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 PDFInfo
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
一种用于测定GLP-1及其功能类似物生物活性的细胞株及其应用，属于药物生物活性检测技术领域。本发明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CGMCCNo.5909。本发明涉及一种表达胰高血糖素样肽-1受体（GLP-1R）基因工程细胞株GLP-1R/HEK293的建立，以及基于GLP-1R/HEK293细胞株受到胰高血糖素样肽-1（GLP-1）及其功能类似物刺激后细胞内环磷酸腺苷（cAMP）水平变化来测定外来刺激中GLP-1及其类似物生物活性的检测方法。该检测方法易标准化，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 invention relates to a cell line for measuring the biological activity of GLP-1 and its functional analogue and its application, belonging to the technical field of drug biological activity detection. The GLP-1R/HEK293 cell strain of the present invention has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the preservation number is CGMCCNo.5909. The present invention relates to the establishment of a genetically engineered cell line GLP-1R/HEK293 expressing glucagon-like peptide-1 receptor (GLP-1R), and based on the GLP-1R/HEK293 cell line receiving glucagon-like peptide- 1 (GLP-1) and its functional analogues stimulated by changes in intracellular cyclic adenosine monophosphate (cAMP) levels to determine the biological activity of GLP-1 and its analogues in external stimuli. The detection method is easy to standardize, has good repeatability, has the characteristics of low cost, convenience and accuracy, and thus has a good application prospect.
Description
Technical field
The present invention relates to a kind of foundation of expressing the proteic genetically engineered cell strain of glucagon-like peptide-1 receptor; And receive glucagon-like-peptide-1 and functional analogue thereof based on this cell strain and stimulate the variation of back cAMP level to measure glucagon-like-peptide-1 and the bioactive detection method of functional analogue thereof, belong to medicine biological activity assay technical field.
Background technology
Glucagon-like-peptide-1 (Glucogan like Peptide-1; GLP-1) act on beta Cell of islet glucagon-like peptide-1 receptor (Glucogan like Peptide-1 Receptor; GLP-1R), promote the synthetic and secretion of the transcribing of insulin gene, Regular Insulin, and can stimulate the propagation and the differentiation of beta Cell of islet; Suppress the beta Cell of islet apoptosis, increase beta Cell of islet quantity.The research proof, GLP-1 can improve diabetes B animal model or patient's blood sugar situation significantly through number of mechanisms, and this provides an extraordinary prospect for the treatment of diabetes B.
Insulin secretion accelerating peptide (Exendin-4) is a kind of peptide material of finding from the Gila monster that is grown in Southwestern United Stares and Mexico desert (Gila monster) saliva, the GLP-1 homology of regulating and controlling blood sugar in 53% and the human blood of aminoacid sequence.Exendin-4 is the GLP-1R agonist equally; With the GLP-1 functional similarity; Has the pancreatic secretion of promotion Regular Insulin; Promote body that utilization, the minimizing liver starch of blood sugar are decomposed, through slowing down GI wriggling the absorption of food slowed down, thereby reduce the peak that the glucose in the food occurs in blood, reach the lowering blood glucose effect.In addition, it controls different physiological roles such as appetite through acting on cns in addition.Because by the dipeptidyl peptidase-IV degraded, its stability is not apparently higher than GLP-1 for Exendin-4.Exendin-4 (Byetta by U.S. Lilly company and the development of Amylin company
), obtained the medicine that drugs approved by FDA is used to treat the brand-new type of diabetes B in 2005.Under physiological condition, Byetta
Similar with the GLP-1 effect; The effect that it stimulates insulin secretion receives the adjusting of blood sugar concentration; Its hormesis alleviated or disappears when blood sugar concentration reduced, thereby the Regular Insulin continuous release can not occur and hypoglycemic phenomenon takes place, and the effect of obvious minimizing diabetes B obese patient body weight is arranged.Yet Exendin-4 is several hours the intravital transformation period the people, and the patient still needs to inject every day Byetta
Twice, still quite inconvenient in the use.Studying at present GLP-1, Exendin-4 medicine with long-acting result of treatment.The long-acting protein medicament of treatment mellitus can the prolong drug transformation period in vivo when keeping hypoglycemic activity, not only can reduce frequency of injection, makes things convenient for the patient to use, and also can reach the purpose that reduces medical expense simultaneously.
