CN102816244A - Fusion protein of exendin-4 peptide and human serum albumin (HSA) and preparation method thereof - Google Patents

Fusion protein of exendin-4 peptide and human serum albumin (HSA) and preparation method thereof Download PDF

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CN102816244A
CN102816244A CN2012103016549A CN201210301654A CN102816244A CN 102816244 A CN102816244 A CN 102816244A CN 2012103016549 A CN2012103016549 A CN 2012103016549A CN 201210301654 A CN201210301654 A CN 201210301654A CN 102816244 A CN102816244 A CN 102816244A
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exendin
hsa
fusion protein
peptide
plasmid
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CN2012103016549A
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杨建良
余传信
孟如杰
冯炜
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无锡和邦生物科技有限公司
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Abstract

The invention discloses a fusion protein of exendin-4 peptide and human serum albumin (HSA) and a preparation method thereof, belonging to the technical field of long-term recombination fusion protein medicine. The invention relates to a preparation method and a product of fusion protein of insulin secretion promoting peptide (Exendin-4) and human serum albumin (HSA). The fusion protein is characterized in that a genetic engineering method is adopted to directly fuse a single Exndin-4 peptide and HAS and without connecting peptide at the middle part to form stable and long half-life period Exendin-4-HAS fusion protein, and is applied in a treatment of non-insulin-dependent diabetes and obesity.

Description

—种Exend in-4肽与人血清白蛋白HSA的融合蛋白及其制备方法 - Exend in-4 species of peptide and human serum albumin fusion proteins and preparation of HSA

技术领域 FIELD

[0001] 一种Exendin-4肽与人血清白蛋白(HSA)的融合蛋白及其制备方法,属于长效重组融合蛋白药物技术领域。 [0001] An Exendin-4 peptide fusion proteins and preparation of human serum albumin (HSA), belonging to the long-acting recombinant fusion protein in the pharmaceutical technical field.

[0002] 本发明通过将单个Exendin-4多肽和人血清白蛋白直接进行融合,在保持Exendin-4的药效的同时使其在体内的半衰期获得延长,提高生物利用度与治疗价值,减轻病人的治疗负担,在药学领域有良好的应用前景。 [0002] By the present invention, a single Exendin-4 and human serum albumin polypeptide fused directly, while maintaining the efficacy of the Exendin-4 is obtained so as to extend in vivo half-life, improved bioavailability and therapeutic value, reduce patient the burden of treatment, there is a good prospect in the pharmaceutical field. 本发明涉及治疗非胰岛素依赖性II型糖尿病长效药物的制备。 The present invention relates to a pharmaceutical depot of non-insulin dependent type II diabetes.

背景技术 Background technique

[0003] 糖尿病是一种严重威胁全世界人民身体健康的慢性病,发病人数在不断增加,至2030年全球糖尿病患者将达到4. 35亿。 [0003] Diabetes is a serious threat to people's health worldwide chronic disease, the incidence is increasing, and 2030 with diabetes worldwide will reach 435 million. 我国的糖尿病的发病率高达9. 7%,全国的糖尿病人将超过一个亿,已成为世界上糖尿病第一大国,而且糖尿病的发展呈低龄化趋势。 Our incidence of diabetes as high as 9.7 percent, the country of people with diabetes will more than one million diabetes has become the world's superpower, and the development of diabetes showed a younger age trend. 因此,研发有效的糖尿病治疗与控制手段具有重要意义与紧迫性。 Therefore, the development of effective diabetes treatment and control of the means of great significance and urgency.

[0004] 糖尿病分为胰岛素依赖性I型糖尿病和非胰岛素依赖性II型糖尿病,II型糖尿病占总病人数的90%以上。 [0004] Diabetes is divided into more than 90% of insulin-dependent Type I diabetes and Type II non-insulin dependent diabetes mellitus, the total number of patients with type II diabetes. II糖尿病是由于各种原因导致患者胰腺β细胞功能减退,分泌的胰岛素不足,或胰岛素受体功能障碍不能正常利用胰岛素,导致糖代谢紊乱,血糖升高所致。 II diabetic patients due to various causes loss of pancreatic β-cell function, insulin secretion, insulin receptor dysfunction or not properly use insulin, glucose metabolism leading to elevated blood glucose caused. 采取各种治疗手段,恢复胰岛功能,增加胰岛素分泌或提高其利用效率是目前治疗II型糖尿病的主要手段之一。 Take a variety of treatments to restore islet function, increase insulin secretion or enhance their efficiency one of the primary means of treatment of type II diabetes is present.

[0005] Exendin-4 (又称促胰岛素分泌肽),是从生长在美国西南部和墨西哥沙漠的希拉毒蜥唾液中发现含39个氨基酸的短肽,其氨基酸序列与人类血液中调控血糖的胰高血糖素类似肽GLP-I有53%的同源性,是胰高血糖素类似肽受体(Glucogan like Peptide IR,GLP-1R)的激动剂,与GLP-I功能相似,具有直接降血糖,促进胰腺分泌胰岛素的作用,还可以通过减缓胃肠道的蠕动使食物的吸收减慢,从而降低食物中的葡萄糖在血液内出现的高峰。 [0005] Exendin-4 (also known as exendin), was found to contain 39 amino acid peptide from the growth southwestern United States and Mexico desert Gila lizard saliva, whose amino acid sequence of human blood glucose regulation the glucagon analogue peptide GLP-I 53% homology, is a peptide similar to glucagon receptor (Glucogan like peptide IR, GLP-1R) agonists, GLP-I and functionally similar, having a direct down blood sugar, promote the role of the pancreas to secrete insulin, it can also slow down the absorption of food by slowing the gastrointestinal tract motility, thereby reducing the peak glucose in food appear in the blood. 由于不被二肽基肽酶一IV(DPP-IV)降解,Exendin-4具有更高更稳定的生物学活性,已成为糖尿病治疗药物开发热点。 Since not degraded by a dipeptidyl peptidase IV (DPP-IV), Exendin-4 has a higher and more stable biological activity, it has become a diabetes drug development of hot spots.

[0006] 商品化用于II型糖尿病治疗的短效Exendin-4肽(又名Byetta®)已获得了美国FDA批准上市,成为治疗II型糖尿病的一种新药。 [0006] fugitive commercial Exendin-4 peptides for the treatment of type II diabetes (also known Byetta®) has received FDA approval listed as a new drug in the treatment of type II diabetes. 在生理条件下,Byetta® (Exendin-4)和GLP-I作用相似,在刺激胰岛素分泌的作用时依赖于血糖浓度,不会因为使用过量而发生低血糖,避免了胰岛素因使用剂量不当会引起低血糖反应的缺点。 Under physiological conditions, Byetta® (Exendin-4) and GLP-I a similar effect, upon stimulation of insulin secretion is dependent on glucose concentration, hypoglycemia and will not be used in excess, to avoid the use of insulin doses can cause improper disadvantage of hypoglycemia. 然而,由于Exendin-4在体内的半衰期为2-3小时,患者仍需要每天注射两次才能获得预期的疗效,不仅费用高,亦较烦琐。 However, due to Exendin-4 in vivo half-life of 2-3 hours, patients still need to be injected twice a day to get the desired effect, not only costly, cumbersome Yijiao.

[0007] 近年来,国内、外均致力于糖尿病长效治疗药的开发,如Lilly公司采用基因工程方法将Exendin-4与免疫球蛋白(IgG)的Fe段融合,加拿大ConjuChem公司采用人工藕联的方法在体外将Exendin-4与人血清白蛋白结合产生CJC-1134-PC,均可能有效延长Exendin-4在体内的半衰期,提高其治疗效果。 [0007] In recent years, domestic and foreign are committed to developing long-acting diabetes treatment drugs, such as Lilly's genetic engineering methods Exendin-4 immunoglobulin (IgG) of paragraph Fe fusion, Canadian ConjuChem company with artificial lotus the in vitro method of Exendin-4 binding to human serum albumin produced CJC-1134-PC, are likely to prolong the half-life of Exendin-4 in vivo, to improve its therapeutic effect. 但在体外将Exendin-4与HSA进行化学藕联的生产成本高,效能低。 However, high Exendin-4 in vitro chemical coupling associated with the HSA production costs, low efficiency.

