为2-8个。Wherein, N is 2-8.

Exendin-4，其为执行治疗糖尿病的主要药效靶点，其生物活性较稳定，不易分解，通过和体内的GLP-1受体结合来促进胰岛素的释放，从而达到治疗糖尿病的目的。Exendin-4, which is the main pharmacological target for the treatment of diabetes, has relatively stable biological activity and is not easy to decompose. It promotes the release of insulin by binding to the GLP-1 receptor in the body, so as to achieve the purpose of treating diabetes.

Exendin-4的核酸序列如SEQ ID NO：1所示。Exendin-4的氨基酸序列如SEQ ID NO：2所示。The nucleic acid sequence of Exendin-4 is shown in SEQ ID NO:1. The amino acid sequence of Exendin-4 is shown in SEQ ID NO:2.

Linker（连接肽）为一组由疏水氨基酸组成的柔性肽段，其主要作用是将Exendin-4分子和IgG Fc突变体连接且更好地降低Exendin-4功能区和IgG Fc突变体功能区之间的空间位阻，避免由于直接连接造成的空间位阻效应而使Exendin-4的活性下降，从而最大程度地保证Exendin-4与受体结合而发挥其生物学活性。Linker (connecting peptide) is a group of flexible peptides composed of hydrophobic amino acids. Its main function is to connect Exendin-4 molecules and IgG Fc mutants and better reduce the gap between the Exendin-4 functional region and IgG Fc mutant functional region. The steric hindrance between them avoids the decrease of the activity of Exendin-4 due to the steric hindrance effect caused by the direct connection, so as to ensure the binding of Exendin-4 to the receptor to the greatest extent and exert its biological activity.

Linker，通俗地讲即为5-15个氨基酸（或者少于25个氨基酸）的柔性肽段，Linker的存在是为了防止各活性区域直接连接后，由于空间位阻效应而使各活性区不能很好地与相应受体结合，而造成的活性下降，因此Linker的存在理论上要比没有Linker更能很好地保留各活性区的活性功能，那么Linker的长短也是有一定限制的，该领域已经发表的文献普遍支持氨基酸长度为小于25个氨基酸范围内。Linker, in layman's terms, is a flexible peptide segment of 5-15 amino acids (or less than 25 amino acids). It binds to the corresponding receptors well, resulting in decreased activity. Therefore, the existence of Linker can theoretically preserve the activity function of each active region better than the absence of Linker. Then the length of Linker is also limited. This field has already Published literature generally supports amino acid lengths in the range of less than 25 amino acids.

IgG Fc突变体是本发明达到长效目的的主要功能区，其主要作用是通过Exendin-4与其连接后，增大多肽的分子量，从而减少肾小球的滤过作用；同时由于Fc段可以与人体内FcRn受体结合而有效地延长Exendin-4的半衰期，同时为了降低天然IgG Fc段带来的不必要的ADCC效应和CDC效应，本发明根据IgG亚型中裂解活性最低的（即ADCC效应和CDC效应最低）IgG2型Fc段的氨基酸序列进行了相应的突变。The IgG Fc mutant is the main functional region for the long-acting purpose of the present invention, and its main function is to increase the molecular weight of the polypeptide after being connected to it by Exendin-4, thereby reducing the filtration of the glomerulus; The binding of FcRn receptors in the human body effectively prolongs the half-life of Exendin-4. At the same time, in order to reduce the unnecessary ADCC effect and CDC effect brought by the natural IgG Fc segment, the present invention is based on the lowest cleavage activity in the IgG subtype (i.e. ADCC effect The amino acid sequence of the IgG2 type Fc segment was mutated accordingly.

本发明的作用机理为：Exendin-4与IgG Fc突变体融合可以有效提高多肽的分子量，避免了不必要的ADCC效应和CDC效应，同时IgG Fc段可以通过和体内的FcRn受体结合而延长其在体内的半衰期。The mechanism of action of the present invention is: the fusion of Exendin-4 and the IgG Fc mutant can effectively increase the molecular weight of the polypeptide, avoid unnecessary ADCC effect and CDC effect, and at the same time, the IgG Fc segment can be combined with the FcRn receptor in the body to prolong its molecular weight. half-life in vivo.

进一步地，所述融合蛋白为2个Exendin-4-Linker与人IgG1 Fc突变体重组成的融合蛋白。由于有2个Exendin-4-Linker分子，在同等摩尔浓度下，该融合蛋白表现出比单个Exendin-4-Linker更高的生物学活性。Further, the fusion protein is a fusion protein composed of two Exendin-4-Linker and human IgG1 Fc mutants. Since there are two Exendin-4-Linker molecules, the fusion protein exhibits higher biological activity than a single Exendin-4-Linker at the same molar concentration.

其中，由于IgG1是人体中含量最多，从安全的角度考虑，所述人IgG Fc突变体优选为IgG1 Fc突变体；IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236/Ala327Gly/Ala330Ser/Pro331Ser中的一个突变位置或者至少两个位置的组合突变位置， 其中突变位置编号依照EU编号系统(氨基酸残基编号是按Kabat等人所述的EU编号体系《SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST》第五版，United States Department of Health and Human Services,1991)。Among them, since IgG1 is the most abundant in the human body, from the perspective of safety, the human IgG Fc mutant is preferably an IgG1 Fc mutant; the amino acid mutation position of the IgG1 Fc mutant is Glu233Pro/Leu234Val/Leu235Ala/ΔGly236/Ala327Gly/ A mutation position in Ala330Ser/Pro331Ser or a combined mutation position of at least two positions, wherein the numbering of the mutation position is according to the EU numbering system (the amino acid residue numbering is according to the EU numbering system "SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST" described by Kabat et al. Fifth Edition, United States Department of Health and Human Services, 1991).

具体地，人IgG1 Fc突变体的突变位置可以是Glu233Pro/Leu234Val/Leu235Ala/ΔGly236 四个位置的组合突变位置，其能有效避免天然IgG Fc段带来的不必要的ADCC效应。人IgG1 Fc突变体的突变位置也可以是Ala327Gly/Ala330Ser/Pro331Ser三个位置的组合突变位置，其有效避免天然IgG Fc段带来的不必要的CDC效应。Specifically, the mutation position of the human IgG1 Fc mutant can be the combined mutation position of Glu233Pro/Leu234Val/Leu235Ala/ΔGly236 four positions, which can effectively avoid the unnecessary ADCC effect brought by the natural IgG Fc segment. The mutation position of the human IgG1 Fc mutant can also be the combined mutation position of the three positions of Ala327Gly/Ala330Ser/Pro331Ser, which can effectively avoid the unnecessary CDC effect brought by the natural IgG Fc segment.

进一步优选地，人IgG1 Fc突变体的突变位置是Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser七个位置的组合突变位置，其能有效避免天然IgG Fc段带来的不必要的ADCC效应和CDC效应。其IgG1 Fc突变体的核酸序列为如SEQ ID NO：3所示。Further preferably, the mutation position of the human IgG1 Fc mutant is Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; Ala327Gly/Ala330Ser/Pro331Ser The combined mutation position of the seven positions can effectively avoid the unnecessary ADCC effect brought by the natural IgG Fc segment and the CDC effect. The nucleic acid sequence of its IgG1 Fc mutant is shown in SEQ ID NO: 3.

氨基酸突变位置的密码子位置是按照和天然的IgG1 Fc的核酸序列对比，但是，由于本序列是经过密码子优化之后的序列，虽然每个密码子对应翻译出来的氨基酸还和天然的相同（突变位置除外），但是整体核苷酸已经优化为最适宜于在哺乳动物类细胞CHO中表达。那么按照本行业所熟知的技术，即使上述各个功能区的天然核酸序列或者通过不同方式适度优化密码子后的核酸序列，或者针对不同表达宿主如大肠杆菌、酵母、昆虫和其它哺乳动物细胞而进行的密码子优化，只要其对应的氨基酸未改变，都应视为和本发明相同，都属于本发明的保护范围。The codon position of the amino acid mutation position is compared with the natural IgG1 Fc nucleic acid sequence. However, since this sequence is a codon-optimized sequence, although each codon corresponds to the translated amino acid is still the same as the natural one (mutation position), but the overall nucleotides have been optimized for expression in mammalian cell-like CHO. Then, according to the well-known techniques in this industry, even if the natural nucleic acid sequences of the above-mentioned various functional regions or the nucleic acid sequences after codons are moderately optimized in different ways, or for different expression hosts such as Escherichia coli, yeast, insects and other mammalian cells. As long as the codon optimization of the corresponding amino acid is not changed, it should be regarded as the same as the present invention, and all belong to the protection scope of the present invention.

其IgG1 Fc突变体的氨基酸序列如SEQ ID NO：4所示。The amino acid sequence of its IgG1 Fc mutant is shown in SEQ ID NO: 4.

突变后的氨基酸及其位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser， 其中突变位置编号依照EU编号系统。The mutated amino acids and their positions are Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; Ala327Gly/Ala330Ser/Pro331Ser, where the numbering of the mutation positions follows the EU numbering system.

当然，人IgG Fc突变体的突变位置可以是上述七个位置中的任意一个位置或任意两个位置或任意两个位置以上结合的组合突变位置。Of course, the mutation position of the human IgG Fc mutant can be any one of the above-mentioned seven positions or any two positions or a combined mutation position of any two or more positions.

本发明的融合蛋白的氨基酸序列以4个Exendin-4-Linker分子来表示时，该融合蛋白为4（Exendin-4-Linker）-人IgG Fc突变体，当连接肽Linker分别为（Gly₄Ser）3和（Gly₄Ser）1时，且人IgG Fc突变体为IgG1 Fc突变体，其氨基酸序列如SEQ ID NO：5所示。When the amino acid sequence of the fusion protein of the present invention is represented by four Exendin-4-Linker molecules, the fusion protein is 4 (Exendin-4-Linker)-human IgG Fc mutant, when the connecting peptide Linker is (Gly₄ Ser ) 3 and (Gly₄ Ser) 1, and the human IgG Fc mutant is an IgG1 Fc mutant, the amino acid sequence of which is shown in SEQ ID NO:5.