Setting up the bioactive measuring method of effective GLP-1 and functional analogue thereof, is one of key measure of estimating the new drug drug effect.At present, the active traditional zoometry method that still depends on that detects of domestic GLP-1 and functional analogue thereof.Its detection method exists laboratory animal to be difficult for obtaining, complicated operation, and the data poor repeatability, detection method is difficult to shortcomings such as stdn.The present invention adopts genetic engineering technique; Genetically engineered Human Embryonic Kidney HEK 293 cell strains (GLP-1R/HEK293) of an ability stably express GLP-1R have been made up; Set up based on detecting GLP-1R/HEK293 cell strain cyclic monophosphate (Cyclic Adenosine monophosphate in the cell after receiving external irritant; CAMP) the bioactive detection method of GLP-1 in the external irritant, Exendin-4 and functional analogue thereof is measured in the variation of content, and detection method is prone to stdn, good reproducibility; Have that cost is low, convenient, characteristic of accurate, thereby have a good application prospect.
Summary of the invention
The purpose of this invention is to provide a kind of being used at external test GLP-1 and the bioactive genetically engineered cell strain of functional analogue GLP-1R/HEK293 thereof.The principle of work of GLP-1R/HEK293 cell strain is: the GLP-1R/HEK293 cell strain can be on cytolemma stably express GLP-1R albumen; This receptor albumen receives the time spent of doing of GLP-1 and functional analogue thereof; The intracellular cAMP content of GLP-1R/HEK293 can raise rapidly, and the biological activity of GLP-1 and analogue thereof is proportionate in level that cAMP raises and the stimulator.Through with the bioactive comparison of GLP-1 standard substance, measure GLP-1 and the biological activity of analogue thereof in the unknown sample.
For making up the HEK293 cell strain of ability stably express GLP-1R, the present invention realizes through following technical scheme.
Technical scheme of the present invention: a strain GLP-1R/HEK293 cell strain; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; Deposit number CGMCC No.5909 uses glucagon-like peptide-1 receptor, is abbreviated as GLP-1R; Transfection Human Embryonic Kidney HEK 293 cells, a strain GLP-1R/HEK293 cell strain of acquisition.
The application of said GLP-1R/HEK293 cell strain, transfection under the GLP-1R gene condition, stimulate at receptor stimulant and can induce second messenger cAMP content increase in the GLP-1R/HEK 293 cell born of the same parents down, the GLP-1R gene of transfection can stably express;
Said receptor stimulant is GLP-1 and analogue thereof, and cAMP content increases and the activity of receptor stimulant is proportionate, and measures the intracellular cAMP content of GLP-1R/HEK293, is the biological activity of measuring receptor stimulant.
The application of said GLP-1R/HEK293 cell strain, GLP-1 and analogue thereof are GLP-1, GLP-1 coupling matter and fusion rotein thereof; Exendin-4, Exendin-4 coupling matter and fusion rotein thereof.
The invention provides a kind of gene fragment method that adopts PCR method to prepare coding human GLP-1R.Particularly; Dna sequence dna SEQ ID NO:1 according to coding human GLP-1R gene ORFs; Design a pair of gene-specific primer: primer GLP-1R1, its nucleotide sequence and SEQ ID NO:3 are 100% homology, and its 5 ' end has the restriction endonuclease sites of Hind III; Primer GLP-1R2 and SEQ ID NO:4 have 100% homology, and its 5 ' end has the restriction endonuclease sites of Xho I.Use primer GLP-1R1, GLP-1R2; From plasmid pReceiver-M02 DNA, amplify the proteic dna fragmentation of coding GLP-1R through PCR method; This segmental nucleotide sequence and SEQ ID NO:1 have 100% homology, and proteic aminoacid sequence of the GLP-1R of its coding and SEQ ID NO:2 have 100% homology.The present invention also with pcr amplification to the proteic dna fragmentation subclone of coding human GLP-1R in TA cloning vector pGEM-T carrier, carry out dna sequence analysis, to confirm the accuracy of pcr amplification product dna sequence dna.
