CN102649947A - Cell strain for measuring bioactivity of GLP-1 and functional analogue thereof and application of cell strain - Google Patents

Cell strain for measuring bioactivity of GLP-1 and functional analogue thereof and application of cell strain Download PDF

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CN102649947A
CN102649947A CN2012101173174A CN201210117317A CN102649947A CN 102649947 A CN102649947 A CN 102649947A CN 2012101173174 A CN2012101173174 A CN 2012101173174A CN 201210117317 A CN201210117317 A CN 201210117317A CN 102649947 A CN102649947 A CN 102649947A
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glp
1r
ir
hek293
cells
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CN2012101173174A
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杨建良
王军志
饶春明
陆晶
王兰
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无锡和邦生物科技有限公司
中国食品药品检定研究院
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Abstract

The invention relates to a cell strain for measuring the bioactivity of GLP-1 and functional analogue thereof and an application of the cell strain and belongs to the technical field of measurement of drug bioactivity. The GLP-1R/HEK293 cell strain is preserved in the common microorganism center of China microorganism culture preservation management committee and the preservation number is CGMCCNo.5909. The invention relates to a measuring method for establishing express glucagon peptide-1 receptor (GLP-1R) genetic engineering cell strain GLP-1R/HEK293 and measuring the bioactivity of GLP-1 and analogue thereof in external stimulation on the basis of the change in the cAMP level in the cell after the GLP-1R/HEK293 cell strain is stimulated by the glucagon peptide-1 receptor (GLP-1R) and the functional analogue thereof. The measuring method is easily standardized, is excellent in repeatability and has the characteristics of low cost, convenience and accuracy, thereby being excellent in application prospect.

Description

—种用于测定GLP-1及其功能类似物生物活性的細胞株及其应用 - for determining the kind of GLP-1 analogs and biologically active functional cell line and its application

技术领域 FIELD

[0001] 本发明涉及ー种表达胰高血糖素样肽-I受体蛋白的基因工程细胞株的建立,以及基于此细胞株受到胰高血糖素样肽-I及其功能类似物刺激后CAMP水平的变化来测定胰高血糖素样肽-I及其功能类似物生物活性的检测方法,属于药物生物活性检测技术领域。 [0001] The present invention relates to the establishment of the expression ー species genetically engineered cell line glucagon-like peptide -I receptor protein, and based on this cell line by glucagon-like peptide analogs and functional stimulation of CAMP -I variation detection levels determined glucagon-like peptide -I and functional analogs of biologically active, biologically active drugs belonging to the technical field of detection.

背景技术 Background technique

[0002] 胰高血糖素样肽-I (Glucogan like Peptide-1, GLP-1)作用于胰岛β细胞胰高血糖素样肽_1受体(Glucogan like Peptide-1 Receptor, GLP-1R),促进胰岛素基因的转录、胰岛素的合成和分泌,并可刺激胰岛β细胞的増殖和分化,抑制胰岛β细胞凋亡,增加胰岛β细胞数量。 [0002] glucagon-like peptide -I (Glucogan like Peptide-1, GLP-1) act on the pancreatic β cell glucagon-like peptide receptor _1 (Glucogan like Peptide-1 Receptor, GLP-1R), promote transcription of the insulin gene, the insulin synthesis and secretion, would also stimulate proliferation and enlargement of pancreatic β cell differentiation, inhibiting islet β cell apoptosis, increasing the number of islet β cells. 研究证明,GLP-I可通过多种机制明显地改善2型糖尿病动物模型或患者的血糖情况,这为2型糖尿病的治疗提供了ー个非常好的前景 Studies have shown, GLP-I can significantly improve blood sugar in diabetic animal models or in patients with type 2 through a variety of mechanisms, which provides ー very good prospects for the treatment of type 2 diabetes

[0003] 促胰岛素分泌肽(Exendin-4)是从生长在美国西南部和墨西哥沙漠的希拉毒蜥(Gila monster)唾液发现的ー种多肽物质,氨基酸序列的53%与人类血液中调控血糖的GLP-I同源。 [0003] Exendin (Exendin-4) is ー polypeptides substance from the growth in the southwestern United States and Mexico desert Gila lizard (Gila monster) saliva found 53% amino acid sequence of human blood regulation of glucose GLP-I homolog. Exendin-4同样是GLP-IR激动剂,与GLP-I功能相似,具有促进胰腺分泌胰岛素,促进机体对血糖的利用、減少肝糖原分解、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,达到降低血糖作用。 Exendin-4 also GLP-IR agonist, GLP-I with similar functions, with the promotion of pancreatic secretion of insulin, blood glucose facilitate use of the body, reducing the breakdown of glycogen, by slowing the gastrointestinal tract motility slows absorption of food , thereby reducing the peak glucose in food appear in the blood, to reduce blood sugar. 此外,它还有通过作用于中枢神经系统控制食欲等多种生理功能。 In addition, it also by the action of the control of appetite and other physiological functions in the central nervous system. 由于Exendin-4不被ニ肽基肽酶ーIV降解,其稳定性明显高于GLP-1。 Since Exendin-4 is not degraded Ni ー IV peptidase, which is significantly higher than the stability of GLP-1. 由美国Lilly公司和Amylin公司研制的Exendin_4(Byetta® ),2005年获得了美国FDA批准用于治疗2型糖尿病全新类型的药物。 Developed by the American company and Lilly Amylin's Exendin_4 (Byetta®), in 2005 a new type of diabetes type 2 drugs approved by the FDA for the treatment. 在生理条件下,Byetta®和GLP-I作用相似,它刺激胰岛素分泌的作用受血糖浓度的调节,血糖浓度降低时其刺激作用减轻或消失,因而不会出现胰岛素持续分泌而发生低血糖的现象,并有明显减少2型糖尿病肥胖患者体重的作用。 Under physiological conditions, and GLP-I Byetta® similar effect, it stimulates insulin secretion by regulating the blood glucose concentration, which stimulates action to reduce or disappear when the blood glucose concentration decreases, and thus will not continue to secrete insulin and hypoglycemia phenomenon and significantly reduce the role of obesity in patients with type 2 diabetes weight. 然而,Exendin-4在人体内的半衰期为数小时,患者还是需要每天注射Byetta®两次,使用上仍然相当不方便。 However, Exendin-4 in the half-life in the human body for a few hours, the patient still requires twice daily injections Byetta®, still quite inconvenient to use. 目前正在研究具有长效治疗效果的GLP-1、Exendin-4药物。 Currently studying GLP-1, Exendin-4 treatment drugs have long-lasting effects. 治疗糖尿病的长效蛋白质药物在保持降糖活性的同时可以延长药物在体内的半衰期,不仅可以减少注射频率,方便患者使用,同时还可达到降低治疗费用之目的。 Long-acting agent for treating diabetes proteins while maintaining the activity of hypoglycemic drugs may prolong the half-life in the body, not only can reduce the frequency of injections, conveniently patients, but can also achieve the purpose of reducing the cost of treatment.