本发明的融合蛋白的氨基酸序列以6个Exendin-4-Linker分子来表示时，该融合蛋白为6（Exendin-4-Linker）-人IgG Fc突变体，当连接肽Linker分别为（Gly₄Ser）3和（Gly₄Ser）1时，且人IgG Fc突变体为IgG1 Fc突变体，其氨基酸序列如SEQ ID NO：6所示。When the amino acid sequence of the fusion protein of the present invention is represented by 6 Exendin-4-Linker molecules, the fusion protein is 6 (Exendin-4-Linker)-human IgG Fc mutant, when the connecting peptide Linker is (Gly₄ Ser ) 3 and (Gly₄ Ser) 1, and the human IgG Fc mutant is an IgG1 Fc mutant, the amino acid sequence of which is shown in SEQ ID NO:6.

因此，可以按照上面的示例依此类推出本发明所保护的融合蛋白N（Exendin-4-Linker）-人IgG Fc突变体的结构式，N=2-8。Therefore, the structural formula of the fusion protein N (Exendin-4-Linker)-human IgG Fc mutant protected by the present invention can be deduced according to the above example, N=2-8.

其中，所述Linker为柔性肽段（Gly₄Ser）n，n=1-5或柔性肽段（Ala₃Ser₂）n，n=1-5。当然，本发明中采用的柔性肽段Linker还可以是本领域的其他常用的Linker。Wherein, the Linker is a flexible peptide (Gly₄ Ser) n, n=1-5 or a flexible peptide (Ala₃ Ser₂ ) n, n=1-5. Certainly, the flexible peptide segment Linker used in the present invention can also be other commonly used Linkers in the field.

Linker为柔性肽段Gly₄Ser时,其氨基酸序列如SEQ ID NO：7所示。When the Linker is a flexible peptide Gly₄ Ser, its amino acid sequence is shown in SEQ ID NO:7.

Linker为柔性肽段Ala₃Ser₂时，其氨基酸序列如SEQ ID NO：8所示。When the Linker is a flexible peptide Ala₃ Ser₂ , its amino acid sequence is shown in SEQ ID NO:8.

其中，所述融合蛋白为2个Exendin-4-Linker与人IgG1 Fc Fc突变体重组成的融合蛋白；Linker为柔性肽段（Gly₄Ser）n，n=1-5；IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置 ，其中突变位置编号依照EU编号系统。Wherein, the fusion protein is a fusion protein composed of two Exendin-4-Linkers and a human IgG1 Fc Fc mutant; the Linker is a flexible peptide (Gly₄ Ser) n, n=1-5; the amino acid of the IgG1 Fc mutant The mutation position of is the combined mutation position of Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; Ala327Gly/Ala330Ser/Pro331Ser, wherein the numbering of the mutation position is according to the EU numbering system.

通过Linker柔性多肽链将Exendin-4二聚体和IgG1 Fc突变体连接，最大程度保留了Exendin-4的生物学活性，同时最大程度降低了Fc段的裂解活性造成的不良反应。The Exendin-4 dimer and the IgG1 Fc mutant are connected through the Linker flexible polypeptide chain, which retains the biological activity of Exendin-4 to the greatest extent, and at the same time minimizes the adverse reactions caused by the cleavage activity of the Fc segment.

利用作用靶点明确的Exendin-4二聚体通过柔性肽Linker与人IgG Fc1Fc突变体连接，通过基因工程的方法高效表达该融合蛋白，用于Ⅰ型、Ⅱ型糖尿病治疗。通过IgG1 Fc突变体可以有效的延长药物半衰期的作用，避免不必要的ADCC效应和CDC效应带来的不良反应，提高病人的依从性，减少病人的给药次数，治疗效果好。The Exendin-4 dimer with a clear target is connected to the human IgG Fc1Fc mutant through a flexible peptide Linker, and the fusion protein is highly expressed by genetic engineering for the treatment of type I and type II diabetes. The IgG1 Fc mutant can effectively prolong the half-life of the drug, avoid unnecessary adverse reactions caused by the ADCC effect and CDC effect, improve the patient's compliance, reduce the number of drug administration of the patient, and have a good therapeutic effect.

其作用机理为：Exendin-4与IgG1 Fc突变体融合可以有效提高多肽的分子量，从而减少肾小球的滤过作用；同时IgG Fc段可以通过和体内的FcRn受体结合而延长其在体内的半衰期。Its mechanism of action is: the fusion of Exendin-4 and IgG1 Fc mutant can effectively increase the molecular weight of the polypeptide, thereby reducing the filtration of glomeruli; at the same time, the IgG Fc segment can prolong its life in vivo by binding to the FcRn receptor in vivo. half life.

IgG Fc段是执行延长该融合蛋白半衰期的重要活性区域，该片段可以与体内的FcRn受体结合通过再循环的方式来延长药物的半衰期，理论上天然的IgG Fc（四个亚型）均具有该功能，然而由于天然IgG Fc片段还具有ADCC效应和CDC效应（该效应通常在治疗性抗体上是关键的），所以本发明对天然的IgG Fc片段进行了突变，以降低其ADCC效应和CDC效应所带来的不良反应。另外，IgG1 Fc是目前使用最多的，研究也是最透彻的，而且它在体内的含量最高，所以在药物使用方面也是最安全的，因此本发明采用人IgG1 Fc进行突变。The IgG Fc segment is an important active region that prolongs the half-life of the fusion protein. This segment can bind to the FcRn receptor in the body to prolong the half-life of the drug through recycling. In theory, natural IgG Fc (four subtypes) have However, since natural IgG Fc fragments also have ADCC effects and CDC effects (this effect is usually critical on therapeutic antibodies), the present invention mutates natural IgG Fc fragments to reduce their ADCC effects and CDC Adverse reactions caused by effects. In addition, IgG1 Fc is currently the most used and most thoroughly studied, and it has the highest content in the body, so it is also the safest in terms of drug use. Therefore, the present invention uses human IgG1 Fc for mutation.

其中，所述融合蛋白为2个Exendin-4-Linker与人IgG1 Fc突变体重组成的融合蛋白；其中，两端分别与Exendin-4连接的Linker为柔性肽段（Gly₄Ser）n，n=3；其中，一端与Exendin-4连接且另一端与人IgG1 Fc突变体连接的Linker为柔性肽段（Gly₄Ser）n，n=1；其中，IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置，其中突变位置编号依照EU编号系统。Wherein, the fusion protein is a fusion protein composed of two Exendin-4-Linkers and a human IgG1 Fc mutant; wherein, the Linker connected to Exendin-4 at both ends is a flexible peptide (Gly₄ Ser) n, n= 3; Among them, the Linker whose one end is connected to Exendin-4 and the other end is connected to the human IgG1 Fc mutant is a flexible peptide (Gly₄ Ser) n, n=1; where the amino acid mutation position of the IgG1 Fc mutant is Glu233Pro The combined mutation positions of /Leu234Val/Leu235Ala/ΔGly236; Ala327Gly/Ala330Ser/Pro331Ser, where the mutation positions are numbered according to the EU numbering system.

该融合蛋白Exendin-4-Linker-Exendin-4-Linker-IgG1 Fc突变体的核苷酸序列如SEQ ID NO：9所示。The nucleotide sequence of the fusion protein Exendin-4-Linker-Exendin-4-Linker-IgG1 Fc mutant is shown in SEQ ID NO: 9.

该融合蛋白的氨基酸序列如SEQ ID NO：10所示。The amino acid sequence of the fusion protein is shown in SEQ ID NO: 10.

其中，所述融合蛋白的表达载体为pOptiVEC-TOPO，所述融合蛋白在哺乳动物细胞中表达，所述细胞选自CHO细胞、HeLa细胞、BHK细胞。pOptiVEC-TOPO TA cloning Kit （Invitrogen产品，catalog no.12744-017）。Wherein, the expression vector of the fusion protein is pOptiVEC-TOPO, and the fusion protein is expressed in mammalian cells, and the cells are selected from CHO cells, HeLa cells, and BHK cells. pOptiVEC-TOPO TA cloning Kit (product of Invitrogen, catalog no. 12744-017).

优选地，所述CHO细胞为CHO DG44细胞（Invitrogen产品，catalog no.12609-012）。Preferably, the CHO cells are CHO DG44 cells (Invitrogen product, catalog no. 12609-012).

其中，所述融合蛋白在治疗Ⅰ型、Ⅱ型糖尿病的应用。利用基因工程的方法制备了一种具有明显长效化特征的治疗Ⅰ型、Ⅱ型糖尿病的重组蛋白质药物，该药物能够有效地降低糖尿病人的血糖和糖化血红蛋白（HbA1c），同时明显延长其半衰期，拟定一周用药一次，比现有市场上的糖尿病药物（每天注射1-2次）具有更好的依从性，可以减少病人的心理和生理负担。同时该融合蛋白具有突变的Fc片段，能够最大程度降低天然IgG Fc所带来的ADCC效应和CDC效应，从而有效降低药物的不良反应。该发明与现有技术相比，具有更高的安全性和实用性。与其他糖尿病类药物相比具有很多优势：①可以显著降低糖基化血红蛋白水平和控制血糖水平；②由于其调控功能依赖血糖水平，因此不会导致低血糖风险；③增强饱食感，降低食物过量摄入及减肥；④改善β细胞功能；⑤控制餐后高血糖的发生。Wherein, the application of the fusion protein in the treatment of type I and type II diabetes. A recombinant protein drug with obvious long-acting characteristics for the treatment of type Ⅰ and type Ⅱ diabetes has been prepared by genetic engineering. The drug can effectively reduce blood sugar and glycosylated hemoglobin (HbA1c) in diabetic patients, and at the same time significantly prolong its half-life , it is planned to be administered once a week, which has better compliance than existing diabetes drugs on the market (injection 1-2 times a day), and can reduce the psychological and physical burden of patients. At the same time, the fusion protein has a mutated Fc fragment, which can minimize the ADCC effect and CDC effect brought by the natural IgG Fc, thereby effectively reducing the adverse reactions of the drug. Compared with the prior art, the invention has higher safety and practicability. Compared with other diabetes drugs, it has many advantages: ①It can significantly reduce the level of glycosylated hemoglobin and control the blood sugar level; ②Because its regulation function depends on the blood sugar level, it will not cause the risk of hypoglycemia; ③Enhance the feeling of satiety and reduce the risk of food Excessive intake and weight loss; ④ Improve β-cell function; ⑤ Control the occurrence of postprandial hyperglycemia.