The present invention provides a kind of construction process that is structured in the proteic recombinant expression plasmid of expressing human GLP-1R in the eukaryotic cell.The employed eukaryon expression plasmid of described construction of recombinant expression plasmid is pcDNA3.1 (+) (purchasing the company in Invitrogen), is characterized in ability self-replacation in eukaryotic cell, and expresses the G418 resistance.But, can also be the eukaryon expression plasmid GLP-1R of other kind, not only be confined to plasmid pcDNA3.1 (+).Describedly be used for the proteic gene of expressing human GLP-1R and SEQ ID NO:1 has 100% homology.Described plasmid pcDNA3.1 (+) and SEQ ID NO:5 have 100% homology.Concrete construction process is following: adopt restriction enzyme Hind III and Xho I (giving birth to the worker available from Shanghai) that recombinant plasmid GLP-1R/pGEM-T and the eukaryon expression plasmid pcDNA3.1 that contains the GLP-1R gene carried out restriction enzyme digestion; Prepare 5 ' end respectively and have Hind III, 3 ' end has the GLP-1R gene fragment and the linearizing pcDNA3.1 (+) of Xho I restriction enzyme site.Employing glue recovery method prepares the GLP-1R gene fragment of purifying and the enzyme of linearizing pcDNA3.1 (+) is cut product.The GLP-1R sheet segment DNA of enzyme being cut purifying mixes by 3:1 with plasmid pcDNA3.1 (+) DNA that enzyme is cut purifying; Under the effect of T4 DNA ligase, link; Make up recombinant expression plasmid GLP-1R/pcDNA3.1 (+), its nucleotide sequence should have 100% homology with SEQ ID NO:6.Adopt the pcr amplification method to identify the recombinant expression plasmid GLP-1R/pcDNA3.1 (+) that whether contains the GLP-1R gene fragment in the transformant bacterium.Adopt restriction enzyme Hind III and Xho I that recombinant expression plasmid GLP-1R/pcDNA3.1 (+) is carried out the double digestion analysis; And send Invitrogen company to carry out dna sequence analysis the recombinant plasmid, with the direction of GLP-1R gene and the exactness of sequence in the proof recombinant expression plasmid.
The present invention provides a kind of expression GLP-1R proteic eucaryotic cell strain construction process.
Particularly; Human Embryonic Kidney HEK 293 cells (Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences buys) are spread 6 orifice plates previous day in transfection; When treating that cell grows to 80% degrees of fusion; Linearizing GLP-1R/pcDNA3.1 (+) recombinant plasmid is mixed with Fugene 6 transfection reagents (purchasing the company in Roche); Join then and carry out gene transfection in the HEK293 culture hole, with the screening of medium that contains G418, the recombinant cell strain that screening can be grown in containing high density G418 substratum.
The present invention also provides a kind of GLP-1R of evaluation whether to be expressed in the method for HEK293 cell surface.Particularly, the anti-GLP-1R antibody of rabbit of employing FITC mark dyes to the HEK293 cell of transfection, and the rabbit igg antibody with the FITC mark contrasts as homotype simultaneously.Whether adopt the flow cytometry method to detect the cells transfected surface has GLP-1R albumen to exist.
The present invention provides a kind of method that RT-PCR detects GLP-1R genetic expression stability among the recombined engineering cell strain GLP-1R/HEK293 of partly measuring.Particularly, collect the GLP-1R/HEK293 cell in different generation times, extracting RNA, the mRNA content that produces when adopting RT-PCR to measure the expression of GLP-1R in this cell.Be the confidential reference items contrasts with house-keeping gene GAPDH simultaneously, sequence is SEQ ID NO:7.Observe whether the GLP-1R expression level changes in the different generation time GLP-1R/HEK293 cells.The result shows that GLP-1R albumen can stably be expressed in recombination engineering cell GLP-1R/HEK293 surface.
The present invention provides a kind of GLP-1R/HEK293 cell GLP-1R active measuring method.