[0004] 建立有效的GLP-I及其功能类似物生物活性的測定方法,是评价新药药效的关键措施之一。 [0004] GLP-I to establish an effective method for measuring functional analogs and biologically active and is a key measure for evaluation of the efficacy of new drugs. 目前,国内GLP-I及其功能类似物活性检测依然依赖于传统的动物测定方法。 At present, GLP-I analogs and functional activity assays still rely on traditional methods of measuring the animal. 其检测方法存在实验动物不易获得,操作复杂,数据重复性差,检测方法难于标准化等缺点。 The presence of readily available experimental animal, complicated operation, poor reproducibility data, the method is difficult to standardize detection method of detection and other shortcomings. 本发明采用基因工程技术,构建了一个能稳定表达GLP-IR的基因工程人胚肾HEK293细胞株(GLP-1R/HEK293),建立了基于检测GLP-1R/HEK293细胞株在受到外来刺激后细胞中环磷酸腺苷(Cyclic Adenosine monophosphate,cAMP)含量的变化来测定外来刺激中GLP-1、Exendin-4及其功能类似物生物活性的检测方法,检测方法易标准化,重复性好,具有成本低、方便、准确的特点,因而具有良好的应用前景。 The present invention uses genetic engineering techniques to construct a stable GLP-IR expressing a genetically engineered human embryonic kidney HEK293 cell line (GLP-1R / HEK293), was established based on the detection GLP-1R / HEK293 cell lines upon stimulation by external change in cyclic adenosine monophosphate (cyclic adenosine monophosphate, cAMP) levels of external stimuli is measured in GLP-1, Exendin-4 and its functional analogs method for detecting biological activity, easily standardized detection methods, good repeatability, low cost, easy, and accurate, and thus has a good prospect.

发明内容[0005] 本发明的目的是提供一种用于在体外測定GLP-I及其功能类似物生物活性的基因工程细胞株GLP-1R/HEK293。 SUMMARY OF THE INVENTION [0005] The object of the present invention is to provide a method for measuring genetic engineering functional analogs and GLP-I biological activity of cell lines GLP-1R / HEK293 in vitro. GLP-1R/HEK293细胞株的工作原理是:GLP_1R/HEK293细胞株能在细胞膜上稳定表达GLP-IR蛋白,该受体蛋白受到GLP-I及其功能类似物的作用吋,GLP-1R/HEK293细胞内的cAMP含量会迅速升高,cAMP升高的水平与刺激物中GLP-I及其类似物的生物活性呈正相关。 Works GLP-1R / HEK293 cell line is: GLP_1R / HEK293 cell lines stably expressing GLP-IR protein in the cell membrane, the receptor protein acted GLP-I analogs and functional inch, GLP-1R / HEK293 intracellular cAMP levels increased rapidly, the biological activity of elevated levels of cAMP and stimulation of GLP-I was positive correlation and the like. 通过与GLP-I标准品生物活性的比较,測定未知样品中的GLP-I及其类似物的生物活性。 By comparison with standard GLP-I biological activity, the biological activity was measured in an unknown sample of GLP-I and analogues thereof.

[0006] 为构建能稳定表达GLP-I R的HEK293细胞株,本发明是通过以下技术方案来实现。 [0006] Construction of cell lines stably expressing HEK293 GLP-I R, and the present invention is achieved by the following technical solutions.

[0007] 本发明的技术方案:一株GLP-1R/HEK293细胞株,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号CGMCC No. 5909,用胰高血糖素样肽-I受体,简写为GLP-1R,转染人胚肾HEK293细胞,获得的一株GLP-1R/HEK293细胞株。 [0007] aspect of the present invention: a GLP-1R / HEK293 cell line has been deposited with the Culture Collection Committee China General Microbiological Center, Accession No. CGMCC No. 5909, a glucagon-like peptide -I by body, abbreviated as GLP-1R, transfected with human embryonic kidney HEK293 cells, obtained from a GLP-1R / HEK293 cell line.

[0008] 所述GLP-1R/HEK293细胞株的应用,转染了GLP-IR基因条件下,在受体激动剂刺激下能诱导GLP-1R/HEK 293细胞胞内第二信使cAMP含量増加,转染的GLP-IR基因能够稳定表达; [0008] Application of the GLP-1R / HEK293 cell line transfected with GLP-IR gene under conditions to induce a second messenger cAMP content to increase in the GLP-1R cells cells / HEK 293 at receptor agonist stimulation, GLP-IR-transfected gene can be stably expressed;

所述受体激动剂为GLP-I及其类似物,cAMP含量増加与受体激动剂的活性呈正相关,测定GLP-1R/HEK293细胞内的cAMP含量,即为测定受体激动剂的生物活性。 , Was a receptor agonist to increase in cAMP levels receptor agonist activity of GLP-I and analogs positive correlation, the cAMP content in GLP-1R / HEK293 cells, i.e. receptor agonist biological activity assay .

[0009] 所述GLP-1R/HEK293细胞株的应用,GLP-I及其类似物为GLP-1、GLP-I耦联物及其融合蛋白;Exendin-4、Exendin-4 I禹联物及其融合蛋白。 [0009] Applications, GLP-I and analogs of the GLP-1R / HEK293 cell line of GLP-1, GLP-I conjugates and fusion proteins thereof; Exendin-4, Exendin-4 I linked and Yu fusion protein.

[0010] 本发明提供了一种采用PCR方法制备了编码人GLP-IR的基因片段方法。 [0010] The present invention provides a method for a gene fragment encoding human GLP-IR prepared by PCR. 具体地,根据编码人GLP-IR基因开放阅读框的DNA序列SEQ ID NO: 1,设计ー对基因特异性引物:引物GLP-1R1,其核苷酸序列与SEQ ID NO: 3为100%的同源,其5'端带有Hind III的限制性内切酶位点;引物GLP-1R2与SEQ ID N0:4有100%的同源性,其5'端带有Xho I的限制性内切酶位点。 In particular, according to the DNA sequence encoding human GLP-IR gene open reading frame of SEQ ID NO: 1, design of gene-specific primers ー: primer GLP-1R1, which is the nucleotide sequence SEQ ID NO: 3 is 100% homologous to the 5 'end with a Hind III restriction enzyme site; GLP-1R2 primer with SEQ ID N0: 4 is 100% homologous, at its 5' end with a Xho I restriction of endonuclease sites. 使用弓I物GLP-IRl、GLP-1R2,经PCR方法从质粒pReceiver_M02 DNA中扩增出编码GLP-IR蛋白的DNA片段,该片段的核苷酸序列与SEQ ID NO: I有100%的同源性,其编码的GLP-IR蛋白的氨基酸序列与SEQ ID NO: 2有100%的同源性。 I was used bow GLP-IRl, GLP-1R2, was amplified by PCR from plasmid pReceiver_M02 DNA a DNA fragment encoding the GLP-IR protein, the nucleotide sequence of the fragment and SEQ ID NO: I with 100% derived, which encoded amino acid sequence of GLP-IR protein and SEQ ID NO: 2 has 100% homology. 本发明还将PCR扩增到的编码人GLP-IR蛋白的DNA片段亚克隆到TA克隆载体pGEM_T载体中进行DNA序列分析,以证实PCR扩增产物DNA序列的准确性。 DNA fragments will be amplified by PCR to the present invention encoding human GLP-IR protein was subcloned into the TA cloning vector pGEM_T vector DNA sequence analysis to confirm accuracy of the PCR amplification products of DNA sequences.