为实现上述另一目的，本发明采用如下技术方案。In order to achieve the above another purpose, the present invention adopts the following technical solutions.

提供一种用于治疗糖尿病的融合蛋白的制备方法，该方法包括在适于表达该融合蛋白的条件下培养权利要求6的宿主细胞，宿主细胞经细胞培养、离心后，上清液分别用亲和层析、离子交换层析和分子筛层析纯化；其中，亲和层析中使用的是rProtein A Sepharose FF亲和填料；其中，离子交换层析中使用的是Q Sepharose FF填料；其中，分子筛层析中使用的是Superdex 200填料。A method for preparing a fusion protein for treating diabetes is provided, the method comprising culturing the host cell according toclaim 6 under conditions suitable for expressing the fusion protein, after the host cell is cultured and centrifuged, the supernatant is respectively used with And chromatography, ion exchange chromatography and molecular sieve chromatography purification; Among them, rProtein A Sepharose FF affinity filler is used in affinity chromatography; Among them, Q Sepharose FF filler is used in ion exchange chromatography; Among them,molecular sieve Superdex 200 packing material was used in the chromatography.

具体地，该融合蛋白的制备方法包括以下步骤：Specifically, the preparation method of the fusion protein comprises the following steps:

步骤（1）目的基因的获得：将Exendin-4的DNA序列、Linker的DNA序列和IgG1 Fc突变体的DNA序列采用全基因合成的方式获得目的基因DiExendin-4-Ig；Step (1) Obtaining the target gene: the DNA sequence of Exendin-4, the DNA sequence of Linker and the DNA sequence of the IgG1 Fc mutant were obtained by whole gene synthesis to obtain the target gene DiExendin-4-Ig;

步骤（2）克隆到合适的表达载体上：目的基因DiExendin-4-Ig合成后，经PCR扩增后将其克隆入表达载体pOptiVEC-TOPO中；Step (2) Cloning into an appropriate expression vector: After the target gene DiExendin-4-Ig is synthesized, it is amplified by PCR and cloned into the expression vector pOptiVEC-TOPO;

步骤（3）转染合适的宿主细胞，进行表达：宿主细胞为CHO DG44细胞，待PCR、双酶切和测序鉴定正确后，将正确的克隆大量扩增并进行线性化以进行CHO DG44细胞转染；Step (3) Transfect appropriate host cells for expression: the host cells are CHO DG44 cells. After PCR, double enzyme digestion and sequencing are identified correctly, a large number of correct clones are amplified and linearized for CHO DG44 cell transfection. dye;

步骤（4）分离纯化目的蛋白：依次通过表达上清液预处理、亲和层析、离子交换层析和分子筛层析相结合的纯化工艺，经SDS-PAGE和HPLC检测，其纯度大于98%。 Step (4) Separation and purification of the target protein: through the purification process of expression supernatant pretreatment, affinity chromatography, ion exchange chromatography and molecular sieve chromatography, the purity is greater than 98% as detected by SDS-PAGE and HPLC . the

本发明中采用的技术手段均参照《分子克隆实验指南》和相关产品的《操作手册》，试剂均采用该领域所认可的生物试剂公司，如NEB、invitrogen、上海生工等。本发明中Exendin-4的DNA序列、Linker的DNA序列和IgG1 Fc突变体的DNA序列均采用全基因合成的方式获得，并进行了密码子优化以利于在宿主细胞CHO DG44细胞中高效表达。The technical means adopted in the present invention refer to the "Molecular Cloning Experiment Guide" and the "Operation Manual" of related products, and the reagents are all approved biological reagent companies in this field, such as NEB, Invitrogen, Shanghai Sangong, etc. In the present invention, the DNA sequence of Exendin-4, the DNA sequence of Linker and the DNA sequence of IgG1 Fc mutant are all obtained by whole gene synthesis, and codon optimization is carried out to facilitate high-efficiency expression in host cell CHO DG44 cells.

本发明的有益效果为：The beneficial effects of the present invention are:

本发明的一种用于治疗糖尿病的融合蛋白，融合蛋白为N个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白；Linker为一组由疏水氨基酸组成的柔性肽段，其氨基酸的个数少于25个；人IgG Fc突变体为IgG1 Fc、IgG2 Fc或IgG4 Fc突变体；N为2-8个。通过Linker柔性多肽链将Exendin-4和IgG1 Fc突变体连接，最大程度保留了Exendin-4的生物学活性，其不仅具有Exendin-4可刺激胰岛素的分泌、抑制餐后胰高血糖素的释放的生物活性，同时最大程度降低了Fc段的裂解活性造成的不良反应，具有延长其在血清中半衰期的特点，用于Ⅰ型和Ⅱ型糖尿病的治疗，病人可以一周用药一次，依从性好，有效减少病人的心理和生理负担，使用安全且实用性强。A fusion protein for treating diabetes of the present invention, the fusion protein is a fusion protein composed of N Exendin-4-Linkers and human IgG Fc mutants; Linker is a group of flexible peptides composed of hydrophobic amino acids, the amino acid The number is less than 25; human IgG Fc mutants are IgG1 Fc, IgG2 Fc or IgG4 Fc mutants; N is 2-8. Linking Exendin-4 and IgG1 Fc mutants through Linker flexible polypeptide chains retains the biological activity of Exendin-4 to the greatest extent, and not only has Exendin-4 stimulate insulin secretion and inhibit postprandial glucagon release Biological activity, while minimizing the adverse reactions caused by the cleavage activity of the Fc segment, it has the characteristics of prolonging its half-life in serum. It is used for the treatment of type Ⅰ and type Ⅱ diabetes. Patients can take medicine once a week, with good compliance and effective Reduce the psychological and physical burden of patients, safe and practical to use.

本发明的用于治疗糖尿病的融合蛋白的制备方法，该方法包括在适于表达该融合蛋白的条件下培养权利要求6的宿主细胞，宿主细胞经细胞培养、离心后，上清液分别用亲和层析、离子交换层析和分子筛层析纯化；其中，亲和层析中使用的是rProtein A Sepharose FF亲和填料；其中，离子交换层析中使用的是Q Sepharose FF填料；其中，分子筛层析中使用的是Superdex 200填料。The preparation method of the fusion protein for treating diabetes of the present invention, the method comprises culturing the host cell ofclaim 6 under conditions suitable for expressing the fusion protein, after the host cell is cultured and centrifuged, the supernatant is respectively washed with the And chromatography, ion exchange chromatography and molecular sieve chromatography purification; Among them, rProtein A Sepharose FF affinity filler is used in affinity chromatography; Among them, Q Sepharose FF filler is used in ion exchange chromatography; Among them,molecular sieve Superdex 200 packing material was used in the chromatography.

本发明的工艺成熟，有利于实现产业化生产和销售，制得的融合蛋白不仅具有Exendin-4可刺激胰岛素的分泌、抑制餐后胰高血糖素的释放的生物活性，还具有延长其在血清中半衰期的特点，用于Ⅰ型和Ⅱ型糖尿病的治疗，病人可以一周用药一次，依从性好，有效减少病人的心理和生理负担，使用安全且实用性强。The process of the present invention is mature, which is conducive to the realization of industrial production and sales. The fusion protein not only has the biological activity of Exendin-4, which can stimulate the secretion of insulin and inhibit the release of postprandial glucagon, but also has the function of prolonging its release in serum. The medium half-life is used for the treatment of type Ⅰ and type Ⅱ diabetes. Patients can take medicine once a week, with good compliance, effectively reducing the psychological and physical burden of patients, and it is safe and practical to use.

本发明的优点在于：（1）本发明对融合的IgG Fc段基因进行突变修饰从而降低ADCC效应、CDC效应，降低其裂解活性，有效避免天然IgG Fc带来的不良反应。提高病人的依从性，减少病人给药次数。The advantages of the present invention are: (1) The present invention mutates and modifies the fused IgG Fc segment gene to reduce the ADCC effect, CDC effect, reduce its cleavage activity, and effectively avoid adverse reactions caused by natural IgG Fc. Improve patient compliance and reduce patient dosing times.

（2）本发明利用作用靶点明确机制清楚的多聚Exendin-4通过柔性肽Linker与人IgG Fc突变体连接，通过基因工程的方法高效表达该融合蛋白，用于Ⅰ型、Ⅱ型糖尿病治疗。最大程度保留了Exendin-4的生物学活性，其不仅具有Exendin-4可刺激胰岛素的分泌、抑制餐后胰高血糖素的释放的生物活性，还具有延长其在血清中半衰期的特点，用于Ⅰ型和Ⅱ型糖尿病的治疗。(2) In the present invention, poly-Exendin-4 with a clear target and clear mechanism is connected to human IgG Fc mutants through a flexible peptide Linker, and the fusion protein is efficiently expressed by genetic engineering methods for the treatment of type I and type II diabetes . The biological activity of Exendin-4 is retained to the greatest extent. It not only has the biological activity that Exendin-4 can stimulate the secretion of insulin and inhibit the release of glucagon after meals, but also has the characteristics of prolonging its half-life in serum. It is used for Treatment oftype 1 andtype 2 diabetes.