[0008] HSA是人体的一个分子量为66KD的大分子蛋白,具有多种生物学功能,是许多生物因子的载体蛋白,因其不能被肾小球滤过,血液中半衰期长达14-21天,通常被用于药物的载体,以延长药物的半衰期。 [0008] HSA is a large protein of molecular weight of 66KD body, with a variety of biological functions, many carrier protein biological agents, because it can not be glomerular filtration, the blood half-life of 14-21 days , it is commonly used half-life drug carriers to prolong the drug. 本发明采用基因工程技术构建了能表达Exendin-4与人血清白蛋白的融合蛋白的重组酵母表达质粒,表达产物中Exendin-4与HSA直接融合,可增加Exendin-4的稳定性,延长其在人体内的半衰期,提高对糖尿病的治疗效果。 The present invention is constructed using genetic engineering techniques and a recombinant yeast capable of expressing human serum albumin fusion protein expression plasmid Exendin-4, Exendin-4 expression products of HSA fused directly, can increase the stability of Exendin-4, which extend half-life in the human body, improve the treatment of diabetes. 构建的酵母外分泌表达系统能大批量制备该融合蛋白,降低了生产成本。 Exocrine yeast expression system constructs can be prepared in large quantities of the fusion protein, reducing production costs.

发明内容 SUMMARY

[0009] 本发明提供一种促胰岛素分泌肽(Exendin-4)与人血清白蛋白(HSA)的融合蛋白长效药物及其制备方法。 [0009] The present invention provides a long acting pharmaceutical preparation and an insulin secretion peptide fusion protein (Exendin-4) pro with human serum albumin (HSA) is. 本发明的目的还在于开发一类具有高稳定性、低副作用的降血糖药物,在保持Exendin-4的生理特性的基础上,延长其半衰期,使之成为新一代长效治疗糖尿病药物。 Object of the present invention is to develop a class of hypoglycemic agents with high stability and low side effects, while maintaining physiological characteristics of Exendin-4 on the extension of half-life, making a new generation long-acting medicament for treating diabetes. [0010] 本发明的技术方案:一种Exendin-4与人血清白蛋白HSA的融合蛋白Exendin-4-HSA,含有2个多肽区,其I区为一个Exendin-4多肽,其2区为人血清白蛋白(HSA),I区通过其C-末端与第2区的N-末端直接连接,中间不加入任何连接肽,其结构式是:Exendin-4-HSA。 [0010] aspect of the present invention: one Exendin-4 HSA human serum albumin fusion protein Exendin-4-HSA, comprising two polypeptide region, which region I Exendin-4 is a polypeptide, which region is human serum 2 albumin (HSA), I N- terminal area directly connected via its C- terminus of the second region, without adding any intermediate connecting peptide, the structural formula is: Exendin-4-HSA.

[0011] I区Exendin-4多肽氨基酸序列与序列SEQ ID NO :1保持有至少90%的同源性。 [0011] I region Exendin-4 polypeptide sequence and amino acid sequence of SEQ ID NO: 1 retaining at least 90% homology.

[0012] 2区多肽选自人血清白蛋白HSA的成熟肽,其氨基酸序列与序列SEQ ID NO :2保持有至少90%的同源性。 [0012] 2 polypeptide region selected from human serum albumin HSA mature peptide, which is an amino acid sequence SEQ ID NO: 2 retaining at least 90% homology.

[0013] 所述的Exendin-4-HSA融合蛋白,其核苷酸序列编码为SEQ ID NO :3。 [0013] according to Exendin-4-HSA fusion protein, which nucleotide sequences encode SEQ ID NO: 3.

[0014] 所述的Exendin-4-HSA融合蛋白,其氨基酸序列为SEQ ID NO :7。 [0014] according to Exendin-4-HSA fusion protein, the amino acid sequence of SEQ ID NO: 7.

[0015] 所述的Exendin-4-HSA融合蛋白,其重组核苷酸序列插入在一种表达载体和细胞宿主系统。 [0015] according to Exendin-4-HSA fusion protein, which recombinant nucleotide sequence is inserted in an expression vector and host cell systems.

[0016] 所述的Exendin-4-HSA融合蛋白的制备方法,包括转录,翻译,蛋白分离和纯化,以及鉴定步骤。 [0016] the preparation of Exendin-4-HSA fusion proteins, including transcription, translation, protein separation and purification, and the identifying step.

[0017] 所述Exendin-4-HSA融合蛋白的应用,其特征是使哺乳动物血糖水平正常化,用于制备非胰岛素依赖性II型糖尿病及肥胖者的治疗制剂。 [0017] Application of the Exendin-4-HSA fusion protein, wherein the normalizing, formulations for treating non-insulin dependent Type II diabetes and obesity in mammalian blood glucose levels was prepared. 给药方式选用注射,口服,鼻内,呼吸道吸服。 Mode of administration selected injection, oral, nasal, respiratory inhale.

[0018] 为了提高用药效果,Exendin-4-HSA融合蛋白可以制成不同的剂型,包括但不限于使用各种酸碱所形成的盐形式。 [0018] In order to improve the effect of medication, Exendin-4-HSA fusion protein may be made in different forms, including but not limited to various pH using the formed salt form.

[0019] 为了提高用药效果,Exendin-4-HSA融合蛋白也可以与其他药物共同使用。 [0019] In order to improve the effect of medication, Exendin-4-HSA fusion protein may also be used together with other drugs.

[0020] 本发明采用基因工程技术,制备Exendin-4-HSA融合蛋白长效蛋白质药物,其长效方案包括:一是该融合蛋白中含有Exendin-4多肽;二是该融合蛋白含有在人血液中具有长半衰期的人血清白蛋白HSA肽段。 [0020] The present invention uses genetic engineering technique, preparation of Exendin-4-HSA fusion protein protein drug depot, which includes a long-term program: First, the fusion protein Exendin-4 polypeptide comprising; two in the fusion protein containing human blood having a long half-life of human serum albumin HSA peptide. 通过生化以及细胞和动物实验验证,Exendin-4-HSA融合蛋白比Exendin-4多肽药物具有更好的稳定性和体内半衰期。 Biochemical and cellular and animal experiments, Exendin-4-HSA fusion protein has better stability and in vivo half-life of Exendin-4 polypeptide drugs.

[0021] 本发明中的Exendin-4多肽氨基酸序列是为39个氨基酸,与序列SEQ ID N0:1保持有至少90%的同源性,优选同源性是95%。 [0021] Exendin-4 amino acid sequence of the polypeptide of the present invention is 39 amino acids, with sequence SEQ ID N0: 1 retaining at least 90% homology, preferably 95% homology. 其中可以有多种氨基酸位点的替代,目的是使该活性肽更稳定,活性更高。 Which can replace a variety of amino acid positions, the purpose of making active peptide more stable and active. [0022] 本发明中的HSA氨基酸序列与序列SEQ ID NO : 2保持有至少90%的同源性,优选同源性是95%。 [0022] HSA amino acid sequence of the present invention in sequence of SEQ ID NO: 2 retains at least 90% homology, preferably 95% homology. 其中可以有多种氨基酸位点的替代,目的是使该活性肽更稳定,活性更高。 Which can replace a variety of amino acid positions, the purpose of making active peptide more stable and active.

[0023] 本发明为了使获得表达的融合蛋白正确折叠,并且能够进行外分泌,Exendin-4-HSA融合蛋白的N-末端与HSA的信号肽序列相连。 [0023] In order to make the present invention to obtain correct folding of the fusion protein, and can be secreted outside, N- terminus of the fusion protein Exendin-4-HSA connected to the signal peptide sequence of HSA. 具体地,在进行酵母重组表达质粒的构建时,将Exendin-4-HSA融合基因插入到表达质粒pWX530中HSA信号肽基因的下游,并使两者发生阅读框内融合。 Specifically, when performing a recombinant yeast expression plasmid construction, the Exendin-4-HSA fusion gene inserted into the expression plasmid pWX530 downstream of HSA signal peptide gene, and the two-frame fusion occurs.

[0024] 本发明优选的质粒是pWX530,适合在酵母工程菌中进行蛋白外分泌表达。 [0024] The plasmid of the present invention is preferably pWX530, suitable for the expression of foreign proteins in yeast secretory engineering bacteria. 该质粒多克隆位点的上游带有编码HSA蛋白信号肽的基因序列,其核苷酸序列为:atg aag tgggta acc ttt att tcc ctt ctt ttt ctc ttt age tcg get tat tcc agg ggt gtg ttt cgtCga0该信号肽基因能与插入的外源基因片段进行框内融合。 Upstream of the cloning site of the plasmid carrying the gene sequence encoding the signal peptide of HSA, which nucleotide sequence: atg aag tgggta acc ttt att tcc ctt ctt ttt ctc ttt age tcg get tat tcc agg ggt gtg ttt cgtCga0 signal peptide gene can be fused in frame with the inserted exogenous gene fragment. 但是,本发明中的融合蛋白也可选择利用其它质粒来表达,这些表达质粒包括但是不局限于原核、真核表达系统常用的质粒。 However, fusion proteins of the present invention may be selected using other expression plasmids, the expression plasmids include, but are not limited to, prokaryotic, commonly plasmid eukaryotic expression systems. [0025] 具体地,该表达质粒含有适当的启动子,用以控制融合蛋白的表达。 [0025] In particular, the expression plasmid containing an appropriate promoter for controlling the expression of the fusion protein. 这些启动子包括但是不局限于以下所述的启动子PRB1,优选的启动子为PRB1。 These promoters include, but are not limited to the following of the promoters PRB1, preferred promoters PRB1.