[0011] 本发明提供一种构建在真核细胞中表达人GLP-IR蛋白的重组表达质粒的构建方法。 [0011] The present invention provides a construct expressing recombinant human GLP-IR protein expression construct a plasmid in eukaryotic cells. 所述的重组表达质粒构建所使用的真核表达质粒是pcDNA3. I ( + )(购于Invitrogen公司),其特点是在真核细胞中能自我复制,并表达G418抗性。 Construction of eukaryotic expression plasmids used for the expression of the recombinant pcDNA3. I (+) (purchased from Invitrogen), which is characterized by self-replicating in eukaryotic cells, and express G418 resistance. 但是,亦可以是其它种类的真核表达质粒GLP-1R,不仅局限于质粒pcDNA3. 1( + )。 However, it may also be other kinds of eukaryotic expression plasmid GLP-1R, it is not limited to plasmids pcDNA3. 1 (+). 所述的用于表达人GLP-IR蛋白的基因与SEQ ID NO: I有100%的同源性。 The gene for human GLP-IR protein and SEQ ID NO: I 100% homology. 所述的质粒pcDNA3. I ( + )与SEQ ID N0:5有100%的同源性。 The plasmid pcDNA3 I (+) and SEQ ID N0:. 5 100% homology. 具体构建方法如下:采用限制性内切酶Hind III和Xho I (购自上海生エ)对含有GLP-IR基因的重组质粒GLP-lR/pGEM-T和真核表达质粒pcDNA3. I进行限制性酶切,分别制备5'端带有Hind III,3'端带有Xho I酶切位点的GLP-IR基因片段与线性化的pcDNA3. I ( + )。 DETAILED constructed as follows: The restriction enzyme Hind III and Xho I (purchased from Shanghai Ester) GLP-lR recombinant plasmid containing the gene of GLP-IR / pGEM-T and eukaryotic expression plasmid pcDNA3 I restriction. digestion, respectively, were prepared 5 'end with Hind III, 3' GLP-IR gene fragment and the linearized pcDNA3 end with a Xho I restriction site. I (+). 采用胶回收方法制备纯化的GLP-IR基因片段与线性化的pcDNA3. I ( + )的酶切产物。 Preparation method of recovering purified by gel GLP-IR gene fragment and the linearized pcDNA3. Cleavage product I (+) is. 将酶切纯化的GLP-IR片段DNA与酶切纯化的质粒pcDNA3. I ( + ) DNA按3:1混合,在T4 DNA连结酶的作用下进行连结,构建重组表达质粒GLP-lR/pcDNA3. 1(+),其核苷酸序列应与SEQ ID N0:6有100%的同源性。 The purified digested DNA fragment of GLP-IR and purified digested plasmid pcDNA3 I (+) DNA at 3: 1 are mixed and connected to the action of T4 DNA ligase to construct a recombinant expression plasmid GLP-lR / pcDNA3. 1 (+), the nucleotide sequence should SEQ ID N0: 6 100% homology. 采用PCR扩增方法鉴定转化子细菌中是否含有含GLP-IR基因片段的重组表达质粒GLP-lR/pcDNA3. 1(+)。 PCR amplification using a method of identifying a transformant containing the recombinant bacterium whether the gene fragment containing GLP-IR expression plasmid GLP-lR / pcDNA3. 1 (+). 采用限制性内切酶Hind III和Xho I对重组表达质粒GLP-lR/pcDNA3. 1(+)进行双酶切分析,并将重组质粒送Invitrogen公司进行DNA序列分析,以证明重组表达质粒中GLP-IR基因的方向与序列的正确性。 Using restriction enzymes Hind III and Xho I to recombinant plasmid GLP-lR / pcDNA3. 1 (+) for double digestion analysis, and recombinant plasmids were subjected to DNA sequence analysis Invitrogen Corporation, to demonstrate that the recombinant plasmid of GLP -IR direction of the correctness of the gene sequence.

[0012] 本发明提供一种表达GLP-IR蛋白的真核细胞株构建方法。 [0012] The present invention provides a eukaryotic cell line expressing GLP-IR protein construction method.

[0013] 具体地,将人胚肾HEK293细胞(中国科学院上海生命科学研究院生物化学与细胞生物学研究所购得)在转染前一天铺6孔板,待细胞生长至80%的融合度时,将线性化的GLP-lR/pcDNA3. I (+)重组质粒与Fugene 6转染试剂(购于Roche公司)混合,然后加入到HEK293培养孔中进行基因转染,用含G418的培养基筛选,筛选能在含高浓度G418培养基中生长的重组细胞株。 [0013] In particular, the human embryonic kidney HEK293 cells (Shanghai Institutes for Biological Sciences, Institute of Biochemistry and Cell Biology, Academia Sinica available) spread in 6-well plates the day before transfection, cells were grown to be 80% confluence when the linearized GLP-lR / pcDNA3. I (+) plasmid with Fugene 6 transfection reagent (purchased by Roche) were mixed, then added to the HEK293 culture wells for gene transfection, the medium containing G418 screening, screening for growth in medium containing a high concentration of the recombinant cell line G418.

[0014] 本发明还提供一种鉴定GLP-IR是否表达在HEK293细胞表面的方法。 [0014] The present invention further provides a method of identifying whether a HEK293 cell surface expression of GLP-IR. 具体地,采用FITC标记的兔抗GLP-IR抗体对转染的HEK293细胞进行染色,同时以FITC标记的兔IgG抗体作为同型対照。 In particular, FITC-labeled rabbit anti-GLP-IR antibody on transfected HEK293 cells were stained, while FITC-labeled rabbit IgG antibody as isotype as Dui. 采用流式细胞分析方法检测转染的细胞表面是否有GLP-IR蛋白存在。 Using flow cytometry to detect whether the transfected cell surface protein present GLP-IR.