（3）本发明创造了一种针对糖尿病治疗的具有明显长效化特征的基因工程重组蛋白药物，该药物能够有效地降低糖尿病人的血糖和糖化血红蛋白（HbA1c），同时具有明显延长的半衰期，拟定一周用药一次，比现有市场上的糖尿病药物（每天注射1-2次）具有更好的依从性，可以减少病人的心理和生理负担。(3) The present invention has created a genetically engineered recombinant protein drug with obvious long-acting characteristics for the treatment of diabetes. The drug can effectively reduce blood sugar and glycosylated hemoglobin (HbA1c) of diabetic patients, and has a significantly extended half-life. It is planned to take medication once a week, which has better compliance than existing diabetes drugs on the market (1-2 injections per day), and can reduce the psychological and physical burden of patients.

（4）本发明的用于治疗糖尿病的融合蛋白的制备方法，工艺成熟，原料来源广泛，有利于实现规模化生产和销售，具有广泛的社会价值，经济价值高，生产安全且实用性强。(4) The preparation method of the fusion protein for treating diabetes of the present invention has a mature process and a wide range of sources of raw materials, which is conducive to the realization of large-scale production and sales, has extensive social value, high economic value, safe production and strong practicability.

（5）本发明制得的用于治疗糖尿病的融合蛋白具有：①可以显著降低糖基化血红蛋白水平和控制血糖水平；②由于其调控功能依赖血糖水平，因此不会导致低血糖的危险；③增强饱食感，降低食物过量摄入及减肥；④改善β细胞功能；⑤控制餐后高血糖的发生。(5) The fusion protein prepared by the present invention for the treatment of diabetes has: ① can significantly reduce the level of glycosylated hemoglobin and control the blood sugar level; ② since its regulation function depends on the blood sugar level, it will not cause the risk of hypoglycemia; ③ Enhance satiety, reduce excessive food intake and lose weight; ④ improve β-cell function; ⑤ control the occurrence of postprandial hyperglycemia.

附图说明Description of drawings

图1是IgG分子的结构和主要功能区图。Figure 1 is a diagram of the structure and main functional regions of an IgG molecule.

图2是本发明一种用于治疗糖尿病的融合蛋白的四种融合蛋白的结构简图。Fig. 2 is a simplified structure diagram of four fusion proteins of a fusion protein used for treating diabetes in the present invention.

图3是本发明一种用于治疗糖尿病的融合蛋白的pOptiVEC-DiExendin-4-Ig质粒PCR验证产物电泳图，其中， 1：DL2000 Marker；2：阴性对照（未加质粒）；3-10分别是编码1-8质粒的PCR结果。Fig. 3 is the pOptiVEC-DiExendin-4-Ig plasmid PCR verification product electrophoresis figure of a kind of fusion protein that is used for the treatment of diabetes of the present invention, wherein, 1: DL2000 Marker; 2: Negative control (without adding plasmid); 3-10 respectively are PCR results of plasmids encoding 1-8.

图4是本发明一种用于治疗糖尿病的融合蛋白的质粒pOptiVEC-DiExendin-4-Ig双酶切电泳结果图，其中，1：DL2000 Marker；2：1kbp ladder marker；3：pOptiVEC-DiExendin-4-Ig未酶切质粒；4-11分别是编号为1-8质粒双酶切结果。Fig. 4 is a double-enzyme electrophoresis result of plasmid pOptiVEC-DiExendin-4-Ig of a fusion protein for treating diabetes of the present invention, wherein, 1: DL2000 Marker; 2: 1kbp ladder marker; 3: pOptiVEC-DiExendin-4 -Ig undigested plasmid; 4-11 are the results of double digestion of plasmids numbered 1-8 respectively.

图5是本发明一种用于治疗糖尿病的融合蛋白的重组DiExendin-4-Ig融合蛋白HPLC纯度分析图。Fig. 5 is an HPLC purity analysis chart of a recombinant DiExendin-4-Ig fusion protein used for treating diabetes in the present invention.

图6是本发明一种用于治疗糖尿病的融合蛋白的重组DiExendin-4-Ig融合蛋白SDS-PAGE分析图，从左至右依次为蛋白MARKER、还原电泳和非还原电泳。Fig. 6 is an SDS-PAGE analysis diagram of a recombinant DiExendin-4-Ig fusion protein of a fusion protein used for treating diabetes according to the present invention, from left to right are protein MARKER, reducing electrophoresis and non-reducing electrophoresis.

图7是本发明一种用于治疗糖尿病的融合蛋白的重组DiExendin-4-Ig融合蛋白刺激RIN-5F细胞cAMP生成图。Fig. 7 is a diagram showing that a recombinant DiExendin-4-Ig fusion protein of the present invention, a fusion protein used for treating diabetes, stimulates cAMP production in RIN-5F cells.

图8是本发明一种用于治疗糖尿病的融合蛋白的融合蛋白DiExendin-4-Ig对正常ICR小鼠血糖曲线下面积的第1天的影响。Fig. 8 shows the influence of the fusion protein DiExendin-4-Ig, a fusion protein used for treating diabetes, on the first day of the area under the curve of blood glucose in normal ICR mice.

图9是本发明一种用于治疗糖尿病的融合蛋白的融合蛋白DiExendin-4-Ig对正常ICR小鼠血糖曲线下面积的第2天的影响。Fig. 9 shows the influence of the fusion protein DiExendin-4-Ig, a fusion protein used for treating diabetes, on the area under the curve of blood glucose in normal ICR mice on the second day.

图10是本发明一种用于治疗糖尿病的融合蛋白的融合蛋白DiExendin-4-Ig对正常ICR小鼠血糖曲线下面积的第3天的影响。Fig. 10 shows the influence of the fusion protein DiExendin-4-Ig, a fusion protein used for treating diabetes, on the area under the curve of blood glucose in normal ICR mice on the third day.

图11是本发明一种用于治疗糖尿病的融合蛋白的融合蛋白DiExendin-4-Ig对正常ICR小鼠血糖曲线下面积的第4-6天的影响。Fig. 11 shows the influence of the fusion protein DiExendin-4-Ig, a fusion protein used for treating diabetes, on the area under the curve of blood glucose in normal ICR mice on day 4-6.

图12是本发明一种用于治疗糖尿病的融合蛋白的融合蛋白DiExendin-4-Ig对正常ICR小鼠血糖曲线下面积的第7-9天的影响。Fig. 12 shows the influence of the fusion protein DiExendin-4-Ig, a fusion protein used for treating diabetes, on the area under the curve of blood glucose in normal ICR mice on days 7-9.

图13是本发明一种用于治疗糖尿病的融合蛋白的融合蛋白DiExendin-4-Ig对正常ICR小鼠血糖曲线下面积的第10-14天的影响。Fig. 13 shows the influence of the fusion protein DiExendin-4-Ig, a fusion protein used for treating diabetes, on the area under the curve of blood glucose in normal ICR mice at day 10-14.

图14是本发明一种用于治疗糖尿病的融合蛋白的融合蛋白DiExendin-4-Ig对正常ICR小鼠血糖曲线下面积的第16-20天的影响。Fig. 14 shows the effect of the fusion protein DiExendin-4-Ig, a fusion protein used for treating diabetes, on the area under the curve of blood glucose in normal ICR mice on days 16-20.

具体实施方式Detailed ways

下面结合附图和实施例对本发明作进一步的说明。The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

实施例1Example 1

如图2所示，本实施例中，用于治疗糖尿病的融合蛋白为2个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白；Linker柔性肽段为（Gly₄Ser）n，n=1-5；人IgG Fc突变体为IgG1 Fc突变体，IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置， 其中突变位置编号依照EU索引。As shown in Figure 2, in this example, the fusion protein used to treat diabetes is a fusion protein composed of two Exendin-4-Linker and human IgG Fc mutants; the Linker flexible peptide is (Gly₄ Ser)n,n =1-5; Human IgG Fc mutants are IgG1 Fc mutants, and the amino acid mutation positions of IgG1 Fc mutants are Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; combined mutation positions of Ala327Gly/Ala330Ser/Pro331Ser, where the mutation positions are numbered according to EU index.

其中，两端分别与Exendin-4连接的Linker为柔性肽段（Gly₄Ser）n，n=3；Among them, the Linker connected to Exendin-4 at both ends is a flexible peptide (Gly₄ Ser) n, n=3;

其中，一端与Exendin-4连接且另一端与人IgG1 Fc Fc突变体连接的Linker为柔性肽段（Gly₄Ser）n，n=1。Among them, the Linker whose one end is connected to Exendin-4 and the other end is connected to the human IgG1 Fc Fc mutant is a flexible peptide (Gly₄ Ser) n, n=1.

实施例2Example 2

 如图2所示，本实施例中，用于治疗糖尿病的融合蛋白为2个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白；Linker柔性肽段为（Gly₄Ser）n，n=1-5；人IgG Fc突变体为IgG1突变体，IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置， 其中突变位置编号依照EU索引。As shown in Figure 2, in this example, the fusion protein used to treat diabetes is a fusion protein composed of two Exendin-4-Linker and human IgG Fc mutants; the Linker flexible peptide is (Gly₄ Ser)n,n =1-5; Human IgG Fc mutants are IgG1 mutants, and the amino acid mutation positions of IgG1 Fc mutants are Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; combined mutation positions of Ala327Gly/Ala330Ser/Pro331Ser, where the mutation positions are numbered according to the EU index .