Particularly; Use GLP-1 and functional analogue thereof to stimulate the GLP-1R/HEK293 cell of growing; Adopt the bioluminescent detection test kit of Promega company, measure the variation that receives cyclic monophosphate (cAMP) level in the GLP-1R/HEK293 cell that agonist stimulates and observe whether biologically active function of the GLP-1R that expresses on the GLP-1R/ HEK293 cytolemma.If the GLP-1R biologically active of expressing on the GLP-1R/ HEK293 cytolemma, after it received the stimulation of agonists such as GLP-1, HEK293 cells physiological metabolic activity strengthened, and the energy of consumption increases.Intracellular Adenosine triphosphate purine (ATP) is converted into cAMP; After also GLP-1R activates in other words; Intracellular cAMP level can obviously raise, and after the HEK293 that does not express GLP-1R received the stimulation of agonists such as GLP-1, tangible change did not take place intracellular cAMP level.
The present invention also provides a kind of cAMP level based on genetically engineered cell strain GLP-1R/HEK293 to change and measures GLP-1 and the bioactive measuring method of functional analogue thereof.Particularly, take by weighing the GLP-1 standard substance of different activities unit, be respectively applied for the GLP-1R/HEK293 cell in the stimulating growth, measure the quantity that each GLP-1 of activity unit stimulates the cAMP that produces behind the GLP-1R/HEK293 cell.Activity with GLP-1 is an X-coordinate, is ordinate zou with the quantity of cAMP, makes typical curve.Simultaneously, measure the quantity that unknown sample stimulates the cAMP that the GLP-1R/HEK293 cell produces, look into typical curve, thereby can confirm the biological activity of GLP-1 in the unknown sample and functional analogue thereof with the quantity of this cAMP.
Beneficial effect of the present invention: set up based on detect the GLP-1R/HEK293 cell strain after receiving external irritant in the cell variation of cAMP content measure GLP-1 and the bioactive detection method of functional analogue thereof in the external irritant; Detection method is prone to stdn; Good reproducibility; Have that cost is low, convenient, characteristic of accurate, thereby have a good application prospect.
Biological material specimens preservation: a strain GLP-1R/HEK293 cell strain; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; Be called for short CGMCC; Address: Beijing Institute of Microorganism, Academia Sinica, deposit number CGMCC No.5909, preservation date on March 22nd, 2012.
Description of drawings
The enzyme of Fig. 1 recombinant eukaryon expression vector GLP-1R/pcDNA3.1 (+) is cut qualification result.1,1kb DNA Marker; 2, recombinant plasmid GLP-1R/pcDNA3.1 (+); 3 recombinant plasmid GLP-1R/pcDNA3.1 (+)/
Hind III+Xho IEnzyme is cut product.
The expression result of Fig. 2 flow cytometry analysis clone cell surface GLP-1R.The expression of 6 clone cell surfaces of flow cytometry analysis GLP-1R, what Fig. 2 showed is that No. 2 clone cell surfaces have the proteic expression of GLP-1R.(a) negative control group is not add the detected fluorescence intensity of antibody in non-transfected cells, No. 2 clone cells; Negative antibody group is that the transfectional cell that does not all add GLP-1R antibody in non-transfected cells, No. 2 clone cells, added the rabbit igg antibody staining of FITC mark is the detected fluorescence intensity of antibody control; (b) anti-GLP-1R group is for all adding the detected fluorescence intensity of GLP-1R antibody in non-transfected cells, No. 2 clone cells; The merging group is the observation of putting together for above-mentioned three kinds of detected fluorescence intensities of non-transfected cells, No. 2 clone cells.The result shows that No. 2 detected fluorescence intensities of clone cell of anti-GLP-1R group obviously increase (its X-coordinate moves right), proves that No. 2 clone cells of anti-GLP-1R group have the proteic expression of GLP-1R.
The stability result of Fig. 3 RT-PCR half-quantitative detection rotaring redyeing gene GLP-1R.1,100bp Marker; 2,10 days samples; 3,20 days samples; 4,40 days samples; 5,60 days samples; 6, negatives.
Fig. 4 GLP-1R/HEK293 cell, HEK293 cell be active reaction curve result after GLP-1 stimulates respectively.