[0015] 本发明提供一种半量RT-PCR检测重组工程细胞株GLP-1R/HEK293中GLP-IR基因表达稳定性的方法。 [0015] The present invention provides a method of half the amount of RT-PCR and recombinant cell strain GLP-1R / HEK293 in GLP-IR gene expression stability. 具体地,收集不同传代时间的GLP-1R/HEK293细胞,抽提RNA,采用RT-PCR测定该细胞中GLP-IR的表达时产生的mRNA含量。 Specifically, the generation time of the collection of different GLP-1R / HEK293 cell, an RNA extraction, mRNA levels were determined by the cell produced when the GLP-IR expression RT-PCR. 同时以管家基因GAPDH为内參对照,序列为SEQ ID NO: 7。 While the housekeeping gene GAPDH as a loading control, the sequence of SEQ ID NO: 7. 观察不同传代时间GLP-1R/HEK293细胞中GLP-IR表达水平是否发生变化。 Observe the generation time of GLP-1R / HEK293 cells expressing GLP-IR levels is changed or not. 结果显示GLP-IR蛋白可以稳定地表达在重组基因工程细胞GLP-1R/HEK293表面。 The results show that GLP-IR protein can be stably expressed in cells genetically engineered recombinant GLP-1R / HEK293 surface.

[0016] 本发明提供ー种GLP-1R/HEK293细胞GLP-1R活性的测定方法。 [0016] The present invention provides a method for measuring the activity of GLP-1R cells ー species GLP-1R / HEK293.

[0017] 具体地,使用GLP-I及其功能类似物刺激正在生长中的GLP-1R/HEK293细胞,采用Promega公司的生物发光检测试剂盒,测定受到激动剂刺激的GLP-1R/HEK293细胞中环磷酸腺苷(cAMP)水平的变化来观察GLP-IR/ HEK293细胞膜上表达的GLP-IR是否具有生物活性功能。 [0017] Specifically, using the GLP-1R GLP-I analogs and functional stimulation of growing / HEK293 cells using the Promega bioluminescent detection kit, GLP-1R agonist stimulation was measured by / HEK293 cells Central varying levels of adenosine monophosphate (cAMP) to observe the GLP-IR expressed on the GLP-IR / HEK293 membrane whether biological activity. 如果GLP-IR/ HEK293细胞膜上表达的GLP-IR具有生物活性,其受到GLP-I等激动剂的刺激后,HEK293细胞的生理代谢活性增强,消耗的能量増加。 If the GLP-IR expressed on the GLP-IR / HEK293 membrane having biological activity, after which the like by GLP-I agonist stimulation, the physiological enhanced metabolic activity HEK293 cells, to increase in the energy consumed. 细胞内的三磷酸腺嘌呤(ATP)转化为cAMP,亦就是说GLP-IR激活后,细胞内的cAMP水平会明显升高,而不表达GLP-IR的HEK293受到GLP-I等激动剂的刺激后,细胞内的cAMP水平不发生明显的改变。 Adenine triphosphate (ATP) of cAMP within cells into, that is also after the GLP-IR activation, the level of cAMP will be increased significantly in cells, do not express the GLP-IR stimulated by GLP-I like HEK293 agonist later, cAMP levels in the cells does not change significantly.

[0018] 本发明还提供一种基于基因工程细胞株GLP-1R/HEK293的cAMP水平变化测定GLP-I及其功能类似物生物活性的測定方法。 [0018] The present invention further provides a method for measuring functional analogs and GLP-I biological activity assay based on cell lines genetically engineered changes in cAMP levels in GLP-1R / HEK293's. 具体地,称取不同活性単位的GLP-I标准物,分别用于刺激生长中的GLP-1R/HEK293细胞,测定各活性单位GLP-I刺激GLP-1R/HEK293细胞后产生的cAMP的数量。 Specifically, it weighed different activities. Unit of GLP-I standards were used to stimulate GLP-1R growing / HEK293 cell, determine the amount of produced after each activity unit of GLP-I stimulation of GLP-1R / HEK293 intracellular cAMP. 以GLP-I的活性为横坐标,以cAMP的数量为纵坐标,作标准曲线。 The active GLP-I as abscissa and the amount of cAMP as ordinate, a standard curve. 同时,测定未知样品刺激GLP-1R/HEK293细胞产生的cAMP的数量,以此cAMP的数量查标准曲线,从而可确定未知样品中GLP-I及其功能类似物的生物活性。 Meanwhile, the number of measuring an unknown sample stimulate GLP-1R / HEK293 cells of cAMP produced, in order to check the amount of cAMP standard curve, thereby determining whether the biological activity of the unknown sample and functional GLP-I analogs.

[0019] 本发明的有益效果:建立了基于检测GLP-1R/HEK293细胞株在受到外来刺激后细胞中cAMP含量的变化来测定外来刺激中GLP-I及其功能类似物生物活性的检测方法,检测方法易标准化,重复性好,具有成本低、方便、准确的特点,因而具有良好的应用前景。 [0019] Advantageous effects of the present invention are: to establish a method for detecting functional analogs and GLP-I biological activity of external stimuli is measured in the detection of changes in cAMP levels in GLP-1R / HEK293 cell line after the cells by external stimuli based on, easy detection methods standardized, reproducible, low cost, convenient, and accurate, and thus has a good prospect.

[0020] 生物材料样品保藏:一株GLP-1R/HEK293细胞株,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,简称CGMCCJii :北京中国科学院微生物研究所,保藏编号CGMCC No. 5909,保藏日期2012年3月22日。 [0020] the deposit of biological material: a GLP-1R / HEK293 cell line has been deposited at the Chinese Culture Collection Committee General Microbiology Center, referred to CGMCCJii: Beijing Institute of Microbiology, Chinese Academy of Sciences, accession number CGMCC No. 5909, preserved date March 22, 2012.

附图说明[0021 ] 图I重组真核表达载体GLP-lR/pcDNA3. I (+)的酶切鉴定結果。 BRIEF DESCRIPTION [0021] Figure I eukaryotic expression vector GLP-lR / pcDNA3. I (+) digested identification. I、Ikb DNAMarker ;2、重组质粒GLP_lR/pcDNA3. I (+) ;3 重组质粒GLP_lR/pcDNA3. I (+) /HincIΙΙΙ+Xho J酶切产物。 I, Ikb DNAMarker;. 2, plasmid GLP_lR / pcDNA3 I (+);. 3 plasmid GLP_lR / pcDNA3 I (+) / HincIΙΙΙ + Xho J cleavage product.