[0026] 本发明为使表达的Exendin-4-HSA融合蛋白不带有任何本融合蛋白以外氨基酸残基,在HSA基因的3'未端加入终止密码子,使融合蛋白转译过程中不会带上表达载体上的任何成份。 [0026] Exendin-4-HSA according to the present invention is that the fusion protein does not contain any of the present fusion proteins other than amino acid residues at the 3 'end of the HSA gene is not added to the stop codon, the fusion protein with the translation process does not the expression of any ingredients on the carrier. 完整的Exendin-4-HSA融合蛋白基因序列如SEQ ID NO :6。 Complete Exendin-4-HSA fusion protein gene sequence is shown in SEQ ID NO: 6.

[0027] 本发明提供构建上述优选的Exendin-4-HSA融合蛋白的表达质粒的构建方法及工程菌构建方法。 [0027] The present invention provides a method for constructing an expression plasmid of Exendin-4-HSA fusion protein construct of the above-described preferred method of constructing and engineering bacteria. (图I) (FIG. I)

首先克隆获得一重组多聚核苷酸序列,该序列含有编码Exendin-4多肽区的核苷酸序列区和编码HSA多肽区的核苷酸序列区。 First, a cloned recombinant polynucleotide sequence comprising the sequence of the nucleotide sequence of the nucleotide sequence of the coding region of HSA polypeptide Exendin-4 polypeptide encoding region. 编码Exendin-4多肽区的氨基酸序列与序列SEQID NO :1保持有至少90%的同源性。 Exendin-4 polypeptide encoding region of an amino acid sequence SEQID NO: 1 retaining at least 90% homology. 编码HSA多肽区的氨基酸序列与序列SEQ ID N0:2保持有至少90%的同源性。 An amino acid sequence SEQ ID N0 polypeptide coding region of HSA: holding at least 90% homology.

[0028] 具体地,通过PCR技术,从人胎肝来源的RNA中反转录获得编码HSA多肽区的核苷酸序列,并使HSA成熟肽基因的3/端带有Nhel的酶切位点。 [0028] In particular, with PCR, reverse transcription from RNA derived from human fetal liver obtained nucleotide sequence encoding a polypeptide region of HSA, and 3 / terminal Nhel restriction site with an HSA gene mature peptide . 编码Exendin-4多肽区的核苷酸序列通过人工合成,获得Exendin-4 DNA片段,并在其5'端带有StuI酶切位点,并使其3'端带上部分HSA成熟肽基因的5'端序列。 Exendin-4 nucleotide sequence encoding the polypeptide synthetically region, the DNA fragment obtained Exendin-4, and at its 5 'end with a StuI restriction site, and allowed to 3' end of the mature peptide gene HSA portion of the belt 5 'end sequence. 将合成的Exendin-4基因片段与HSA成熟肽基因片段混合,采用重叠PCR方法制备Exendin-4-HSA融合蛋白基因片段,Exendin-4-HSA融合蛋白基因的Y端带有StuI酶切位点,3'端带有Nhel的酶切位点。 The synthetic gene fragment of Exendin-4 HSA mixed with mature peptide gene fragment, Exendin-4-HSA fusion protein gene fragment was prepared using the overlap PCR method, Exendin-4-HSA Y terminal fusion proteins with the StuI restriction site, 3 'end with a Nhel restriction site of. 将Exendin-4-HSA融合蛋白基因片段进行TA克隆,制备重组质粒Exendin-4-HSA/pEGM-T。 The Exendin-4-HSA fusion protein gene fragments were TA cloning, the recombinant plasmid prepared Exendin-4-HSA / pEGM-T.

[0029] 用Stul、Nhel对重组质粒Exendin-4-HSA/pEGM-T进行双酶切,制备Exendin-4-HSA基因片段。 [0029] with Stul, Nhel recombinant plasmid Exendin-4-HSA / pEGM-T double digested prepared Exendin-4-HSA gene fragment. 同法对酵母表达质粒pWX530进行双酶切,制备线性化质粒PWX530 DNA片段。 Same method pWX530 double digested yeast plasmids, linearized plasmid expression PWX530 DNA fragment was prepared. 将Exendin-4-HSA基因片段与线性化质粒pWX530 DNA片段按摩尔比3-5 : I的比例混合,用T4 DNA连接酶进行连接,构建重组酵母表达质粒EX-HSA/pWX530。 The Exendin-4-HSA gene fragment and the linearized plasmid pWX530 DNA fragment molar ratio of 3-5: I proportions, with T4 DNA ligase to connect recombinant yeast expression plasmid EX-HSA / pWX530. EX-HSA 即为Exendin-4-HSA 的缩写。 EX-HSA is the abbreviation of Exendin-4-HSA.

[0030] 本发明表达Exendin-4-HSA融合蛋白的重组质粒EX_HSA/pWX530可在多种酵母菌中表达,其优选的酵母菌是酿酒酵母,其优选菌株是CICM Y0600 (江南大学中国高校工业微生物资源与信息中心提供)。 [0030] The present invention is a recombinant expression plasmid EX_HSA Exendin-4-HSA fusion protein / pWX530 may be expressed in a variety of yeast, which is preferably the yeast is Saccharomyces cerevisiae, preferably strains which are CICM Y0600 (University Industrial microorganisms southern China University resource and information Center).

[0031] 本发明提供一种工程菌大规模发酵表达Exendin-4-HSA融合蛋白的方法。 [0031] The present invention provides a method of engineering bacteria Exendin-4-HSA fusion protein expression of large-scale fermentation. [0032] 具体地,将含有重组表达质粒EX_HSA/pWX530的单个酵母菌(CICM Y0600)菌落接种到选择性培养基SD[O. 67%YNB,0. 609%十二水合磷酸氢二钠(Na2HPO4 · 12H20),5. 04%Citric acid (C6H8O7), 2%蔗糖]中,30°C振摇过夜。 [0032] Specifically, a recombinant plasmid containing a single yeast EX_HSA / pWX530 of (CICM Y0600) colonies were inoculated into a selective medium SD [O. 67% YNB, 0. 609% disodium hydrogen phosphate dodecahydrate (the Na2HPO4 · 12H20), 5. 04% Citric acid (C6H8O7), 2% sucrose] is, 30 ° C and shaken overnight. 计算获得OD6tltl=O. 4所需要过夜培养物的总量。 Obtained by calculation OD6tltl = O. The required amount was 4 overnight. 离心收集菌体沉淀,并用选择性培养基SD悬浮,并转种到发酵培养基YPD (1%酵母粉,2%蛋白胨,2%蔗糖)中,30°C振摇培养48h,离心收集发酵上清液,_80°C保存用于融合蛋白表达的分析和纯化。 Precipitate was harvested by centrifugation, and resuspended SD selective medium, and transferred to the seed fermentation medium YPD (1% yeast extract, 2% peptone, 2% sucrose), 30 ° C with shaking 48h, collected by centrifugation the Fermentation serum, _80 ° C saved for analysis and purification for the fusion protein.

[0033] 融合蛋白的表达,可以通过一般的蛋白生化手段检测,包括但不局限于:SDS-PAGE, Western blotting 等。 [0033] The fusion protein may be by a general means for detecting protein biochemistry, including but not limited to: SDS-PAGE, Western blotting and the like.

[0034] 本发明也提供一种Exendin-4-HSA融合蛋白的分离纯化方法。 [0034] The present invention also provides a method for separation and purification of Exendin-4-HSA fusion protein.

[0035] 具体地,该分离纯化方法利用了Exendin-4-HSA的等电点pH 4. 7左右的特点,选择阳离子交换层析柱,使用SP Sepharose FF分离效果最好,目的蛋白质洗脱峰最高,含杂最少。 [0035] In particular, the separation and purification method utilizes isoelectric pH Exendin-4-HSA is about 4.7 features, selecting a cation exchange column, SP Sepharose FF using the best separation effect, the protein elution peak maximum, minimum impurity. 选择SP Seharose FF为填料,成功捕获了发酵液中的Exendin-4_HSA融合蛋白质。 Select SP Seharose FF filler, Exendin-4_HSA successfully captured fusion protein in the fermentation broth. 捕获的Exendin-4-HSA融合蛋白质再经过疏水型层析柱Butyl Sepharose FF,阴离子交换层析DEAE Sepharose FF进行精细纯化,目标蛋白质的纯度可以达98%以上。 Captured Exendin-4-HSA fusion protein and then through the hydrophobic chromatography column Butyl Sepharose FF, the anion-exchange chromatography, DEAE Sepharose FF fine purification, purity of the target protein may be 98% or more.