其中，两端分别与Exendin-4连接的Linker为柔性肽段（Gly₄Ser）n，n=4；Among them, the Linker connected to Exendin-4 at both ends is a flexible peptide (Gly₄ Ser) n, n=4;

其中，一端与Exendin-4连接且另一端与人IgG1 Fc突变体连接的Linker为柔性肽段（Gly₄Ser）n，n=2。Among them, the Linker whose one end is connected to Exendin-4 and the other end is connected to the human IgG1 Fc mutant is a flexible peptide segment (Gly₄ Ser) n, n=2.

实施例3Example 3

 如图2所示，本实施例中，用于治疗糖尿病的融合蛋白为2个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白；Linker柔性肽段为（Gly4Ser）n，n=1-5；人IgG Fc突变体为IgG1突变体，IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置， 其中突变位置编号依照EU索引。As shown in Figure 2, in this example, the fusion protein used to treat diabetes is a fusion protein composed of two Exendin-4-Linker and human IgG Fc mutants; the Linker flexible peptide is (Gly4Ser)n, n=1 -5; the human IgG Fc mutant is an IgG1 mutant, and the amino acid mutation position of the IgG1 Fc mutant is the combined mutation position of Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; Ala327Gly/Ala330Ser/Pro331Ser, wherein the mutation position numbering is according to the EU index.

其中，两端分别与Exendin-4连接的Linker为柔性肽段（Gly₄Ser）n，n=3；Among them, the Linker connected to Exendin-4 at both ends is a flexible peptide (Gly₄ Ser) n, n=3;

其中，一端与Exendin-4连接且另一端与人IgG1 Fc突变体连接的Linker为柔性肽段（Gly₄Ser）n，n=3。Among them, the Linker whose one end is connected to Exendin-4 and the other end is connected to the human IgG1 Fc mutant is a flexible peptide (Gly₄ Ser) n, n=3.

实施例4Example 4

 如图2所示，本实施例中，用于治疗糖尿病的融合蛋白为2个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白；Linker柔性肽段为（Gly₄Ser）n，n=1-5；人IgG Fc突变体为IgG1突变体，IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置， 其中突变位置编号依照EU索引。As shown in Figure 2, in this example, the fusion protein used to treat diabetes is a fusion protein composed of two Exendin-4-Linker and human IgG Fc mutants; the Linker flexible peptide is (Gly₄ Ser)n,n =1-5; Human IgG Fc mutants are IgG1 mutants, and the amino acid mutation positions of IgG1 Fc mutants are Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; combined mutation positions of Ala327Gly/Ala330Ser/Pro331Ser, where the mutation positions are numbered according to the EU index .

其中，两端分别与Exendin-4连接的Linker为柔性肽段（Gly₄Ser）n，n=5；Among them, the Linker connected to Exendin-4 at both ends is a flexible peptide (Gly₄ Ser) n, n=5;

其中，一端与Exendin-4连接且另一端与人IgG1 Fc突变体连接的Linker为柔性肽段（Gly₄Ser）n，n=3。Among them, the Linker whose one end is connected to Exendin-4 and the other end is connected to the human IgG1 Fc mutant is a flexible peptide (Gly₄ Ser) n, n=3.

实施例5Example 5

本实施例中，用于治疗糖尿病的融合蛋白为2个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白；Linker柔性肽段为（Gly₄Ser）n，n=1-5；人IgG Fc突变体为IgG1突变体，IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置， 其中突变位置编号依照EU索引。In this example, the fusion protein used to treat diabetes is a fusion protein composed of two Exendin-4-Linker and human IgG Fc mutants; the Linker flexible peptide is (Gly₄ Ser) n, n=1-5; human The IgG Fc mutant is an IgG1 mutant, and the amino acid mutation position of the IgG1 Fc mutant is the combined mutation position of Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; Ala327Gly/Ala330Ser/Pro331Ser, wherein the numbering of the mutation position is according to the EU index.

实施例6Example 6

本实施例中，用于治疗糖尿病的融合蛋白为2个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白；Linker柔性肽段为（Ala₃Ser₂）n，n=1-5；人IgG Fc突变体为IgG1突变体，IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置， 其中突变位置编号依照EU编号系统。In this example, the fusion protein used to treat diabetes is a fusion protein composed of two Exendin-4-Linkers and human IgG Fc mutants; the Linker flexible peptide is (Ala₃ Ser₂ ) n, n=1-5; Human IgG Fc mutants are IgG1 mutants, and the amino acid mutation positions of IgG1 Fc mutants are the combined mutation positions of Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; Ala327Gly/Ala330Ser/Pro331Ser, wherein the mutation positions are numbered according to the EU numbering system.

实施例7Example 7

本实施例中，用于治疗糖尿病的融合蛋白为4个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白；Linker柔性肽段为（Gly₄Ser）n，n=1-5；人IgG Fc突变体为IgG1突变体，IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置， 其中突变位置编号依照EU索引。In this example, the fusion protein used to treat diabetes is a fusion protein composed of four Exendin-4-Linkers and human IgG Fc mutants; the Linker flexible peptide is (Gly₄ Ser) n, n=1-5; human The IgG Fc mutant is an IgG1 mutant, and the amino acid mutation position of the IgG1 Fc mutant is the combined mutation position of Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; Ala327Gly/Ala330Ser/Pro331Ser, wherein the numbering of the mutation position is according to the EU index.

实施例8Example 8

本实施例中，用于治疗糖尿病的融合蛋白为8个Exendin-4-Linker与人IgG Fc突变体重组成的融合蛋白；Linker柔性肽段为（Gly₄Ser）n，n=1-5；人IgG Fc突变体为IgG1突变体，IgG1 Fc突变体的氨基酸的突变位置为Glu233Pro/Leu234Val/Leu235Ala/ΔGly236；Ala327Gly/Ala330Ser/Pro331Ser的组合突变位置， 其中突变位置编号依照EU索引。In this example, the fusion protein used to treat diabetes is a fusion protein composed of 8 Exendin-4-Linker and human IgG Fc mutants; the Linker flexible peptide is (Gly₄ Ser) n, n=1-5; human The IgG Fc mutant is an IgG1 mutant, and the amino acid mutation position of the IgG1 Fc mutant is the combined mutation position of Glu233Pro/Leu234Val/Leu235Ala/ΔGly236; Ala327Gly/Ala330Ser/Pro331Ser, wherein the numbering of the mutation position is according to the EU index.

实施例9Example 9

一种用于治疗糖尿病的融合蛋白的制备方法，该方法包括在适于表达该融合蛋白的条件下培养权利要求6的宿主细胞，宿主细胞经细胞培养、离心后，上清液分别用亲和层析、离子交换层析和分子筛层析纯化；其中，分子筛层析中使用的是Superdex 200填料。其中，亲和层析中使用的是rProtein A Sepharose FF亲和填料；其中，离子交换层析中使用的是Q Sepharose FF填料。A method for preparing a fusion protein for treating diabetes, the method comprising culturing the host cell ofclaim 6 under conditions suitable for expressing the fusion protein, after the host cell is cultured and centrifuged, the supernatant is respectively used with affinity Purification by chromatography, ion exchange chromatography and molecular sieve chromatography; among them,Superdex 200 filler is used in molecular sieve chromatography. Among them, rProtein A Sepharose FF affinity packing is used in affinity chromatography; among them, Q Sepharose FF packing is used in ion exchange chromatography.

具体地，该融合蛋白的制备方法包括以下步骤：Specifically, the preparation method of the fusion protein comprises the following steps:

步骤（1）目的基因的获得：将Exendin-4的DNA序列、Linker的DNA序列和IgG1 Fc突变体的DNA序列采用全基因合成的方式获得目的基因DiExendin-4-Ig；Step (1) Obtaining the target gene: the DNA sequence of Exendin-4, the DNA sequence of Linker and the DNA sequence of the IgG1 Fc mutant were obtained by whole gene synthesis to obtain the target gene DiExendin-4-Ig;

步骤（2）克隆到合适的表达载体上：目的基因DiExendin-4-Ig合成后经PCR扩增后，将其克隆入表达载体pOptiVEC-TOPO中；Step (2) Cloning into an appropriate expression vector: After the target gene DiExendin-4-Ig is synthesized and amplified by PCR, it is cloned into the expression vector pOptiVEC-TOPO;

步骤（3）转染入合适的宿主细胞中进行表达：宿主细胞为CHO DG44细胞，待PCR、双酶切和测序鉴定正确后，将正确的克隆大量扩增并进行线性化以进行CHO DG44细胞细胞转染；Step (3) Transfection into a suitable host cell for expression: the host cell is CHO DG44 cells. After PCR, double enzyme digestion and sequencing are identified correctly, the correct clones are amplified in large quantities and linearized for CHO DG44 cells cell transfection;

步骤（4）分离纯化目的蛋白：依次通过表达上清液预处理、亲和层析、离子交换层析和分子筛层析相结合的纯化工艺，经SDS-PAGE和HPLC检测，其纯度大于98%。Step (4) Separation and purification of the target protein: through the purification process of expression supernatant pretreatment, affinity chromatography, ion exchange chromatography and molecular sieve chromatography, the purity is greater than 98% as detected by SDS-PAGE and HPLC .

实验例1 重组DiExendin-4-Ig融合蛋白的设计及表达载体pOptiVEC-DiExendin-4-Ig的构建Experimental example 1 Design of recombinant DiExendin-4-Ig fusion protein and construction of expression vector pOptiVEC-DiExendin-4-Ig

1.重组DiExendin-4-Ig融合蛋白的设计。1. Design of recombinant DiExendin-4-Ig fusion protein.

本发明根据哺乳动物细胞使用密码子的偏爱性，已知Exendin-4片段和突变后人IgG1 Fc片段的氨基酸序列，设计了一段新的重组DiExendin-4-Ig融合蛋白，该融合蛋白的核苷酸序列如SEQ ID NO：11所示。The present invention designs a new recombinant DiExendin-4-Ig fusion protein according to the preference of codons used by mammalian cells and known the amino acid sequences of Exendin-4 fragment and mutated human IgG1 Fc fragment. The nucleoside of the fusion protein is The acid sequence is shown in SEQ ID NO: 11.