[0022] 图2流式细胞仪分析克隆细胞表面GLP-IR的表达情况结果。 [0022] FIG. 2 Flow cytometric analysis of cell surface expression of cloned results of GLP-IR. 流式细胞仪分析6个克隆细胞表面GLP-IR的表达情況,图2显示的是2号克隆细胞表面有GLP-IR蛋白的表达。 Flow cytometric analysis of the 6 clones expression of cell surface GLP-IR, FIG. 2 shows the clone No. 2 cell surface protein expression of GLP-IR. (a)阴性对照组为未转染细胞、2号克隆细胞中未加抗体所检测到的荧光强度;阴性抗体组为未转染细胞、2号克隆细胞中均未加GLP-IR抗体、加入了FITC标记的兔IgG抗体染色的转染细胞为抗体对照所检测到的荧光强度;(b)抗GLP-IR组为未转染细胞、2号克隆细胞中均加GLP-IR抗体所检测到的荧光强度;合并组为为未转染细胞、2号克隆细胞的上述三种所检测到的荧光强度放在一起观察。 (A) negative control group untransfected cells, clonal cell No. 2 antibody was not added to the detected fluorescence intensity; antibody negative group of untransfected cells, clone 2 cells were not added GLP-IR antibody, was added FITC-labeled rabbit IgG antibody transfected cells were stained for antibody control the detected fluorescence intensity; (b) an anti-GLP-IR group of untransfected, No. 2 clone cells are added GLP-IR antibody detected fluorescence intensity; the three combined group of non-transfected cells, clone # 2 cells detected in fluorescence intensity was observed together. 结果显示抗GLP-IR组的2号克隆细胞所检测到的荧光强度明显增加(其横坐标向右移),证明抗GLP-IR组的2号克隆细胞有GLP-IR蛋白的表达。 The results showed that anti-cell clone No. 2 group of GLP-IR detected a significant increase in fluorescence intensity (abscissa to the right), demonstrating that anti-cell clone No. 2 group of GLP-IR expression GLP-IR protein.

[0023] 图3 RT-PCR半定量检测转染基因GLP-IR的稳定性結果。 [0023] FIG. 3 RT-PCR results of semi-quantitative detection of transfected gene stability of GLP-IR. l、100bp Marker ;2、10天样本;3、20天样本;4、40天样本;5、60天样本;6、阴性样本。 l, 100bp Marker; 2,10-day sample; Sample 3, 20 days; day sample 4,40; 5,60-day sample; 6, negative samples.

[0024] 图4 GLP-1R/HEK293细胞、HEK293细胞分别经GLP-I刺激后活性反应曲线結果。 [0024] FIG. 4 GLP-1R / HEK293 cells, HEK293 cells were activated reactive profile results after GLP-I stimulation.

[0025] 图5 GLP-1R/HEK293细胞经GLP-1、Exendin-4刺激后活性反应曲线結果。 [0025] FIG. 5 GLP-1R / HEK293 cells were treated with GLP-1, active reaction profile results Exendin-4 stimulation.

[0026] 图6 GLP-1R/HEK293细胞经Exendin-4-HSA、HSA刺激后活性反应曲线結果。 [0026] FIG. 6 GLP-1R / HEK293 cells were Exendin-4-HSA, HSA-stimulating activity profile results after the reaction.

具体实施方式 Detailed ways

[0027] 实施例I:重组GLP_lR/pcDNA3. I (+)真核表达质粒的构建1.1 GLP-IR基因的扩增与鉴定 [0027] Example I:. Recombinant GLP_lR / pcDNA3 I (+) was amplified with the eukaryotic expression plasmid containing a 1.1 GLP-IR gene

根据编码人GLP-IR蛋白开放阅读框cDNA序列(SEQ ID NO: I)设计ー对基因特异性引 The encoding human GLP-IR protein open reading frame cDNA sequence (SEQ ID NO: I) the design of gene-specific primers ー

物: Material:

GLP-IRl: 5' -AAAAGCTTATGGCCGGCGCCCCCGG-3' (SEQ ID NO:3); GLP-IRl: 5 '-AAAAGCTTATGGCCGGCGCCCCCGG-3' (SEQ ID NO: 3);

Hind III Hind III

GLP-IR2: 5' -TTCTCGAGTCAGCTGCAGGAGGCCTG-3' (SEQ ID NO:4) GLP-IR2: 5 '-TTCTCGAGTCAGCTGCAGGAGGCCTG-3' (SEQ ID NO: 4)

Xho I Xho I

引物GLP-IRl的5'端带有"ifli/ III的限制性内切酶位点,GLP-1R2的5'端带有Xho /的限制性内切酶位点。以GLP-IR编码基因(pReceiver-M02质粒DNA,江苏省血吸虫病防治研究所科研部提供)为模板,进行PCR扩增。具体条件如下:在O. 2 mL的PCR管中,加入pReceiver_M02 质粒DNA I μί ; 10XTaq Buffer, ΙΟμί ;MgCl2 (2. 5mM) ΙΟμί ;dNTPMixture(IOMm),2μί ;GLP-1R1 2μΐ , GLP-1R2 2μί ;ddH20,72. Ομί。混匀后99°C加热变性10 min,将PCR管置于冰上冷却后,加入Taq DNA Polymerase, I. Ομί (3U)。混匀,短暂离心。然后,把PCR管放到PCR仪孔中,根据下列反应条件进行扩增:94°C,30 sec ;55°C,20 sec,72°C,90 sec,共30个循环;最后,72°C,5min。所产生的PCR反应产物使用琼脂糖电泳方法进行分析,在紫外灯下观察电泳情况。采用低融点琼脂糖凝胶电泳,切胶回收分子量约1392bp的GLP-IR基因的DNA条带。将GLP-IR基因的 GLP-IRl primers 5 'end "the restriction endonuclease site ifli / III a, 5 GLP-1R2' end restriction endonuclease site with Xho / in. To GLP-IR-encoding gene ( pReceiver-M02 plasmid the DNA, Jiangsu Institute of Research schistosomiasis control unit provided) as a template, PCR amplification was specific conditions were as follows: in O. 2 mL PCR tube was added pReceiver_M02 plasmid DNA I μί; 10XTaq Buffer, ΙΟμί ; MgCl2 (2. 5mM) ΙΟμί; dNTPMixture (IOMm), 2μί; GLP-1R1 2μΐ, GLP-1R2 2μί;. ddH20,72 Ομί mixing after 99 ° C heat denaturation 10 min, the PCR tube was placed on ice. after cooling, Taq DNA Polymerase, I. Ομί (3U) mixed, centrifuged briefly and then, the PCR tubes into wells PCR machine, the amplification according to the following reaction conditions: 94 ° C, 30 sec; 55 °.. C, 20 sec, 72 ° C, 90 sec, 30 cycles; and finally, 72 ° 5min C, PCR resulting reaction products were analyzed by agarose electrophoresis, electrophoresis was observed under ultraviolet light in the case of a low melting point. agarose gel electrophoresis, a molecular weight of gel Extraction GLP-IR DNA genes with approximately 1392bp. the GLP-IR gene DNA片段与pGEM_T克隆载体连接,构建GLP-lR/pGEM-T重组质粒,连结产物转化DH5 α感受态细菌,经含Ampicilin的LB平板筛选获得含GLP-lR/pGEM-T重组质粒的菌株。用质粒DNA纯化试剂盒(Promega公司)纯化获得重组GLP-IR基因质粒DNA。 DNA fragments pGEM_T cloning vector was constructed by ligating GLP-lR / pGEM-T plasmid, the coupling product is transformed DH5 α competent bacteria, dried over LB plates containing Ampicilin obtained by screening strain containing GLP-lR / pGEM-T recombinant plasmid. By plasmid DNA purification kit (Promega Corporation) to obtain GLP-IR recombinant plasmid DNA.