[0036] 本发明也提供了一种鉴定Exendin-4-HSA融合蛋白生物活性的方法。 [0036] The present invention also provides a method of identifying Exendin-4-HSA fusion protein bioactivity.

[0037] 本发明也提供了一种测定Exendin-4-HSA融合蛋白体外稳定性和体内半衰期鉴定的方法。 [0037] The present invention also provides a method for determination of Exendin-4-HSA fusion protein in vitro stability and in vivo half-life identification. 具体地,检测方法测定融合蛋白的活性的货架寿命(shelf life)以及动物体内的活性半衰期。 Determination of the activity of the fusion protein specifically, the detection method of shelf life (shelf life) and the half-life of activity in animals. 动物包括但不限制于小鼠,狗,猴子,以及人类。 Animals include, but are not limited to mice, dogs, monkeys, and humans.

[0038] 本发明的Exendin-4-HSA融合蛋白可以用于包括糖尿病等症状的治疗。 [0038] Exendin-4-HSA fusion protein of the present invention may be used in the treatment of other symptoms, including diabetes. 给药方式可以是注射,口服,鼻内,呼吸道吸服等。 Administration may be by injection, oral, nasal, respiratory inhale the like.

[0039] 为了提高用药效果,本发明的Exendin-4-HSA融合蛋白也可以制成不同的剂型,包括但不限于使用各种酸碱所形成的盐形式。 [0039] In order to improve the effect of medication, Exendin-4-HSA fusion protein of the present invention can also be made in different forms, including but not limited to various pH using the formed salt form.

[0040] 为了提高用药效果,本发明的Exendin-4-HSA融合蛋白也可以与其他药物共同使用。 [0040] In order to improve the effect of medication, Exendin-4-HSA fusion protein of the present invention may be used together with other drugs.

[0041] 本发明的有益效果:本发明采用基因重组技术,将Exendin-4多肽和人血清白蛋白直接融合,产生促胰岛素分泌肽融合蛋白Exendin-4-HSA,中间不加入任何连接肽,在保持Exendin-4的药理特性的基础上延长其在体内的半衰期,在药学领域包括II型糖尿病及肥胖症治疗将有良好的应用前景。 [0041] Advantageous Effects of Invention: The present invention uses genetic recombination technique, and the Exendin-4 polypeptide fused directly to human serum albumin, exendin generating fusion protein Exendin-4-HSA, does not add any intermediate connecting peptide, in in vivo extended half-life of Exendin-4 based holding the pharmacological properties, the pharmaceutical arts including type II diabetes and obesity treatment will have a good prospect.

附图说明 BRIEF DESCRIPTION

[0042] 图I Exendin-4-HSA融合蛋白重组表达质粒构建图。 [0042] FIG I Exendin-4-HSA fusion protein recombinant plasmid FIG.

[0043] 图2 DNA电泳图(1%琼脂糖);左A为PCR扩增产物状况图;右B为质粒DNA限制性酶切分析图。 [0043] FIG. 2 DNA electrophoresis (1% agarose); PCR product A is left of FIG condition; B is a right restriction analysis of plasmid DNA FIG.

[0044] 图3 10%SDS-PAGE电泳图和Western bloting结果图;左为A蛋白质发酵表达产物SDS-PAGE (10%)电泳图;右为B Western bloting 结果图。 [0044] FIG. 3 10% SDS-PAGE electrophoresis and Western bloting FIG results; left for the A protein expression product of fermentation SDS-PAGE (10%) electrophoresis; B Western bloting right is the result of FIG.

[0045] 图4纯化Exendin-4-HSA融合蛋白SDS-PAGE (10%)电泳图;1、蛋白质分子量标志物,2、Exendin-4-HSA融合蛋白纯化产物。 [0045] FIG purified Exendin-4-HSA fusion protein SDS-PAGE (10%) electrophoresis; 1, protein molecular weight markers, 2, Exendin-4-HSA fusion protein was purified product.

[0046] 图5小鼠糖耐量试验血糖浓度曲线图。 [0046] FIG. 5 glucose tolerance test in mice blood glucose concentration graph.

[0047] 图6血浆胰岛素测定图。 [0047] FIG 6 FIG plasma insulin assay. [0048] 图7 Exendin-4-HSA融合蛋白的长效活性观察图。 [0048] FIG. 7 Exendin-4-HSA fusion protein depot activity observed in FIG.

[0049] 图8糖尿病模型鼠BSK db/db第1-11天给药后的各种变化图;A、糖尿病模型鼠BSK db/db第1-11天血糖变化;B、糖尿病模型鼠BSK db/db第1-11天体重变化;C、糖尿病模型鼠BSK db/db第1-11天摄食变化;D、糖尿病模型鼠BSK db/db第1-11天饮水变化。 [0049] FIG. 8 diabetic mice models BSK db / db 1-11 days after the administration FIG variations; A, diabetic model rats BSK db / db 1-11 day changes in blood sugar; B, BSK db diabetic mouse model / db 1-11 days weight change; C, diabetic model rats BSK db / db 1-11 days feeding change; D, diabetic mice models BSK db / db 1-11 days water changes.

[0050] 图9食蟹猴单次给药测得的血药浓度-时间曲线图;A、食蟹猴单次皮下给药250μδ^δ^时测得的血药浓度-时间曲线;Β、食蟹猴单次皮下给药SOOPg^kg—1时测得的血药浓度-时间曲线;C、食蟹猴单次皮下给药时测得的血药浓度-时间曲线;D、食蟹猴单次静脉给药soogg.kg—1时测得的血药浓度-时间曲线。 [0050] FIG. 9 cynomolgus monkeys single dose measured plasma concentration - time curve; A, cynomolgus measured when administered subcutaneously 250μδ ^ δ ^ single plasma concentration - time curve; Β, cynomolgus monkeys administered a single subcutaneous SOOPg ^ kg-1 when measured plasma concentration - time curve; measured when administered subcutaneously C, cynomolgus monkeys single plasma concentration - time curve; D, cynomolgus single intravenous administration soogg.kg-1 measured at the plasma concentration - time curve.

具体实施方式 Detailed ways

[0051] 以下所提供的实施例,可以更详细地解释本发明内容。 [0051] Example embodiments provided below, the present invention can be explained in more detail. 但是,本发明的内容并不限于这些实施例所阐述的内容。 However, the present invention is not limited to the contents of these examples set forth herein. [0052] 实施例I :人白蛋白基因克隆 [0052] Example I: Cloning of human albumin

采用RT-PCR制备人HSA成熟肽基因,引物设计如下: RT-PCR was prepared using HSA human mature peptide gene, primers were designed as follows:

HSAl:5/ -GATGCACACA AGAGTGAGGT TGCTCA-3', HSAl: 5 / -GATGCACACA AGAGTGAGGT TGCTCA-3 ',

HSA2:5; -AAGCTAGCTT ATAAGCCTAA GGCAGCTTG ACTTGC-3'。 HSA2: 5; -AAGCTAGCTT ATAAGCCTAA GGCAGCTTG ACTTGC-3 '.

[0053] 取人胎肝组织,用Trizol法制备总RNA。 [0053] taken from human fetal liver tissue, total RNA was prepared with Trizol method. RT-PCR扩增:一个O. 2mL的PCR管中,加入2XRT PCR 缓冲液25μί,总RNA 3μΐ (50ng),引物HSAl O. 5μΐ (25μΜ)、HSA2 O. 5μΐ(25μΜ),RT-Phusion 混合酶ΐμί,加DEPC 水到终体积50μί。 RT-PCR amplification: PCR O. 2mL a tube was added 2XRT PCR buffer 25μί, total RNA 3μΐ (50ng), primers HSAl O. 5μΐ (25μΜ), HSA2 O. 5μΐ (25μΜ), RT-Phusion mixed enzyme ΐμί, DEPC water was added to a final volume of 50μί. PCR 条件:45°C 30min ;然后94°C变性2min ;再按94°C、30sec ;55°C、lmin,72°C、2min,一共30 个循环。 PCR conditions were: 45 ° C 30min; 2min and 94 ° C denaturation; then 94 ° C, 30sec; 55 ° C, lmin, 72 ° C, 2min, 30 cycles total. 最后,72°C保温5min。 Finally, 72 ° C incubation 5min. 将PCR产物进行琼脂糖凝胶电泳观察扩增结果与产物大小。 The PCR product was subjected to agarose gel electrophoresis and the amplification product size. 然后采用胶回收法纯化HSA基因片段,具体方法是在电泳后琼脂糖凝胶上切下分子量在1750bp左右DNA条带,再用Promega公司的PCR产物胶回收试剂盒纯化HSA基因DNA片段,方法按操作说明书。 HSA gene fragment was then purified using gel extraction method, a specific method is in the agarose gel was cut at about 1750bp DNA molecular weight band, then the Promega PCR product gel extraction kit HSA gene DNA fragment was purified according to the method operating instructions.