Fig. 5 GLP-1R/HEK293 cell is active reaction curve result after GLP-1, Exendin-4 stimulate.
Fig. 6 GLP-1R/HEK293 cell is active reaction curve result after Exendin-4-HSA, HSA stimulate.
Embodiment
Embodiment 1: the structure of reorganization GLP-1R/pcDNA3.1 (+) eukaryon expression plasmid
1.1 the amplification of GLP-1R gene and evaluation
Design a pair of gene-specific primer according to coding human GLP-1R albumen ORFs cDNA sequence (SEQ ID NO:1):
GLP-1R1:?5′-AAAAGCTTATGGCCGGCGCCCCCGG-3′（SEQ?ID?NO:3）；
Hind?III
GLP-1R2:?5′-TTCTCGAGTCAGCTGCAGGAGGCCTG-3’?（SEQ?ID?NO:4）
Xho?I
5 ' the end of primer GLP-1R1 has
Hind IIIRestriction endonuclease sites, GLP-1R2 5 ' end have
Xho IRestriction endonuclease sites.With GLP-1R encoding sox (pReceiver-M02 DNA, scientific research department of Jiangsu Prov. Bilharziasis Prevention and Control Inst. provides) is template, carries out pcr amplification.Actual conditions is following: in the PCR of 0.2 mL pipe, add pReceiver-M02 DNA 1 μ L; 10 * Taq Buffer, 10 μ L; MgCl
2(2.5mM) 10 μ L; DNTP Mixture (10Mm), 2 μ L; GLP-1R1 2 μ L, GLP-1R2 2 μ L; DdH
2O, 72.0 μ L.99 ℃ of heat denatured 10 min behind the mixing, PCR pipe placed cooled on ice after, add Taq DNA Polymerase, 1.0 μ L (3U).Mixing, of short duration centrifugal.Then, be put into the PCR pipe in the PCR appearance hole, increase according to following reaction conditions: 94 ℃, 30 sec; 55 ℃, 20 sec, 72 ℃, 90 sec, totally 30 circulations; At last, 72 ℃, 5min.The PCR reaction product that is produced uses the agarose electrophoresis method to analyze, and under uv lamp, observes the electrophoresis situation.Adopt low melting point agarose gel electrophoresis, cut the DNA band that glue reclaims the GLP-1R gene of the about 1392bp of molecular weight.The dna fragmentation of GLP-1R gene is connected with the pGEM-T cloning vector, makes up the GLP-1R/pGEM-T recombinant plasmid, link product and transform DH5 α competence bacterium, obtain to contain the bacterial strain of GLP-1R/pGEM-T recombinant plasmid through the LB plate screening that contains Ampicilin.Obtain reorganization GLP-1R gene plasmid DNA with DNA purification kit (Promega company) purifying.
1.2 the structure of GLP-1R eukaryotic expression recombinant plasmid
It is expression vector that eukaryon expression plasmid pcDNA3.1 (+) is adopted in this experiment, and its preparation method is molecular biology method commonly used.The bacterial classification that promptly contains expression vector through cultivation extracts and obtains this plasmid, the GLP-1R gene is inserted pcDNA3.1 (+) be built into the GLP-1R eukaryotic expression recombinant plasmid.
To expressing vector plasmid pcDNA3.1 (+), carry out earlier
Hind III,
Xho IDouble digestion.Actual conditions is following.Expression vector plasmid pcDNA3.1 (+) 10 μ L;
Hind III1 μ L,
Xho I1 μ L, 10 * Tango damping fluid, 5 μ L, ddH
2O 33 μ L, TV are 50 μ L.37 ℃ of thermostat water bath internal reactions 3 hours reclaim linearizing through agarose gel electrophoresis
Xho IDNA.The GLP-1R/pGEM-T plasmid is made same double digestion, reclaims the GLP-1R gene fragment of about 1392 bp of molecular weight through agarose gel electrophoresis.