[0028] I. 2 GLP-IR重组真核表达质粒的构建 [0028] I. 2 GLP-IR recombinant eukaryotic expression plasmid containing

本实验采用真核表达质粒pcDNA3. 1(+)为表达载体,其制备方法为常用分子生物学方法。 In this experiment the eukaryotic expression plasmid pcDNA3. 1 (+) 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,将GLP-IR基因插入pcDNA3. I (+)构建成GLP-IR重组真核表达质粒。 I.e. by culturing strains containing the expression vector, the plasmid obtained is extracted, the GLP-IR gene into pcDNA3. I (+) GLP-IR constructed recombinant plasmid expressing true. [0029] 先对表达载体质粒pcDNA3. I (+),进行历ii/ III,Xho I双酶切。 [0029] The first expression plasmid vector pcDNA3. I (+), for calendar ii / III, Xho I double digestion. 具体条件如下。 Specific conditions were as follows. 表达载体质粒pcDNA3. I (+) -,Hind III ΐμΐ, Xho I ΐμί,IOXTango 缓冲液5μί,ddH2033μί,总体积为50μί。 Plasmid expression vector pcDNA3 I (+) -., Hind III ΐμΐ, Xho I ΐμί, IOXTango 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Xho J质粒DNA。 37 ° C water bath with constant temperature for 3 hours, Xho J linearized plasmid DNA was recovered by agarose gel electrophoresis. GLP-lR/pGEM-T质粒作同样的双酶切,通过琼脂糖凝胶电泳回收分子量约1392 bp的GLP-IR基因片段。 GLP-lR / pGEM-T plasmids for the same double digestion, recovered by agarose gel electrophoresis the molecular weight of GLP-IR gene fragment of approximately 1392 bp.

[0030] 将回收的GLP-IR DNA与上述双酶切的表达质粒pcDNA3. 1(+) DNA连接构建重组表达质粒GLP-lR/pcDNA3. I (+)。 [0030] The recovery of GLP-IR DNA double digested with the aforementioned expression plasmid pcDNA3. 1 (+) DNA ligation to construct recombinant plasmid GLP-lR / pcDNA3. I (+). 连接体系一般为ΙΟμί,双酶切的pcDNA3. I (+)质粒载体与双酶切GLP-IR DNA 的摩尔比为I : 2_10,10XT4 DNA Iigase 缓冲液ΐμί,Τ4 DNA Iigase1KL,加无菌水至总体积为ΙΟμί。 The connection system is generally ΙΟμί, I molar ratio of double-digested plasmid vector with the GLP-IR DNA double-digested pcDNA3 (+) is the I: 2_10,10XT4 DNA Iigase buffer ΐμί, Τ4 DNA Iigase1KL, sterile water was added to the The total volume of ΙΟμί. 连接混合物在16°C恒温水浴锅内反应16小时。 The ligation mixture was reacted at 16 ° C water bath with constant temperature for 16 hours. 连接产物转化大肠杆菌DH5 α感受态细胞。 The ligation product was transformed into E. coli DH5 α 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。 Positive clones were identified that positive clones by the LB plate containing Ampicilin, plasmids were extracted. 将重组质粒DNA用JJJ、ZAo J内切酶进行双酶切,酶切产物经琼脂糖凝胶电泳,溴化こ啶染色,结果显示,重组质粒被切割成一条分子量约5. 4Kb载体DNA条带和一条分子量约1392bp的GLP-IR基因条带,证明GLP-IR被正确地插入到表达质粒pcDNA3. 1(+)中(图I)。 JJJ recombinant plasmid DNA, within ZAo J enzyme double digestion, the digestion products by agarose gel electrophoresis, ko bromide-stained showed, the molecular weight of the recombinant plasmid was cut into a vector DNA bands of about 5. 4Kb and with a molecular weight of GLP-IR band of about 1392bp gene, demonstrating GLP-IR is properly inserted into the expression vector pcDNA3. 1 (+) (Figure I).

[0031] 实施例2 :表达GLP-IR蛋白基因工程细胞株的构建与鉴定2. I 重组GLP-lR/pcDNA3. I (+)质粒转染HEK293 细胞 [0031] Example 2: Construction of the expression of GLP-IR protein and identification of genetically engineered cell lines 2. I recombinant GLP-lR / pcDNA3 I (+) plasmid was transfected HEK293 cells

HEK293细胞在转染前一天接种于六孔板中(4 X IO5细胞/孔),每孔含100 μ L DMEM培养基(无抗生素),培养18〜24h至80%细胞密度。 HEK293 cells were seeded the day before transfection in six-well plates (4 X IO5 cells / well), each well containing 100 μ L DMEM medium (without antibiotics) and incubated 18~24h to 80% of cell density. 将线性化重组质粒2 μ g,与3 μ L FuGene6混合后进行细胞转染。 The linearized recombinant plasmid 2 μ g, and after mixing 3 μ L FuGene6 transfection of cells.

[0032] 待转染24h后,将转染细胞按1:10传代至新鲜的含300 μ g/mL G418的培养液中(G418起始浓度由预实验确定),放37°C、5%C02孵箱中培养。 [0032] After 24h to be transfected, the transfected cells were passaged to fresh 1:10 containing 300 μ g / mL G418 in the culture medium (G418 initial concentration determined by the preliminary experiment), put 37 ° C, 5% C02 incubator. 每两天换一次液,培养48〜72h后可观察到大部份细胞死亡。 Most of the cells were observed after death every two days for liquid culture 48~72h. 将存活细胞转移到另外ー个六孔板中,逐渐加大G418的浓度(最大浓度达1200 μ g/mL)继续进行筛选,获得表达GLP-IR蛋白基因单细胞克隆。 The surviving cells were transferred to six-well plates ー Further, gradually increasing concentrations of G418 (the maximum concentration of up to 1200 μ g / mL) continue screening, GLP-IR gene expression of single cell clones obtained.