[0054] 实施例2 :Exendin_4基因合成 Gene Synthesis Exendin_4: Example 2 [0054] Embodiment

Exendin-4基因采用人工合成方法制备。 The method of preparing synthetic genes using Exendin-4. 按SEQ ID NO :5的序列由上海生工生物工程技术服务有限公司Exendin-4基因进行合成,并使其5'末端带StuI的酶切位点。 According to SEQ ID NO: 5 is the sequence synthesized by Shanghai Sangon Biological Engineering Technology Services Ltd. genetic engineering Exendin-4 and 5 so-terminal cleavage site of StuI '.

[0055] 实施例3 :Exendin-4-HSA融合基因的构建与克隆 Construction and Cloning of Exendin-4-HSA fusion gene: [0055] Example 3

本实验采用重叠PCR方法将Exendin-4和HSA基因直接融合构成Exendin-4-HSA融合基因为例,说明合成融合基因的方法。 This method of overlap PCR experiment using the method for forming fused directly Exendin-4-HSA fusion gene as an example, it illustrates the synthesis of Exendin-4 fusion gene and HSA gene. 具体方法如下:在不加引物和聚合酶的条件下,按照以下体系混合各成分。 Specific methods are as follows: under the conditions without primers and polymerase, mixing the ingredients according to the following system.

[0056] 5 Xphusion HF buffer 20 μ L, dNTPs(10 mM) 2 μ L, Exendin-4 DNA 片段2 μ L,HSA DNA 片段2 μ L,ddH20 69 μ L,总体积95 μ L。 [0056] 5 Xphusion HF buffer 20 μ L, dNTPs (10 mM) 2 μ L, Exendin-4 DNA fragment 2 μ L, HSA DNA fragment of 2 μ L, ddH20 69 μ L, total volume of 95 μ L.

[0057] 99°C变性10 min,然后置室温自然冷却。 [0057] 99 ° C denaturation 10 min, then allowed to cool in room temperature. 加入Phusion高保真酶O. 5 μ L,72°C保温15min,然后再加入引物 Add Phusion High Fidelity enzyme O. 5 μ L, 72 ° C incubation 15min, then added primer

Exl :(5/ -TAAGGCCTCACGGTGAAGGTACTTTCACTT-3' , Stul) (50 μ M)和HSA2 :(5/ -AAGCTAGCTTATAAGCCTAAGGCAGCTTGACTTGC-3',Nhel) (50 μ Μ)各2 μ L,Phusion高保真酶O. 5 μ L,进行常规PCR扩增: Exl: (5 / -TAAGGCCTCACGGTGAAGGTACTTTCACTT-3 ', Stul) (50 μ M) and HSA2: (5 / -AAGCTAGCTTATAAGCCTAAGGCAGCTTGACTTGC-3', Nhel) (50 μ Μ) each of 2 μ L, Phusion High Fidelity enzyme O. 5 μ L, conventional PCR amplification:

98°C预变性30 sec,98°C变性10 sec,72°C退火30 sec,进行35个循环。 98 ° C denaturation for 30 sec, 98 ° C denaturation 10 sec, 72 ° C annealing 30 sec, 35 cycles. 最后在72 V保温7min。 Finally, 72 V incubated 7min. 将扩增产物通过1%琼脂糖凝胶电泳分析,观察扩增产物状况(图2A)。 The amplified product was purified by 1% agarose gel electrophoresis, the amplified product was observed condition (FIG. 2A). 用PCR产物纯化试剂盒纯化融合基因Exendin-4-HSA DNA片段,操作方法按试剂盒说明书。 Gene Exendin-4-HSA DNA fragment purification kit fusion PCR product, method of operation according to kit instructions.

[0058] 融合基因的TA克隆:将纯化融合基因产物在只含脱氧三磷酸腺嘌呤的PCR反应体系中,在Taq DNA聚合酶的作用下进行5'端加“A”尾。 [0058] TA cloning of the fusion genes: the gene product in the purification of the fusion PCR reaction system containing only deoxy-triphosphates of adenine, the 5 'end of the added "A" at the end of the action of Taq DNA polymerase. 反应体系如下:10XTaq polymerasebuffer 10 μ L, MgCl2 (2.5 mM) 10 μ L,dATP (2 mM) 10 μ L,纯化的PCR 产物60 μ L,灭菌去离子H2O 9μ L, Taq DNA聚合酶I μ L,总体积80 μ L。 The reaction system as follows: 10XTaq polymerasebuffer 10 μ L, MgCl2 (2.5 mM) 10 μ L, dATP (2 mM) 10 μ L, purified PCR product was 60 μ L, sterile deionized H2O 9μ L, Taq DNA polymerase I μ L, a total volume of 80 μ L. 在PCR仪器上72°C反应30 min。 In the PCR reaction the instrument 72 ° C 30 min. 用PCR产物纯化试剂盒纯化加“A”尾的Exendin-4-HSA基因片段。 PCR product was purified using purification kit plus "A" tail Exendin-4-HSA gene fragment. 将Exendin-4-HSA片段与TA clone载体pGEM_T按照分子摩尔数3_5 : I的比例混合,在T4 DNA连接酶的作用下连接,16°C水浴连接过夜,构建含融合基因的重组质粒EX-HSA/pGEM-T。 The Exendin-4-HSA fragment TA clone vector pGEM_T molecule according moles 3_5: I proportions connected under the action of T4 DNA ligase to, 16 ° C water bath ligated overnight recombinant plasmid EX-HSA containing the fusion gene / pGEM-T. 连接产物电转化入感受态E.coli DH5,涂布于含100 μ g/mL氨节青霉素(ΑΜΡ)、40 μ L X-gal (20 mg/mL)的LB平板上,37°C培养过夜。 The ligation product was transformed into competent E.coli DH5, a coating containing 100 μ g / mL ampicillin (ΑΜΡ), a 40 μ L X-gal (20 mg / mL) LB plates, 37 ° C overnight culture . 挑取AMP平板上生长的单个菌落进行质粒DNA限制性酶切分析(图2B)和DNA测序确定融合基因序列的准确性。 Single colonies were picked and grown on AMP plates plasmid DNA restriction analysis (FIG. 2B) and DNA sequencing to determine the accuracy of the fusion gene sequence.

[0059] 实施例4 :Exendin-4_HSA表达质粒的构建 [0059] Example 4: Exendin-4_HSA Construction of expression plasmid

以下以Exendin-4-HSA融合基因为例,说明表达载体质粒的构建。 In the following Exendin-4-HSA fusion gene as an example, a plasmid expression vector described. 本实验采用pWX530为表达载体质粒,其制备方法为常用分子生物学方法。 This experiment pWX530 plasmid is an expression vector, which is a common method of preparing molecular biology methods. 即通过培养含有表达载体的菌种,提取获得该质粒。 I.e. by culturing strains containing the expression vector, the plasmid obtained is extracted. 制备质粒后_20°C保存待用。 After plasmid preparation save _20 ° C until use.

[0060] 具体如下,先对表达载体质粒PWX530进行Stul和Nhel双酶切。 [0060] As follows, the first expression vector plasmid Nhel and Stul PWX530 for double digestion. 具体条件如下。 Specific conditions were as follows. 表达载体质粒PWX530 ΙΟμί ;Stul ΐμί,Nhel ΐμί, 10ΧΤ4 DNA Iigase 缓冲液5μί ;ddH2033μί,总体积为50μί。 Expression vector plasmid PWX530 ΙΟμί; Stul ΐμί, Nhel ΐμί, 10ΧΤ4 DNA Iigase buffer 5μί; ddH2033μί, total volume 50μί. 37°C恒温水浴锅内反应3小时,通过琼脂糖凝胶电泳回收线性化的PWX530质粒DNA。 37 ° C water bath with constant temperature for 3 hours, PWX530 linearized plasmid DNA was recovered by agarose gel electrophoresis. Exendin-4-HSA/pGEM-T DNA作同样的双酶切,通过琼脂糖凝胶电泳回收分子量为1891bp的Exendin-4-HSA基因片段。 Exendin-4-HSA / pGEM-T DNA for the same double digestion, recovered by agarose gel electrophoresis the molecular weight of Exendin-4-HSA gene fragment of 1891bp.