其中前10位碱基是KOZAK序列，第11-67位碱基为小鼠Ig heavy chain的信号肽密码子，第68-184、230-346位碱基为Exendin-4密码子，第185-229、347-361位碱基为连接肽密码子，362-1039为碱基为人IgG1 Fc突变体密码子，最后三位碱基为终止密码子。Among them, the first 10 bases are the KOZAK sequence, the 11th-67th bases are the signal peptide codons of mouse Ig heavy chain, the 68th-184th, 230-346th bases are Exendin-4 codons, and the 185th- Bases 229 and 347-361 are connecting peptide codons, bases 362-1039 are human IgG1 Fc mutant codons, and the last three bases are stop codons.

该融合蛋白的氨基酸序列如SEQ ID NO：12所示，其中前19个氨基酸（划线部分）为信号肽序列。The amino acid sequence of the fusion protein is shown in SEQ ID NO: 12, wherein the first 19 amino acids (underlined part) are signal peptide sequences.

将设计好的上述序列采用全基因合成的方法制备。合成完成后，按照下面的PCR体系扩增目的基因。The designed above-mentioned sequence is prepared by the method of whole gene synthesis. After the synthesis is completed, amplify the target gene according to the following PCR system.

100 μl PCR反应体系：100 μl PCR reaction system:

试剂Reagent含量content多核苷酸片段（2μg/μl）Polynucleotide fragments (2μg/μl)1 μl1μl10×PCR 反应缓冲体系10×PCRreaction buffer system10 μl10 μl2.5 mM dNTPs2.5 mMdNTPs8 μl8 μl引物1(100 mM，SEQ ID NO:13)Primer 1 (100 mM, SEQ ID NO: 13)0.2 μl0.2 μl引物2(100 mM，SEQ ID NO:14)Primer 2 (100 mM, SEQ ID NO: 14)0.2 μl0.2 μlPlatinum Taq polymerasePlatinum Taq polymerase0.4 μl0.4 μlSterile waterSterile water80.2 μl80.2 μl

反应条件：Reaction conditions:

预变性：94℃，2分钟；Pre-denaturation: 94°C, 2 minutes;

主循环：94℃，30秒，55℃，30秒，72℃,1分钟；Main cycle: 94°C, 30 seconds, 55°C, 30 seconds, 72°C, 1 minute;

循环数：30；Number of cycles: 30;

后延伸：72℃，10分钟。Post-extension: 72°C, 10 minutes.

2.电泳鉴定与凝胶回收。2. Electrophoresis identification and gel recovery.

按照上述条件进行PCR后，取5 μl产物在0.8%琼脂糖凝胶电泳上鉴定，发现产物中唯一一条分子量1000bp左右的带（如图3所示）。用QIAGEN QIAquick Gel Extraction Kit进行胶回收纯化，最后得到约为2.5μg目标DNA。After PCR was carried out according to the above conditions, 5 μl of the product was taken for identification on 0.8% agarose gel electrophoresis, and the only band with a molecular weight of about 1000 bp was found in the product (as shown in Figure 3). Use QIAGEN QIAquick Gel Extraction Kit for gel extraction and purification, and finally obtain about 2.5 μg of target DNA.

3.重组DiExendin-4-Ig融合蛋白表达载体的构建。3. Construction of recombinant DiExendin-4-Ig fusion protein expression vector.

按照pOptiVEC-TOPO vector说明书操作，将上述DNA片段克隆到该载体上，转化One Shot Chemically Competent E. coli后，取8个单菌落接种于含有100μg/ml 氨苄青霉素LB培养基中，37℃、250rpm过夜扩增培养，用质粒抽提纯化试剂盒（TIANGEN）抽提质粒DNA并进行酶切鉴定后选出2个大小正确的克隆（如图4所示）进行DNA测序。测序结果显示，2个克隆均正确，即pOptiVEC-DiExendin-4-Ig。According to the instructions of pOptiVEC-TOPO vector, the above DNA fragments were cloned into the vector, transformed into One Shot Chemically Competent E. coli, and 8 single colonies were inoculated in LB medium containing 100 μg/ml ampicillin, 37°C, 250rpm After amplifying and culturing overnight, the plasmid DNA was extracted with a plasmid extraction and purification kit (TIANGEN) and identified by enzyme digestion. After that, two clones with the correct size (as shown in Figure 4) were selected for DNA sequencing. Sequencing results showed that both clones were correct, namely pOptiVEC-DiExendin-4-Ig.

实验例2 重组DiExendin-4-Ig融合蛋白的表达Experimental example 2 Expression of recombinant DiExendin-4-Ig fusion protein

1.表达载体pOptiVEC-DiExendin-4-Ig的提取。1. Extraction of expression vector pOptiVEC-DiExendin-4-Ig.

取鉴定正确的1号质粒菌株，接种于500ml含氨苄青霉素的LB培养基中，37℃、250rpm培养18小时。用质粒抽提纯化试剂盒（TIANGEN）抽提质粒DNA，抽提过程按照厂家提供的试剂盒说明书进行。抽提完成后，将质粒进行PvuⅠ单酶切线性化，以备后续转染。The correctly identified No. 1 plasmid strain was inoculated into 500ml LB medium containing ampicillin, and cultured at 37°C and 250rpm for 18 hours. Plasmid DNA was extracted with a plasmid extraction and purification kit (TIANGEN), and the extraction process was carried out according to the kit instructions provided by the manufacturer. After the extraction is completed, the plasmid is linearized by PvuI single enzyme digestion for subsequent transfection.

2.CHO DG44细胞的转染与表达。2. Transfection and expression of CHO DG44 cells.

采用FreeStyle? MAX转染试剂转染CHO细胞，CHO DG44细胞及转染试剂均购自Invitrogen公司。转染操作程序按照厂家提供的说明书进行。FreeStyle MAX transfection reagent was used to transfect CHO cells, and CHO DG44 cells and transfection reagents were purchased from Invitrogen. The transfection procedure was carried out according to the instructions provided by the manufacturer.

转染后的细胞经HT（-）的CD OptiCHO^TM完全培养基连续培养一个月以获得阳性转染克隆，然后再用含有氨甲喋呤（MTX，工作浓度为100-500nM）的CD OptiCHO^TM完全培养基加压筛选，此过程需两个月。然后将稳定的阳性克隆通过有限稀释法进行单克隆筛选，用半固体培养基CloneMedia-CHO（Genetix公司产品，cat no K8710）将细胞悬液调整至1cell/100μL/孔接种入96孔板中，置于37℃，8%CO₂培养箱中培养3周左右，待单克隆细胞扩增后， 用ELISA方法检测上清中融合蛋白的表达量。选取表达最高的3个克隆扩增后保种，取其中F10号克隆进行放大培养、表达。The transfected cells were continuously cultured with HT (-) CD OptiCHO^TM complete medium for one month to obtain positive transfected clones, and then treated with CD OptiCHO^TM complete medium containing methotrexate (MTX, working concentration 100-500nM) Pressurized screening, a process that takes two months. Then, the stable positive clones were subjected to monoclonal screening by the limiting dilution method, and the cell suspension was adjusted to 1 cell/100 μL/well with a semi-solid medium CloneMedia-CHO (product of Genetix, cat no K8710) and inoculated into a 96-well plate. Place them in a 37°C, 8% CO₂ incubator and culture them for about 3 weeks. After the monoclonal cells are expanded, use the ELISA method to detect the expression of the fusion protein in the supernatant. The three clones with the highest expression were selected to be amplified and preserved, and the F10 clone was selected for amplified culture and expression.

该部分操作完全按照invitrogen公司的操作手册（pOptiVEC-TOPO-TA Cloning Kit, Cat nos. 12744-017, 12745-014, 12762-019），其中所用到的主要试剂也均选自invitrogen公司。This part of the operation is completely in accordance with the operation manual of Invitrogen Company (pOptiVEC-TOPO-TA Cloning Kit, Cat nos. 12744-017, 12745-014, 12762-019), and the main reagents used are also selected from Invitrogen Company.

实验例3重组DiExendin-4-Ig融合蛋白的纯化Purification of experimental example 3 recombinant DiExendin-4-Ig fusion protein

由于该融合蛋白具有IgG Fc片段，优选地可以进行亲和纯化来获得目的蛋白，在本实施实例中，我们采用了亲和层析、离子交换层析和分子筛层析相结合的纯化工艺，经SDS-PAGE和HPLC检测，其纯度可>98%。具体方法如下：Since the fusion protein has an IgG Fc fragment, it is preferable to perform affinity purification to obtain the target protein. In this implementation example, we adopted a purification process combining affinity chromatography, ion exchange chromatography and molecular sieve chromatography. SDS-PAGE and HPLC detection, its purity can be >98%. The specific method is as follows:

1.表达上清液预处理：1. Expression supernatant pretreatment:

用1M的NaOH将上清液缓慢调节pH至7.3，在4℃下4000rpm离心10min，收集上清。The supernatant was slowly adjusted to pH 7.3 with 1M NaOH, centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was collected.

2.亲和层析：2. Affinity chromatography:

用10 mM PB（pH7.3）缓冲液平衡rProtein A-Sepharose F.F.(GE)亲和层析柱后，将上述离心上清上样，再用同样的缓冲液洗涤亲和柱至280nm的OD值小于0.01，用0.1M的柠檬酸缓冲液（pH 4）将融合蛋白洗下，收集洗脱峰。立即将洗脱峰用200 mM Na₂HPO₄精确调节pH至7.5待用。After equilibrating the rProtein A-Sepharose FF (GE) affinity chromatography column with 10 mM PB (pH7.3) buffer, load the above centrifuged supernatant, and then wash the affinity column with the same buffer to an OD value of 280nm If it is less than 0.01, wash the fusion protein with 0.1M citrate buffer (pH 4) and collect the eluted peak. The eluted peak was immediately adjusted to a fine pH of 7.5 with 200 mM Na₂ HPO₄ for use.