The GLP-1R DNA that reclaims is connected structure recombinant expression plasmid GLP-1R/pcDNA3.1 (+) with expression plasmid pcDNA3.1 (+) DNA of above-mentioned double digestion.Linked system is generally 10 μ L, and the mol ratio of pcDNA3.1 (+) plasmid vector of double digestion and double digestion GLP-1R DNA is 1 ︰ 2-10,10 * T4 DNA ligase damping fluid, 1 μ L, and T4 DNA ligase 1 μ L, adding sterilized water to TV is 10 μ L.Connect mixture 16 ℃ of thermostat water bath internal reactions 16 hours.Connect product transformed into escherichia coli DH5 α competent cell.The evaluation of positive colony is to select positive colony through the LB flat board that contains Ampicilin, the extracting plasmid.Recombinant plasmid dna is used
Hind III,
Xho IRestriction endonuclease carries out double digestion; Enzyme is cut product through agarose gel electrophoresis; Bromination second pyridine dyeing; The result shows that recombinant plasmid is cut into the GLP-1R gene band of a molecular weight about 5.4Kb carrier DNA band and an about 1392bp of molecular weight, proves that GLP-1R correctly is inserted into (Fig. 1) among the expression plasmid pcDNA3.1 (+).
Embodiment 2: express the structure and the evaluation of GLP-1R protein gene engineering cell strain
2. 1 reorganization GLP-1R/pcDNA3.1 (+) plasmid transfection HEK293 cell
The HEK293 cell is inoculated in six orifice plates (4 * 10 previous day in transfection
5Cells/well), every hole contains 100 μ L DMEM substratum (antibiotic-free), cultivates 18 ~ 24h to 80% cell density.With linearizing recombinant plasmid 2 μ g, and after mixing, 3 μ L FuGene 6 carry out cell transfecting.
After treating transfection 24h, transfectional cell is passaged to (the G418 initial concentration is confirmed by preliminary experiment) in the fresh nutrient solution that contains 300 μ g/mL G418 by 1:10, puts 37 ℃, 5%CO
2Cultivate in the incubator.Change a not good liquor in per two days, can be observed most necrocytosis behind cultivation 48 ~ 72 h.Survivaling cell is transferred in another one six orifice plates, strengthened the concentration (peak concentration reaches 1200 μ g/mL) of G418 gradually and proceed screening, obtain to express GLP-1R protein gene single cell clone.
2.2 the evaluation of recombinant cell strain surface expression GLP-1R
2.2.1 the proteic expression of flow cytometer showed method checking recombinant cell strain surface GLP-1R
Whether there is GLP-1R to express with anti-GLP-1R antibody test HEK293 cell surface.Concrete grammar is following: get 2 * 10
5Individual transfectional cell, the anti-GLP-1R protein antibodies of rabbit that adds 2 μ L FITC marks dyes, and the transfectional cell of while with the rabbit igg antibody staining of FITC mark is antibody control, with the negative contrast of the transfectional cell that does not add antibody.Detect through flow cytometer, there is the proteic expression of GLP-1R (Fig. 2) on the surface of checking clone cell.To express the proteic single cell clone of GLP-1R and freeze method by conventional cell, storE in liquid nitrogen, carry out frozen for a long time.
2.2.2 the stability observing that reorganization GLP-1R/ HEK293 cell strain GLP-1R expresses
Adopt the RT-PCR method of partly measuring; The expression level that GLP-1R in the GLP-1R/HEK293 cell of cultivating different time is expressed detects; Be interior mark contrast with house-keeping gene GAPDH simultaneously, observe GLP-1R expression of gene stability in the GLP-1R/HEK293 cell.Concrete grammar is following: cultivate the GLP-1R/HEK293 cell, went down to posterity once in per 2 days, respectively at the 10th day, 20 days, 40 days and 60 days, extract total RNA of the reconstitution cell of cultivating different time respectively by the RNA of QIAGEN company extraction agent box specification sheets.Sxemiquantitative RT-PCR detects the expression of GLP-1R, and the result shows, the expression level kept stable of GLP-1R in the cell of cultivation different time (between 10 days-60 days).(Fig. 3).