[0061] 将回收的Exendin-4-HSA DNA与上述双酶切的表达质粒pWX530DNA进行连接,构建融合蛋白表达质粒EX-HSA/pWX530。 [0061] The recovered Exendin-4-HSA DNA double digested with the aforementioned expression plasmid pWX530DNA connected, fusion protein expression plasmid constructed EX-HSA / pWX530. 连接体系一般为ΙΟμί,双酶切的pWX530质粒载体与双酶切Exendin-4-HSA DNA的摩尔比为I : 2_10,10XT4 DNA Iigase缓冲液ΐμί,加无菌水至总体积为10μί。 Connecting system generally ΙΟμί, pWX530 molar ratio of double-digested plasmid vector digested with Exendin-4-HSA DNA is I: 2_10,10XT4 DNA Iigase buffer ΐμί, sterile water added to a total volume 10μί. 连接反应16°C恒温水浴锅内反应16小时。 The ligation reaction 16 ° C water bath with constant temperature for 16 hours. 连接产物转化大肠杆菌DH5a感受态细胞。 The ligation product was transformed into E. coli DH5a competent cells. 阳性克隆的鉴定是通过含Ampicilin的LB平板挑选阳性克隆,抽提质粒,并进行DNA测序确定融合基因序列的准确性。 Positive clones were identified positive clones are selected using the LB plates containing the Ampicilin, plasmids were extracted, and DNA sequencing to determine the accuracy of the fusion gene sequence.

[0062] 实施例5 :Exendin-4-HSA融合蛋白表达工程菌的构建 [0062] Example 5: Exendin-4-HSA fusion protein constructs engineered strain of

本实验利用电转化方法和Exendin-4-HSA融合基因为例,说明Exendin-4-HSA融合蛋白表达工程菌构建的方法。 In this experiment, electroporation method and Exendin-4-HSA fusion gene as an example, a method for constructing bacterial expression of protein engineering Exendin-4-HSA fusion. 具体方法如下。 Specific methods are as follows.

[0063] 首先挑选含有EX_HSA/pWX530表达载体质粒的细菌克隆,提取表达载体质粒。 [0063] First, the selection of bacteria containing EX_HSA / pWX530 expression cloning vector plasmid, a plasmid expression vector extraction. 然后,用电转化的方法把质粒转入酿酒酵母INVScl中。 Then, the transformed electrochemically into the plasmid in Saccharomyces cerevisiae INVScl. 质粒DNA提取方法按Promega公司质粒DNA纯化试剂盒操作说明进行。 Extraction of plasmid DNA Plasmid DNA was purified according to Promega's instructions for the kit.

[0064] 电转化方法转化酿酒酵母具体如下。 [0064] Methods for transforming S. cerevisiae was transformed as follows.

[0065] 先进行菌体的准备。 [0065] to prepare bacterial cells. 挑取酵母单菌落,接种至含有5mL YPD培养基的50mL三角瓶中,30°C、250-300r/min培养过夜。 Single yeast colonies were picked, inoculated into a flask containing 50mL 5mL YPD culture media, 30 ° C, 250-300r / min overnight. 然后取100_500μί的培养物接种至含有500mL新鲜培养基的2L三角摇瓶中,28〜30°C、250-300 r/min培养过夜,至OD6tltl达到1-1. 5。 100_500μί then take the culture was inoculated into fresh medium containing 500mL triangular 2L shake flasks, 28~30 ° C, 250-300 r / min cultured overnight to reach OD6tltl 1-1. 5. 再将细胞培养物于4°C,1500g离心5 min,用500 mL的冰预冷的ddH20将菌体沉淀重悬。 Then the cell culture at 4 ° C, 1500g centrifugation 5 min, with 500 mL of ice-cold ddH20 the cell pellet was resuspended. 离心后,再用250mL的冰预冷的ddH20将菌体沉淀重悬。 After centrifugation, then 250mL of ice-cold ddH20 the cell pellet was resuspended. 再离心,用20mL的冰预冷的I mol的山梨醇溶液将菌体沉淀重悬。 Centrifuged again, 20mL of ice-cold sorbitol solution I mol bacterial pellet was resuspended. 再离心,用ImL的冰预冷的lmol/L的山梨醇溶液将菌体沉淀重悬,其终体积约为I. 5mL。 Centrifuged again with ice-cold ImL of lmol / L sorbitol solution cell pellet was resuspended at a final volume of about I. 5mL.

[0066] 然后用电击方法进行转化。 [0066] The method then transformed with the shock. 具体如下。 details as follows.

[0067] 将5〜2(^g EX-HSA/ pffX530表达质粒DNA溶解在5〜ΙΟμί TE溶液中,与80μί的上述感受态菌体混勻,转至O. 2 cm冰预冷的电转化杯中。将电转化杯冰浴5 min。然后根据相应的电转化仪,采用优化的电压、电流、电容等参数,进行电击。电击完毕后,加入I mL冰预冷的山梨醇溶液将菌体混匀,转至I. 5 mL的EP管中,将菌体悬液涂布于SD选择性平板上,每20(Γ600μί涂布一块平板。将平板置于30°C培养,直至单个菌落出现。本实验推荐电击参数为:电压I. 5kV ;电容25PF ;电阻200 Ω。电击时间为4〜10 msec。 [0067] The 5~2 (^ g EX-HSA / pffX530 expression plasmid DNA was dissolved in 5~ΙΟμί TE solution, the above-described kneading 80μί competent cells, electrically transferred to ice cold O. 2 cm conversion cup. the ice bath was electroporated into the cup 5 min. then transformed according to the corresponding electrical device with an optimized voltage, current, capacitance and other parameters, a shock after the shock is finished, was added I mL of ice-cold sorbitol solution and the bacteria is mixing body, go to I. 5 mL of EP tube, the cell suspension was spread on SD selection plates every 20 (Γ600μί coating a plate. the plate was placed in 30 ° C incubation, single colonies until this experiment appears shock recommended parameters: voltage I. 5kV; 25PF capacitor; shock resistance 200 Ω time 4~10 msec..

[0068] 实施例6 :Exendin-4-HSA融合蛋白表达和纯化 [0068] Example 6: Exendin-4-HSA fusion protein expression and purification

将EX-HSA/pWX530质粒电转化酵母,经SD筛选培养基平板挑选较饱满的菌落接于SD筛选培养基中扩大培养,菌体生长到一定浓度后接于Yro培养基中发酵表达融合蛋白质。 The EX-HSA / pWX530 plasmid was transformed yeast selection medium SD plate was picked colonies than full contact with the culture expanded in SD selection medium, the cells were grown to a concentration of the fermentation medium connected to Yro expression of the fusion protein. 发酵液经离心,取上清液进行SDS-PAGE分析显示70 kDa的蛋白质表达产物条带(图3A),·Western bloting试验显示能与Anti-Exendin_4抗体有特异性结合(图3B)。 Analysis by SDS-PAGE fermentation broth was centrifuged and the supernatant show the protein expression product of 70 kDa band (FIG. 3A), · Western bloting trials have shown to bind specifically (FIG. 3B) and Anti-Exendin_4 antibody.

[0069] 将发酵上清液过SP Sepharose FF柱,捕获的Exendin-4_HSA融合蛋白质,再经过疏水型层析柱Butyl Sepharose FF,阴离子交换层析DEAE Sepharose FF分离精细纯化,获得纯度可以达98%以上的Exendin-4-HSA (图4)。 [0069] The fermentation supernatant was SP Sepharose FF column, the captured fusion protein Exendin-4_HSA, hydrophobic chromatography column and then through the Butyl Sepharose FF, DEAE Sepharose FF anion exchange chromatography separated fine purification, the purity can be 98% above Exendin-4-HSA (FIG. 4).

[0070] 实施例7 :小鼠耐糖试验 [0070] Example 7: glucose tolerance test in mice

C57BL/6 小鼠,雌雄各半,禁食18h, IP 注射Exendin-4 (10 μ g/kg)或Exendin-4-HSA(表达纯化)、Exendin-4-HSA (lmg/kg)。 C57BL / 6 mice, male and female, were fasted for 18 h, (expression and purification) IP injection of Exendin-4 (10 μ g / kg) or Exendin-4-HSA, Exendin-4-HSA (lmg / kg). Ih后注射葡萄糖(I. 5mg/kg, IP),注射葡萄糖后分别于10、20、30、60、1201^11尾静脉采血,用血糖仪测定血样中葡萄糖含量,观察上述待试品对小鼠血糖的影响。 Injection of glucose (I. 5mg / kg, IP), after injection of glucose in the blood vein 10,20,30,60,1201 ^ 11, blood glucose content by the glucose meter ih, respectively, were observed to be the above-described test of the smaller of rat blood sugar. 结果表明给药能有效地降低空腹血糖峰浓度,同时使血糖浓度曲线趋于平稳(图5)。 The results show that the administration can be effectively reduced fasting blood glucose concentration peak, while the blood glucose concentration profile leveled (FIG. 5).