3.离子交换层析：3. Ion exchange chromatography:

用10 mM PB（pH7.5）缓冲液平衡 SP Sepharose F.F.(GE)层析柱后，将上述溶液上样，再用同样的缓冲液洗涤层析柱基线，用含100mM NaCl的磷酸缓冲液（pH7.5）进行洗脱，收集洗脱峰。After equilibrating the SP Sepharose F.F. (GE) chromatography column with 10 mM PB (pH7.5) buffer solution, load the above solution, then wash the column baseline with the same buffer solution, wash with phosphate buffer solution containing 100mM NaCl ( pH7.5) for elution, and the elution peaks were collected.

4.分子筛层析：4. Molecular sieve chromatography:

用10 mM PB（pH7.0）缓冲液平衡 Superdex 200(GE)分子筛层析柱，上样并收集洗脱峰。洗脱峰保存于-20℃。取样检测其纯度和分子量，结果纯度大于98%，表观分子量与理论分子量接近（如图5、6所示）。Equilibrate the Superdex 200 (GE) molecular sieve chromatography column with 10 mM PB (pH7.0) buffer, load the sample and collect the elution peak. Eluted peaks were stored at -20°C. Samples were taken to test its purity and molecular weight, and the result was that the purity was greater than 98%, and the apparent molecular weight was close to the theoretical molecular weight (as shown in Figures 5 and 6).

实验例4重组DiExendin-4-Ig融合蛋白体外活性检测Experimental example 4 In vitro activity detection of recombinant DiExendin-4-Ig fusion protein

本实施实例采用cAMP酶联测定法来检测重组DiExendin-4-Ig的体外生物学活性。所选用的cAMP检测试剂盒为R&D公司产品，操作方法也按照试剂盒说明书进行，细胞系为大鼠胰岛瘤RIN-5F细胞，细胞的传代培养按照常规方法进行，阳性对照品为Exendin-4。具体操作方法如下：In this embodiment, cAMP enzyme-linked assay is used to detect the in vitro biological activity of recombinant DiExendin-4-Ig. The selected cAMP detection kit is the product of R&D Company, and the operation method is also carried out according to the kit instructions. The cell line is rat insulinoma RIN-5F cells, and the subculture of the cells is carried out according to the conventional method. The positive control substance is Exendin-4. The specific operation method is as follows:

RIN-5F细胞于含10%小牛血清、1mol/L丙酮酸钠、0.5%HEPES的RPMI-1640高糖培养液（pH7.2）中，在37℃、5%二氧化碳的条件下培养。取对数生长期的细胞，胰酶消化成细胞悬液，调整细胞至一定浓度。加细胞悬液于96孔培养板中于二氧化碳培养箱培养3天。除去细胞培养上清，加入无血清培养基继续培养2h。取试验品和对照品分别用含一定浓度的IBMX和牛血清白蛋白的细胞培养液对待测样品作一定比例稀释，共4-6个稀释度。除去细胞上清，加入稀释好的不同浓度的试验品和对照品，每个稀释度做3个复孔。在二氧化碳培养箱中放置10-20min，除去细胞上清，用cAMP试剂盒检测，检测波长为405nm，参考波长为570nm。结果：在同等摩尔浓度下，DiExendin-4-Ig较Exendin-4有更高的生物学活性（如图7所示）。RIN-5F cells were cultured in RPMI-1640 high-glucose medium (pH 7.2) containing 10% calf serum, 1mol/L sodium pyruvate, and 0.5% HEPES at 37°C and 5% carbon dioxide. Take the cells in the logarithmic growth phase, digest with trypsin to form a cell suspension, and adjust the cells to a certain concentration. The cell suspension was added to a 96-well culture plate and cultured in a carbon dioxide incubator for 3 days. The cell culture supernatant was removed, and serum-free medium was added to continue culturing for 2 h. Take the test product and the reference product and make a certain proportion dilution of the test sample with the cell culture fluid containing certain concentration of IBMX and bovine serum albumin respectively, a total of 4-6 dilutions. Remove the cell supernatant, add the diluted test substance and control substance with different concentrations, and make 3 duplicate holes for each dilution. Place in a carbon dioxide incubator for 10-20 minutes, remove the cell supernatant, and detect with a cAMP kit, the detection wavelength is 405 nm, and the reference wavelength is 570 nm. Results: At the same molar concentration, DiExendin-4-Ig had higher biological activity than Exendin-4 (as shown in Figure 7).

实验例5 建立ELISA方法检测表达上清中DiExendin-4-Ig融合蛋白的含量Experimental example 5 Establishing an ELISA method to detect the content of DiExendin-4-Ig fusion protein in the expression supernatant

用包被液稀释Anti human IgG(γ-chain specific)（1：2000），以100μl /孔加至96孔酶标板内，置4℃过夜，PBS-T洗板3次，用0.1%BSA封闭液200ul/ml加至酶标板内，置37℃，2小时，PBS-T洗板3次，将稀释好的标准品（10ng/ml 、5ng/ml 、2.5ng/ml、 1.25ng/ml、 0.625ng/ml 、0.3125ng/ml）和表达上清以100μl/孔加入板中，置37℃，2小时。PBS-T洗板4次，用0.1%BSA-PBS-T按1：1000稀释Anti human IgG（whole molecule ）-HRP，以100μl /孔加入板内，置37℃，1小时。PBS-T洗板4次，以100μl /孔加入临时配制的OPD显色底物，室温，避光显色10min，加入1mol/L硫酸50μl终止反应，用酶标仪检测A490值。以标准品A值和对应的标准品浓度做标准曲线，以待检样品A值和稀释倍数，计算出上清中DiExendin-4-Ig融合蛋白的含量。Dilute Anti human IgG (γ-chain specific) (1:2000) with coating solution, add 100 μl/well to 96-well ELISA plate, place at 4°C overnight, wash theplate 3 times with PBS-T, and wash the plate with 0.1% BSA Add 200ul/ml blocking solution to the microplate, place at 37°C for 2 hours, wash theplate 3 times with PBS-T, and dilute the diluted standard (10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml ml, 0.625ng/ml, 0.3125ng/ml) and expression supernatant were added to the plate at 100μl/well, and placed at 37°C for 2 hours. Wash theplate 4 times with PBS-T, dilute Anti human IgG (whole molecule)-HRP 1:1000 with 0.1% BSA-PBS-T, add 100 μl/well into the plate, and place at 37°C for 1 hour. Wash theplate 4 times with PBS-T, add temporarily prepared OPD chromogenic substrate at 100 μl/well, develop color at room temperature for 10 minutes in the dark, add 50 μl of 1 mol/L sulfuric acid to terminate the reaction, and detect the A490 value with a microplate reader. Use the A value of the standard substance and the corresponding concentration of the standard substance to make a standard curve, and use the A value of the sample to be tested and the dilution factor to calculate the content of the DiExendin-4-Ig fusion protein in the supernatant.

实验例6重组DiExendin-4-Ig融合蛋白初步药效学分析Experimental Example 6 Preliminary Pharmacodynamic Analysis of Recombinant DiExendin-4-Ig Fusion Protein

正常ICR小鼠在SPF级动物房适应性饲养3天后，按照体重随机分为4组，分别为正常对照组(Nor)、1 mg/kg、2 mg/kg和4 mg/kg，每组7只。各组动物禁食过夜（自由饮水），次日尾尖采血，然后皮下注射给药（0.1 ml/10 g，正常对照组给予生理盐水）。在给药后30 min灌胃给予葡萄糖（0.1 ml/10 g），并分别于糖负荷后30、60、120 min尾尖采血，测定血糖（葡萄糖氧化酶法）； Normal ICR mice were adaptively fed for 3 days in the SPF animal room, and were randomly divided into 4 groups according to body weight, namely normal control group (Nor), 1 mg/kg, 2 mg/kg and 4 mg/kg, eachgroup 7 Only. Animals in each group were fasted overnight (drinking water freely), blood was collected from the tip of the tail the next day, and then administered by subcutaneous injection (0.1 ml/10 g, normal control group was given normal saline). Glucose (0.1 ml/10 g) was given by intragastric administration 30 minutes after the administration, and blood was collected from the tip of the tail at 30, 60, and 120 minutes after the glucose load, and blood glucose was measured (glucose oxidase method);

在给药后第2天和第3天，各组动物尾尖采血后禁食4 h（自由饮水），然后灌胃给予葡萄糖（同上），并分别于糖负荷后30和60 min尾尖采血，测定血糖； On the 2nd and 3rd day after the administration, the animals in each group were fasted for 4 hours after blood collection from the tail tip (drinking water freely), and then intragastrically administered glucose (same as above), and blood was collected from the tail tip 30 and 60 min after the glucose load, respectively , measure blood sugar;

在给药后第4天至第14天，各组动物禁食4 h（自由饮水）后灌胃给予葡萄糖（同上），于糖负荷后30 min尾尖采血，测定血糖。From the 4th day to the 14th day after the administration, the animals in each group were fasted for 4 hours (free drinking water), and then given glucose (as above) by intragastric administration, and the blood was collected from the tip of the tail 30 minutes after the sugar load, and the blood glucose was measured.