[0033] 2. 2重组细胞株表面表达GLP-IR的鉴定 [0033] 2.2 Identification of recombinant cell lines expressing the surface of the GLP-IR

2. 2. I流式分析法验证重组细胞株表面GLP-IR蛋白的表达 2. 2. Verify the cell surface expression of recombinant proteins GLP-IR analysis flow strain I

用抗GLP-IR抗体检测HEK293细胞表面是否有GLP-IR表达。 Is there a GLP-IR cell surface expression of antibodies with HEK293 GLP-IR antibody. 具体方法如下:取2 X IO5个转染细胞,加入2 μ L FITC标记的兔抗GLP-IR蛋白抗体染色,同时以FITC标记的兔IgG抗体染色的转染细胞为抗体対照,以不加抗体的转染细胞为阴性对照。 Specific methods are as follows: Take 2 X IO5 two transfected cells, added to rabbit anti-GLP-IR antibody stained protein 2 μ L FITC labeled, while the transfected cells with FITC-labeled rabbit IgG antibody staining antibody Dui according to without antibody transfected cells as a negative control. 经过流式细胞仪检測,验证克隆细胞的表面有GLP-IR蛋白的表达(图2)。 After flow cytometry, clonal cell surface verify expression of the GLP-IR protein (FIG. 2). 获得表达GLP-IR蛋白的单细胞克隆按常规细胞冻方法,存置于液氮中进行长期冻存。 GLP-IR protein expression is obtained in a conventional single-cell clones the cells in frozen, placed in long term storage in liquid nitrogen cryopreservation.

[0034] 2. 2. 2重组GLP-IR/ ΗΕΚ293细胞株GLP-1R表达的稳定性观察 [0034] 2. Stability of 2.2 and expression of the recombinant GLP-IR / ΗΕΚ293 GLP-1R cell lines

采用半量RT-PCR方法,对培养不同时间的GLP-1R/HEK293细胞中GLP-IR表达的表达水平进行检测,同时以管家基因GAPDH为内标对照,观察GLP-1R/HEK293细胞中GLP-IR基因的表达稳定性。 Using half the amount of RT-PCR method, cultured at different times of GLP-1R for detecting the expression level expression / HEK293 cells GLP-IR, while the housekeeping gene GAPDH as an internal standard control, was observed GLP-1R / HEK293 cells GLP-IR expression of genetic instability. 具体方法如下:培养GLP-1R/HEK293细胞,每2天传代一次,分别在第10天、20天、40天和60天,按QIAGEN公司RNA抽提试剂盒说明书分别提取培养不同时间的重组细胞的总RNA。 Specific methods are as follows: culture GLP-1R / HEK293 cells were passaged once every two days, respectively, at 10 days, 20 days, 40 days and 60 days, according to the company QIAGEN RNA extraction kit instructions recombinant cell culture were extracted at different times the total RNA. 半定量RT-PCR检测GLP-IR的表达情況,结果表明,培养不同时间(10天-60天之间)的细胞中GLP-IR的表达水平基本保持稳定。 Semi-quantitative RT-PCR to detect the expression of GLP-IR results showed that the expression levels of different culture times (between 10 days -60 days) GLP-IR cells remained stable. (图3)。 (image 3).

[0035] 实施例3: GLP-1R/HEK293细胞表达GLP-1受体活性分析 [0035] Example 3: GLP-1R / HEK293 cell GLP-1 receptor activity Expression

当重组基因工程GLP-1R/HEK293细胞表达的GLP-I受体蛋白具有生理活性时,在其受激动剂GLP-I或其功能类似物的刺激吋,GLP-1R/HEK293细胞的生理代谢活动增强,表现为细胞内cAMP含量増加。 When recombinant expression of genetically engineered GLP-1R / HEK293 cells GLP-I receptor protein having a physiological activity, it stimulates GLP-I agonist by inch or functional analogs, GLP-1R / HEK293 cell physiological metabolic activity enhanced performance add to intracellular cAMP levels zo. 因此,通过测定受到GLP-I等激动剂刺激后GLP-1R/HEK293细胞内cAMP含量的变化,即可判定GLP-1R/HEK293细胞表达的GLP-IR蛋白是否具有生理活性。 Thus, changes in GLP-1R / HEK293 cells by measuring cAMP levels after subjected to other GLP-I agonist stimulation, the expression can be determined in GLP-1R / HEK293 cells whether GLP-IR protein having physiological activity. 具体方法如下: Specific methods are as follows:

GLP-1R/HEK293细胞按100 μ L/孔(30,000细胞/孔)培养与96孔培养板,用DMEM培养基,37°C、5%C02条件下培养24小吋。 GLP-1R / HEK293 cells by 100 μ L / hole (30,000 cells / well) and cultured in 96-well culture plates with DMEM medium, 37 ° C, cultured for 24 hours under 5% C02 conditions inch. 第二天撤去完全培养基,加入基础培养基培养过夜(15小时左右)。 Complete medium was removed the following day, the basal medium was added and cultured overnight (about 15 hours). 移弃基础培养基,加入待测定样本GLP-1,37°C,5% CO2条件下培养I小时。 Remove and discard the basal medium, the culture to be assayed is added at I hour sample GLP-1,37 ° C, 5% CO2 conditions. 用cAMP试剂检测盒(购于AB公司)测定上述GLP-I刺激GLP-1R/HEK293细胞后胞内的cAMP含量。 (Available from AB Company) measurement of the GLP-I stimulated cAMP levels in cells after GLP-1R / HEK293 cells using cAMP detection reagent cartridge. 结果表明,GLP-1R/HEK293细胞的cAMP含量显著增加,而未转染GLP-IR基因的HEK293细胞未有任何剂量效应趋势(图4)。 The results show, the content of cAMP GLP-1R / HEK293 cells was significantly increased, whereas the GLP-IR gene transfected HEK293 cells No dose effect any trend (FIG. 4). 以上实验结果证实重组GLP-1R/HEK293细胞表达的GLP-IR蛋白具有天然GLP-IR蛋白相似的生理活性,且具有特异性。 The above experimental results confirmed the expression of the recombinant GLP-1R / HEK293 cells with similar GLP-IR protein of native GLP-IR physiologically active proteins, and specific.