[0071] 实施例8 :Exendin-4-HSA融合蛋白的促胰岛素分泌作用 [0071] Example 8: insulin secretion Exendin-4-HSA fusion protein

应用糖耐量试验模型,C57BL/6J小鼠注射Exendin-4-HSA融合蛋白,共有20(^g/kg和lmg/kg两个剂量组,给药I. Oh后,腹腔注射葡萄糖I. 5mg/g,于20min时取尾静脉血,采用酶联免疫吸附方法(ELISA,—抗Anti-insulin antibody D6C4 ;配对二抗Anti-insulinbiotinlated antibody D3E7,由ABC Antibody, com供应)对小鼠血清中的膜岛素进行检测,观察Exendin-4-HSA是否能有效地促进胰岛素分泌。与对照组(注射生理盐水)相比,Exendin-4-HSA融合蛋白组的胰岛素含量均显著升高,与Exendin-4有类似的作用(图6)。由此可见,注射Exendin-4-HSA融合蛋白能有效地促进胰岛素分泌。 Application of glucose tolerance test model, C57BL / 6J mice were injected Exendin-4-HSA fusion protein, a total of 20 (^ g / kg and lmg / kg dose of two groups after administration I. Oh, intraperitoneal injection of glucose I. 5mg / g, when taken in the tail vein 20min, enzyme-linked immunosorbent assay (ELISA, - anti-anti-insulin antibody D6C4; paired secondary antibody anti-insulinbiotinlated antibody D3E7, by the ABC Antibody, com supply) film in mouse serum insulin is detected, Exendin-4-HSA was observed whether or not effective in promoting insulin secretion, compared with the control group (normal saline), Exendin-4-HSA fusion protein groups insulin levels were significantly increased, and Exendin-4 has a similar effect (FIG. 6). Thus, injection of Exendin-4-HSA fusion protein effective to promote insulin secretion.

[0072] 实施例9 :Exendin-4-HSA融合蛋白的长效活性观察 9 [0072] Example: Exendin-4-HSA fusion protein activity observed depot

给小鼠注射Exendin-4-HSA (1.0mg/kg, IP),然后于1、24、48、72、96小时注射葡萄糖(IP),注射葡萄糖后0、10、20、30、60、120min时间点取血样,用血糖仪测定血样中葡萄糖含量,观察Exendin-4-HSA在不同注射葡萄糖时间点对小鼠血糖的影响。 Mice were injected after 0,10,20,30,60,120min Exendin-4-HSA (1.0mg / kg, IP), and then 1,24,48,72,96 hours in glucose injection (IP), the injection of glucose time points blood samples were taken, blood glucose content, Exendin-4-HSA was observed effect on blood glucose in mice at different time points was determined by glucose injection the blood glucose meter. 与阴性对照HSA相比,Exendin-4-HSA明显降低血糖峰值,血糖浓度波动较小,显示明显降低血糖作用,并具有长效特性(图7)。 Compared with the negative control HSA, Exendin-4-HSA decreased peak glucose, blood glucose levels fluctuate less, show significantly lower blood glucose, and having a long-term characteristic (FIG. 7).

[0073] 实施例10 :Exendin-4-HSA融合蛋白在糖尿病模型鼠药效的观察 [0073] Example 10: Protein observed in diabetic mice models efficacy Exendin-4-HSA fusion

糖尿病模型鼠BSK db/db,其特征为高血糖(19-32 mmol/L,血糖为正常鼠的2-3倍),肥胖(40-56克/只,体重为正常鼠的2-3倍),本实验通过小鼠注射Exendin-4-HSA (I. Omg/kg, IP),每两天一次,共12 天,观察给药后30min,60 min, 2h, 24h,48h 血糖值;1、3、5、7、9、11天饮水,饮食,体重变化。 Diabetic mice models BSK db / db, which is characterized by high blood sugar (19-32 mmol / L, glucose 2-3 times normal mice), obesity (40-56 g /, weighing 2-3 times the normal mice ), this experiment mice were injected with Exendin-4-HSA (I. Omg / kg, IP), once every two days, 12 days, 30min observed after administration, 60 min, 2h, 24h, 48h blood glucose level; 1 , 3,5,7,9,11 day drinking, diet, weight changes. 与阴性对照药HSA相比,Exendin-4-HSA明显降低糖尿病模型鼠BSK db/db血糖峰值,具有显著降低血糖浓度的作用,并具有长效特性,结果见图8A ;明显减少糖尿病模型鼠BSK db/db的体重、摄食及饮水量结果见图8 B、8C、8D。 Compared to the negative control drug HSA, Exendin-4-HSA significantly reduced diabetic mice models BSK db / db peak glucose, with significantly reduced blood glucose concentration effect, and have long-lasting properties, the results shown in Figure. 8A; significantly reduced diabetic mice models BSK db / db of body weight, food and water intake results shown in Figure 8 B, 8C, 8D.

[0074] 实施例11 :Exendin-4_HSA融合蛋白的体内药代动力学 [0074] Example 11: Exendin-4_HSA fusion protein in vivo pharmacokinetics

给食蟹猴采用单剂量静脉注射Exendin-4-HSA融合蛋白500Pg/kg或皮下注射250Pg/kg、500Pg/kg、75(^g/kg低、中、高三个剂量组。二房室模型(weight=l/C2)分析获得的药代动力学参数显示,静脉注射Exendin-4-HSA融合蛋白(500Pg/kg)时,消除半衰期大约为140. 6小时。皮下注射Exendin-4-HSA融合蛋白低、中、高三个剂量组消除半衰期分别为98. 8、100. 1、102. 8小时,组间无显著差异(P > O. 05)(图9A,9B,9C)。皮下注射Tmax为12. 5-27. 3h (图9D)。给药剂量与AUC和Cmax呈线形关系。清除率CLz/F为I. OmL/h/kg。生物利用度为82. 1%。食蟹猴的血样中Exendin-4-HSA融合蛋白生物活性检测结果表明皮下注射50(^g/kg的Exendin-4-HSA融合蛋白药物活性维持时间长达72〜96小时。 To cynomolgus monkeys by single-dose intravenous injection of Exendin-4-HSA fusion protein 500Pg / kg or subcutaneous 250Pg / kg, 500Pg / kg, 75 (^ g / kg of low, medium, and high dose groups. Two compartment model (weight time = l / C2) analysis of pharmacokinetic parameters obtained show that intravenous injection of Exendin-4-HSA fusion proteins (500Pg / kg), elimination half-life of approximately 140.6 hours. subcutaneous injection of Exendin-4-HSA fusion protein low , medium and high dose groups respectively 98. the elimination half life of 8,100. 1,102. 8 hours, no significant difference (P> O. 05) (FIGS. 9A, 9B, 9C) between the two groups. Tmax is 12 subcutaneous . 5-27. 3h (FIG. 9D). AUC and Cmax with dose was linear relationship clearance CLz / F to I. OmL / h / kg. bioavailability of 82.1%. cynomolgus monkey blood samples of Exendin-4-HSA fusion protein bioassay results showed that subcutaneous injection 50 (^ g / kg of Exendin-4-HSA fusion protein of the pharmaceutical active is maintained for up to 72~96 hours.

Claims (6)