2.结果：2. Results:

1) DiExendin-4-Ig单次给药后第1天对正常ICR小鼠血糖变化的影响 1) Effect of DiExendin-4-Ig on blood glucose changes in normal ICR mice onday 1 after single administration

与正常对照组相比，DiExendin-4-Ig样品各剂量单次给药后都能够显著降低正常ICR小鼠口服葡萄糖负荷后的血糖曲线下面积（如图8所示）。 Compared with the normal control group, each dose of DiExendin-4-Ig sample can significantly reduce the area under the curve of blood glucose after oral glucose load in normal ICR mice (as shown in Figure 8). the

2) DiExendin-4-Ig单次给药后第2天对正常ICR小鼠血糖变化的影响            2) Effect of DiExendin-4-Ig on blood glucose changes in normal ICR mice onday 2 after single administration

与正常对照组相比， DiExendin-4-Ig样品各剂量单次给药后第2天还能够显著降低正常ICR小鼠的非禁食血糖和口服葡萄糖负荷后的血糖曲线下面积（如图9所示），说明其作用时间至少为1天。Compared with the normal control group, DiExendin-4-Ig samples can also significantly reduce the non-fasting blood glucose and the area under the blood glucose curve after oral glucose load in normal ICR mice on the second day after a single dose of each dose (as shown in Figure 9 shown), indicating that its action time is at least 1 day.

3) DiExendin-4-Ig单次给药后第3天对正常ICR小鼠血糖变化的影响 3) The effect of DiExendin-4-Ig on blood glucose changes in normal ICR mice on the third day after a single administration

与正常对照组比较， DiExendin-4-Ig样品各剂量单次给药后第3天还能够显著降低正常ICR小鼠的非禁食血糖和口服葡萄糖负荷后的血糖曲线下面积（如图10所示），说明其作用时间至少为2天。Compared with the normal control group, DiExendin-4-Ig samples can also significantly reduce the non-fasting blood glucose and the area under the blood glucose curve after oral glucose load in normal ICR mice on the 3rd day after each dose of single administration (as shown in Figure 10 shown), indicating that its action time is at least 2 days.

4) DiExendin-4-Ig单次给药后第4-6天对正常ICR小鼠血糖变化的影响 4) Effects of DiExendin-4-Ig on blood glucose changes in normal ICR mice on day 4-6 after a single administration

与正常对照组相比，DiExendin-4-Ig样品各剂量单次给药后第4-6天都能够显著降低正常ICR小鼠的非禁食血糖和口服葡萄糖负荷后30 min的血糖曲线下面积（如图11所示），说明其作用时间至少为5天。 Compared with the normal control group, each dose of DiExendin-4-Ig samples can significantly reduce the non-fasting blood glucose and the area under the curve of blood glucose 30 min after oral glucose load in normal ICR mice on the 4th to 6th day after a single administration (as shown in Figure 11), indicating that its action time is at least 5 days. the

5) DiExendin-4-Ig单次给药后第7-9天对正常ICR小鼠血糖变化的影响 5) Effects of DiExendin-4-Ig on blood glucose changes in normal ICR mice on day 7-9 after a single administration

与正常对照组比较，DiExendin-4-Ig各剂量单次给药后第7-9天都能够显著降低正常ICR小鼠口服葡萄糖负荷后30 min的血糖曲线下面积（如图12所示），但对ICR小鼠非禁食血糖的降低作用逐渐减弱，说明其作用时间至少为8天。 Compared with the normal control group, each dose of DiExendin-4-Ig can significantly reduce the area under the blood glucose curve 30 minutes after oral glucose load in normal ICR mice on the 7th to 9th day after a single administration (as shown in Figure 12). However, the lowering effect on non-fasting blood sugar of ICR mice gradually weakened, indicating that the effect time was at least 8 days. the

6) DiExendin-4-Ig单次给药后第10-14天对正常ICR小鼠血糖变化的影响 6) Effects of DiExendin-4-Ig on blood glucose changes in normal ICR mice on day 10-14 after a single administration

与正常对照组相比，DiExendin-4-Ig各剂量单次给药后第10-14天都能够显著降低正常ICR小鼠口服葡萄糖负荷后30 min的血糖曲线下面积（如图13所示），对ICR小鼠的非禁食血糖已没有明显影响，但仍然说明其作用时间至少为13天。 Compared with the normal control group, each dose of DiExendin-4-Ig can significantly reduce the area under the blood glucose curve 30 minutes after oral glucose load in normal ICR mice on the 10th to 14th day after a single administration (as shown in Figure 13) , has no obvious effect on the non-fasting blood sugar of ICR mice, but still shows that its effect time is at least 13 days. the

7) DiExendin-4-Ig单次给药后第16-20天对正常ICR小鼠血糖变化的影响 7) Effects of DiExendin-4-Ig on blood glucose changes in normal ICR mice at day 16-20 after single administration

与正常对照组比较，DiExendin-4-Ig 4 mg/kg单次给药后第16-20天都能够显著降低正常ICR小鼠口服葡萄糖负荷后30 min的血糖曲线下面积（如图14所示），说明其作用时间约为19 天，而2 mg/kg和1 mg/kg的作用时间均约为15天。 Compared with the normal control group, DiExendin-4-Ig 4 mg/kg can significantly reduce the area under the blood glucose curve 30 minutes after oral glucose load in normal ICR mice on the 16th to 20th day after a single administration (as shown in Figure 14 ), indicating that its action time is about 19 days, while the action time of 2 mg/kg and 1 mg/kg is about 15 days. the

3. 实验小结： 3. Experiment summary:

    DiExendin-4-Ig样品4 mg/kg、2 mg/kg和1 mg/kg单次给药后都能够显著降低正常ICR小鼠的非禁食血糖和口服葡萄糖负荷后的血糖，具有良好的量效关系，并且2mg/kg和1mg/kg作用时间为15天，4mg/kg作用时间至少为19天。 After a single administration of 4 mg/kg, 2 mg/kg and 1 mg/kg, DiExendin-4-Ig samples can significantly reduce the non-fasting blood glucose and blood glucose after oral glucose load in normal ICR mice, with a good amount Effect relationship, and the 2mg/kg and 1mg/kg action time is 15 days, and the 4mg/kg action time is at least 19 days. the

当然，以上所述仅是本发明的较佳实施方式，故凡依本发明专利申请范围所述的构造、特征及原理所做的等效变化或修饰，均包括于本发明专利申请范围内。Of course, the above descriptions are only preferred implementations of the present invention, so all equivalent changes or modifications made according to the structures, features and principles described in the scope of the patent application of the present invention are included in the scope of the patent application of the present invention.

Claims (9)

1. a fusion rotein that is used to treat mellitus is characterized in that, said fusion rotein is the fusion rotein that N Exendin-4-Linker and human IgG Fc two mutants reassemble into;

Wherein, Linker is one group of flexible peptide section of being made up of hydrophobic amino acid, and its amino acid whose number is less than 25;

Wherein, human IgG Fc two mutants is IgG1 Fc, IgG2 Fc or IgG4 Fc two mutants;

Wherein, N is 2-8.

2. a kind of fusion rotein that is used to treat mellitus according to claim 1 is characterized in that: said fusion rotein is the fusion rotein that 2 Exendin-4-Linker and human IgG1 Fc two mutants reassemble into.

3. a kind of fusion rotein that is used to treat mellitus according to claim 1 is characterized in that: said human IgG Fc two mutants is an IgG1 Fc two mutants; The amino acid whose sudden change position of IgG1 Fc two mutants is the combinatorial mutagenesis position of among the Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236/Ala327Gly/Ala330Ser/Pro331Ser one sudden change position or at least two positions, and the Position Number that wherein suddenlys change is according to the EU numbering system.

4. a kind of fusion rotein that is used to treat mellitus according to claim 1 is characterized in that: said Linker is flexible peptide section (Gly₄Ser) n, n=1-5 or flexible peptide section (Ala₃Ser₂) n, n=1-5.

5. a kind of fusion rotein that is used to treat mellitus according to claim 2 is characterized in that: said fusion rotein is the fusion rotein that 2 Exendin-4-Linker and human IgG1 Fc two mutants reassemble into;

Wherein, Linker is flexible peptide section (Gly₄Ser) n, n=1-5;

Wherein, the amino acid whose sudden change position of IgG1 Fc two mutants is the combinatorial mutagenesis position of Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236/Ala327Gly/Ala330Ser/Pro331Ser, and the Position Number that wherein suddenlys change is according to the EU numbering system.

6. a kind of fusion rotein that is used to treat mellitus according to claim 5 is characterized in that: said fusion rotein is the fusion rotein that 2 Exendin-4-Linker and human IgG1 Fc two mutants reassemble into;

Wherein, the Linker that is connected with Exendin-4 respectively of two ends is flexible peptide section (Gly₄Ser) n, n=3;

Wherein, the Linker that an end is connected with Exendin-4 and the other end is connected with human IgG1 Fc two mutants is flexible peptide section (Gly₄Ser) n, n=1;

Wherein, the amino acid whose sudden change position of IgG1 Fc two mutants is the combinatorial mutagenesis position of Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236/Ala327Gly/Ala330Ser/Pro331Ser, and the Position Number that wherein suddenlys change is according to the EU numbering system.

7. a kind of fusion rotein that is used to treat mellitus according to claim 1; It is characterized in that: said Expression of Fusion Protein carrier is pOptiVEC-TOPO; Said fusion rotein is expressed in mammalian cell, and said cell is selected from Chinese hamster ovary celI, HeLa cell and bhk cell.

8. according to any described a kind of fusion rotein that is used to treat mellitus among the claim 1-7, it is characterized in that: said fusion rotein is in the application of treatment I type, type II diabetes.

9. preparation method who is used to treat the fusion rotein of mellitus; It is characterized in that: this method is included in the host cell of cultivating claim 6 under the condition that is suitable for expressing this fusion rotein; Host cell through cell cultures, centrifugal after, supernatant is used affinity chromatography, ion exchange chromatography and sieve chromatography purifying respectively;

What wherein, use in the affinity chromatography is the affine filler of rProtein A Sepharose FF;

What wherein, use in the ion exchange chromatography is Q Sepharose FF filler;

What wherein, use in the sieve chromatography is Superdex 200 fillers.

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