[0036] 实施例4 :胰高血糖素样肽-I及其功能类似物生物学活性测定方法 4 [0036] Example: glucagon-like peptide -I and functional analogs biological activity determination

称取不同浓度的GLP-I及其功能类似物Exendin-4,用实施例3方法分别用于刺激生长中的GLP-1R/HEK293细胞,测定各样本刺激GLP-1R/HEK293细胞后产生的cAMP的数量。 cAMP production weighed after different concentrations of GLP-I analogs and functional Exendin-4, respectively, by the method of Example 3 for stimulating growth of GLP-1R / HEK293 cells, each sample was measured stimulate GLP-1R / HEK293 cells quantity. 以样本浓度为横坐标,以cAMP的数量为纵坐标,作曲线。 A sample concentration of abscissa and the amount of cAMP as ordinate plotted. 结果显示,经Exendin-4、GLP-1刺激后胞内的cAMP含量曲线呈典型的反“S”型(图5),不同浓度的GLP-1、Exendin-4刺激GLP-1R/HEK293细胞产生的cAMP含量存在剂量效应相关性,并通过标准曲线可以测定未知样品刺激GLP-1R/HEK293细胞产生的cAMP的数量,以此cAMP的数量查标准曲线,从而可确定未知样品中GLP-I及其功能类似物的生物活性。 The results show that, by Exendin-4, the stimulation of GLP-1 cAMP content curve intracellular form of typical anti "S" type (FIG. 5), different concentrations of GLP-1, Exendin-4 stimulation of GLP-1R / HEK293 cells cAMP levels in the presence of a dose-related effects, and unknown samples can be determined number of stimulated GLP-1R / HEK293 cells of cAMP produced, in order to check the amount of cAMP standard curve of the standard curve, thereby determining the unknown sample and GLP-I biologically active functional analogs. 以上实验结果进一歩表明重组GLP-IR/HEK293细胞用于测定GLP-I及其功能类似物的生物活性具有特异性,据此可以提供ー种基于基因工程细胞株GLP-1R/HEK293的cAMP水平变化测定GLP-I及其功能类似物生物活性的測定方法。 Ho into the above experimental results show that a recombinant GLP-IR / HEK293 cells were used to determine the biological activity of GLP-I analogs and its specific function, whereby cAMP levels may be provided ー species genetically engineered cell line based GLP-1R / HEK293 of Determination of GLP-I analogs and functional changes in the biological activity assay.

[0037] 实施例5 :促胰岛素分泌肽-I及其功能类似物生物学活性測定方法 5 [0037] Example: -I exendin analogs and functional biological activity determination

称取不同浓度的Exendin-4功能类似物长效Exendin-4-HSA融合蛋白和人血清白蛋白(HSA),用实施例3方法分别用于刺激生长中的GLP-1R/HEK293细胞,測定各样本刺激GLP-1R/HEK293细胞后产生的cAMP的数量。 Weigh the different concentrations of long-term functional analog Exendin-4 Exendin-4-HSA fusion proteins and human serum albumin (HSA), using the method of Example 3 are used to stimulate the growth of GLP-1R in / HEK293 cells, each assay the number of samples produced after stimulation of GLP-1R / HEK293 intracellular cAMP. 以样本浓度为横坐标,以cAMP的数量为纵坐标作曲线。 A sample concentration of abscissa and the ordinate is the amount of cAMP plotted. 结果显示,经不同浓度的Exendin-4-HSA融合蛋白刺激GLP-1R/HEK293细胞产生的cAMP含量存在剂量效应相关性,而人血清白蛋白未有任何剂量效应趋势(图6)。 The results show, with different concentrations of Exendin-4-HSA fusion protein in the presence of stimulated cAMP levels in a dose-related effects of GLP-1R / HEK293 cells, and human serum albumin No dose effect any trend (FIG. 6). 以上实验结果进ー步表明重组GLP-1R/HEK293细胞用于测定GLP-I及其功能类似的生物活性具有特异性,据此可以提供一种基于基因工程细胞株GLP-1R/HEK293的cAMP水平变化测定GLP-I及其功能类似物生物活性的測定方法。 Into the above experimental results further showed that the recombinant ー GLP-1R / HEK293 cells were used to determine the GLP-I biological activity and the like having a specific function, whereby cAMP levels can be provided based on the genetically engineered cell line GLP-1R / HEK293 of Determination of GLP-I analogs and functional changes in the biological activity assay.

Claims (3)

1. 一株GLP-1R/HEK293细胞株,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号CGMCC No. 5909,其特征在于:用胰高血糖素样肽_1受体,简写为GLP-1R,转染人胚肾HEK293细胞,获得的一株GLP-1R/HEK293细胞株。 1. a GLP-1R / HEK293 cell line has been deposited with the Culture Collection Committee China General Microbiological Center, Accession No. CGMCC No. 5909, wherein: a glucagon-like peptide receptor _1, abbreviated for the GLP-1R, transfected with human embryonic kidney HEK293 cells, obtained from a GLP-1R / HEK293 cell line.
2.权利要求I所述GLP-1R/HEK293细胞株的应用,其特征在于:转染了 GLP-IR基因条件下,在受体激动剂刺激下能诱导GLP-1R/HEK 293细胞胞内第二信使cAMP含量増加,转染的GLP-IR基因能够稳定表达; 所述受体激动剂为GLP-I及其类似物,cAMP含量増加与受体激动剂的活性呈正相关,测定GLP-1R/HEK293细胞内的cAMP含量,即为测定受体激动剂的生物活性。 2. Application I The GLP-1R / HEK293 cell line of claim, wherein: the GLP-IR-transfected genes under conditions in agonist induced stimulation of 293 cells intracellular GLP-1R / HEK content to increase in two messenger cAMP, GLP-IR-transfected gene can be stably expressed; receptor agonist is a GLP-I and analogues thereof, and to increase in cAMP content receptor agonist activity is positively correlated, measurement GLP-1R / cAMP levels in HEK293 cells, is the determination of the biological activity of receptor agonists.
3.根据权利要求2所述GLP-1R/HEK293细胞株的应用,其特征在于:GLP_1及其类似物为:胰高血糖素样肽-I、GLP-I耦联物及其融合蛋白;促胰岛素分泌肽Exendin-4、Exendin-4 I禹联物及其融合蛋白。 The application / HEK293 cell line of the claimed in claim 2 GLP-1R, wherein: GLP_1 and the like as: glucagon-like peptide -I, GLP-I conjugates and fusion proteins thereof; pro insulin secretion peptide Exendin-4, Exendin-4 I Yu and fusion proteins linked.
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CN103344764A (en) * 2013-06-19 2013-10-09 天津美德太平洋科技有限公司 Reagent, method and kit for detection of biological activity of glucagon-like peptide-1 (GLP-1)
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US9789165B2 (en) 2013-12-13 2017-10-17 Sanofi Exendin-4 peptide analogues as dual GLP-1/GIP receptor agonists
US9750788B2 (en) 2013-12-13 2017-09-05 Sanofi Non-acylated exendin-4 peptide analogues
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US9775904B2 (en) 2014-04-07 2017-10-03 Sanofi Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists
CN103966171A (en) * 2014-05-29 2014-08-06 昆明贝尔吉科技有限公司 Cell line for screening peptide and non-peptide GLP-1 (Glucagon-Like Peptide 1) analogs as well as preparation method and application of cell line
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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