1. 一种Exendin-4肽与人血清白蛋白HSA的融合蛋白Exendin-4-HSA,其特征在于该融合蛋白含有2个多肽区,其I区为一个Exendin-4多肽,其2区为人血清白蛋白HSA,I区通过其C-末端与第2区的N-末端直接连接,中间不加入任何连接肽,其结构式是:Exendin-4-HSA ; I区Exendin-4多肽氨基酸序列与序列SEQ ID NO :1保持有至少90%的同源性; 2区多肽选自人血清白蛋白HSA的成熟肽,其氨基酸序列与序列SEQ ID NO :2保持有至少90%的同源性; 所述的Exendin-4-HSA融合蛋白,其核苷酸序列编码为SEQ ID NO :3 ; 所述的Exendin-4-HSA融合蛋白,其氨基酸序列为SEQ ID NO :7 ; 所述的Exendin-4-HSA融合蛋白,其重组核苷酸序列插入在一种表达载体和细胞宿主系统。 A peptide of Exendin-4 HSA human serum albumin fusion protein Exendin-4-HSA, characterized in that the fusion protein contains two polypeptide region, the I region is a Exendin-4 polypeptide, which region is human serum 2 albumin HSA, I region via its C- terminus to the N- terminus of the second region is directly connected, without adding any intermediate connecting peptide, the structural formula is: Exendin-4-HSA; I region amino acid sequence of Exendin-4 polypeptide with the sequence SEQ ID NO: 1 retaining at least 90% homology; mature peptide region 2 polypeptide selected from human serum albumin HSA, which is an amino acid sequence SEQ ID NO: 2 retaining at least 90% homology; the the Exendin-4-HSA fusion protein, which nucleotide sequences encode SEQ ID NO: 3; the Exendin-4-HSA fusion protein, the amino acid sequence of SEQ ID NO: 7; the Exendin-4- HSA fusion protein, which recombinant nucleotide sequence is inserted in an expression vector and host cell systems.
2.权利要求I所述Exendin-4-HSA融合蛋白的制备方法,其特征在于包括转录,翻译,蛋白分离和纯化,以及鉴定步骤;步骤为: 通过PCR技术,从人胎肝来源的RNA中反转录获得编码HSA多肽区的核苷酸序列,并使HSA成熟肽基因的3'端带有Nhel的酶切位点;编码Exendin-4多肽区的核苷酸序列通过人工合成,获得Exendin-4 DNA片段,并在其5'端带有StuI酶切位点,并使其3'端带上部分HSA成熟肽基因的5'端序列;将合成的Exendin-4基因片段与HSA成熟肽基因片段混合,采用重叠PCR方法制备Exendin-4-HSA融合蛋白基因片段,Exendin-4-HSA融合蛋白基因的Y端带有StuI酶切位点,3'端带有Nhel的酶切位点;将Exendin-4-HSA融合蛋白基因片段进行TA克隆,制备重组质粒Exendin-4-HSA/pEGM-T ; 用Stul、Nhel 对重组质粒Exendin-4-HSA/pEGM-T 进行双酶切,制备Exendin-4-HSA 基因片段; 同法对酵母表达质粒PWX530进行双酶切,制备 2. I claim the preparation of protein Exendin-4-HSA fusion, comprising transcription, translation, protein separation and purification, and the identifying step; step: by PCR technique derived from human fetal liver RNA, reverse transcription to obtain a nucleotide sequence encoding HSA polypeptide region, and 3 'end with a Nhel restriction site in the mature peptide HSA gene; nucleotide sequence encoding Exendin-4 polypeptide region synthetically obtained Exendin -4 DNA fragment, and its 5 'end with a StuI restriction site, and allowed to 3' 5 'end of the sequence of the mature peptide band portion HSA gene; Exendin-4 gene fragment with a synthetic peptide of the mature HSA gene fragments were mixed, prepared using the overlap PCR method Exendin-4-HSA fusion protein gene fragment, Exendin-4-HSA Y terminal fusion proteins with the StuI restriction site, 3 'end with a Nhel restriction site of; the Exendin-4-HSA fusion protein gene fragments were TA cloning, the recombinant plasmid prepared Exendin-4-HSA / pEGM-T; with Stul, Nhel recombinant plasmid Exendin-4-HSA / pEGM-T double digested, prepared Exendin -4-HSA gene fragment; the same method digested yeast expression plasmid PWX530, prepared 性化质粒pWX530 DNA片段;将Exendin-4-HSA基因片段与线性化质粒pWX530 DNA片段按摩尔比3-5 : I的比例混合,用T4 DNA连接酶进行连接,构建重组酵母表达质粒EX-HSA/pWX530 ;EX-HSA即为Exendin-4-HSA 的缩写; 重组质粒EX-HSA/pWX530可在多种酵母菌中表达,其优选的酵母菌是酿酒酵母,其优选菌株是CICM Y0600。 PWX530 DNA fragment of the plasmid; to Exendin-4-HSA gene fragment and the linearized plasmid pWX530 DNA fragment molar ratio of 3-5: I proportions, with T4 DNA ligase to connect recombinant yeast expression plasmid EX-HSA / pWX530; EX-HSA is the abbreviation of Exendin-4-HSA; recombinant plasmid EX-HSA / pWX530 may be expressed in a variety of yeast, which is preferably the yeast is Saccharomyces cerevisiae, preferably strains which are CICM Y0600.
3. 一种权利要求I所述Exendin-4-HSA融合蛋白的发酵表达方法,其特征在于将含有重组表达质粒EX-HSA/pWX530的单个酵母菌CICM Y0600菌落接种到选择性培养基SD中,30°C振摇过夜,计算获得OD6tltl=O. 4所需要过夜培养物的总量;离心收集菌体沉淀,并用选择性培养基SD悬浮,并转种到发酵培养基YPD中,30°C振摇培养48h,离心收集发酵上清液,_80°C保存用于融合蛋白表达的分析和纯化; 选择性培养基SD为:0. 67%YNB,0. 609%十二水合磷酸氢二钠,5. 04% Citric acidC6H8O7, 2%蔗糖的溶液; 发酵培养基YPD为1%酵母粉,2%蛋白胨,2%蔗糖的溶液。 I 3. The method of Claim Fermentation Expression of the fusion protein-4-HSA Exendin, characterized by containing a single recombinant yeast expression plasmid CICM Y0600 colonies EX-HSA / pWX530 seeded in selective medium SD, shaken overnight at 30 ° C, OD6tltl = O 4 obtained by calculation amount needed was an overnight culture;. the precipitate harvested by centrifugation, and resuspended SD selective medium, and transferred to the fermentation medium of YPD species, 30 ° C shaking culture 48h, fermentation supernatant was collected by centrifugation, _80 ° C and stored for analysis of the purified fusion protein expression; selective medium SD is: 0 67% YNB, 0,609% Sodium disodium hydrogen phosphate dihydrate. , 5 04% Citric acidC6H8O7, 2% sucrose solution; fermentation YPD medium 1% yeast extract, 2% peptone, 2% sucrose solution.
4.权利要求I所述Exendin-4-HSA融合蛋白的应用,其特征在于用于制备非胰岛素依赖性II型糖尿病及肥胖者的治疗制剂;给药方式选用注射,口服,鼻内,呼吸道吸服。 I 4. Claim applications Exendin-4-HSA fusion protein, wherein the formulations for the treatment of non-insulin dependent Type II diabetes and obesity prepared; mode of administration selected injection, oral, nasal, respiratory suction clothes.
5.根据权利要求4所述Exendin-4-HSA融合蛋白的应用,其特征在于为了提高用药效果,Exendin-4-HSA融合蛋白可以制成不同的剂型,包括但不限于使用各种酸碱所形成的盐形式。 5. The 4 Exendin-4-HSA fusion applications as claimed in claim proteins, characterized in that in order to improve the effect of medication, Exendin-4-HSA fusion protein may be made in different forms, including but not limited to the use of various pH salt formation.
6.根据权利要求4所述Exendin-4-HSA融合蛋白的应用,其特征在于为了提高用药效果,Exendin-4-HSA融合蛋白也可以与其他药物共同使用。 6. The 4 Exendin-4-HSA fusion applications as claimed in claim proteins, characterized in that in order to improve the effect of medication, Exendin-4-HSA fusion protein may also be used together with other drugs.
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CN103204944A (en) * 2013-03-26 2013-07-17 江苏健德生物药业有限公司 Long-acting immune fusion protein for treating diabetes mellitus
CN103613670A (en) * 2013-11-21 2014-03-05 中国人民解放军军事医学科学院生物工程研究所 HSA (Human Serum Albumin) long-acting fusion protein and rapid assembly, separation and purification method thereof
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US9745360B2 (en) 2012-12-21 2017-08-29 Sanofi Dual GLP1/GIP or trigonal GLP1/GIP/glucagon agonists
US10253079B2 (en) 2012-12-21 2019-04-09 Sanofi Functionalized Exendin-4 derivatives
US9670261B2 (en) 2012-12-21 2017-06-06 Sanofi Functionalized exendin-4 derivatives
CN103204944A (en) * 2013-03-26 2013-07-17 江苏健德生物药业有限公司 Long-acting immune fusion protein for treating diabetes mellitus
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US9751926B2 (en) 2013-12-13 2017-09-05 Sanofi Dual GLP-1/GIP receptor agonists
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US9789165B2 (en) 2013-12-13 2017-10-17 Sanofi Exendin-4 peptide analogues as dual GLP-1/GIP receptor agonists
US9771406B2 (en) 2014-04-07 2017-09-26 Sanofi Peptidic dual GLP-1/glucagon receptor agonists derived from exendin-4
US9775904B2 (en) 2014-04-07 2017-10-03 Sanofi Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists
US9758561B2 (en) 2014-04-07 2017-09-12 Sanofi Dual GLP-1/glucagon receptor agonists derived from exendin-4
US9932381B2 (en) 2014-06-18 2018-04-03 Sanofi Exendin-4 derivatives as selective glucagon receptor agonists
US9982029B2 (en) 2015-07-10 2018-05-29 Sanofi Exendin-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists

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