Embodiment 3:GLP-1R/HEK293 cell expressing GLP-1 receptor active is analyzed
When the GLP-1 receptor protein of recombination engineering GLP-1R/HEK293 cell expressing has physiologically active; When it receives the stimulation of agonist GLP-1 or its functional analogue; GLP-1R/HEK293 cells physiological Metabolic activity strengthens, and shows as cAMP content increase in the cell.Therefore, receive the variation that agonist such as GLP-1 stimulates cAMP content in the GLP-1R/HEK293 cell of back through mensuration, promptly whether the GLP-1R albumen of decidable GLP-1R/HEK293 cell expressing has physiologically active.Concrete grammar is following:
The GLP-1R/HEK293 cell is cultivated and 96 well culture plates by 100 μ L/ holes (30,000 cells/well), uses the DMEM substratum, 37 ℃, 5%CO
2Cultivated 24 hours under the condition.Remove perfect medium in second day, add base culture base spend the night (about 15 hours).Move and abandon basic medium, add sample GLP-1 to be determined, 37 ℃, 5% CO
2Cultivated 1 hour under the condition.With the cAMP content in the born of the same parents behind the above-mentioned GLP-1 stimulation of cAMP reagent detection box (purchasing company) the mensuration GLP-1R/HEK293 cell in AB.The result shows that the cAMP content of GLP-1R/HEK293 cell significantly increases, and the HEK293 cell of untransfected GLP-1R gene does not have any dosage effect trend (Fig. 4).Above experimental result confirms that the GLP-1R albumen of reorganization GLP-1R/HEK293 cell expressing has the physiologically active of natural GLP-1R protein similar, and has specificity.
Embodiment 4: glucagon-like-peptide-1 and functional analogue Determination of biological activity method thereof
Take by weighing the GLP-1 and the functional analogue Exendin-4 thereof of different concns, be respectively applied for the GLP-1R/HEK293 cell in the stimulating growth, measure the quantity that each sample stimulates the cAMP that produces behind the GLP-1R/HEK293 cell with embodiment 3 methods.With the concentration of specimens is X-coordinate, is ordinate zou with the quantity of cAMP, makes curve.The result shows; CAMP after Exendin-4, GLP-1 stimulate in the born of the same parents contains discharge curve and is typical anti-" S " type (Fig. 5); There is the dosage effect dependency in the cAMP content that the GLP-1 of different concns, Exendin-4 stimulate the GLP-1R/HEK293 cell to produce; And can measure the quantity that unknown sample stimulates the cAMP that the GLP-1R/HEK293 cell produces through typical curve; Quantity with this cAMP is looked into typical curve, thereby can confirm the biological activity of GLP-1 in the unknown sample and functional analogue thereof.Above experimental result shows that further the biological activity that reorganization GLP-1R/HEK293 cell is used to measure GLP-1 and functional analogue thereof has specificity, can provide a kind of cAMP level based on genetically engineered cell strain GLP-1R/HEK293 to change in view of the above and measure GLP-1 and the bioactive measuring method of functional analogue thereof.
Embodiment 5: insulin secretion accelerating peptide-1 and functional analogue Determination of biological activity method thereof
Take by weighing the long-acting Exendin-4-HSA fusion rotein of Exendin-4 functional analogue and the human serum albumin (HSA) of different concns; Be respectively applied for the GLP-1R/HEK293 cell in the stimulating growth with embodiment 3 methods, measure the quantity that each sample stimulates the cAMP that produces behind the GLP-1R/HEK293 cell.With the concentration of specimens is X-coordinate, is that ordinate zou is made curve with the quantity of cAMP.The result shows that there is the dosage effect dependency in the cAMP content that stimulates the GLP-1R/HEK293 cell to produce through the Exendin-4-HSA of different concns fusion rotein, and human serum albumin does not have any dosage effect trend (Fig. 6).Above experimental result shows that further reorganization GLP-1R/HEK293 cell is used to measure GLP-1 and functionally similar biological activity has specificity, can provide a kind of cAMP level based on genetically engineered cell strain GLP-1R/HEK293 to change in view of the above and measure GLP-1 and the bioactive measuring method of functional analogue thereof.
<160>?9
?
<210> SEQ?ID?NO:?1
<211> 1392
<212> DNA
<213> GLP-1R
<400> 1
atggccggcg?cccccggccc?gctgcgcctt?gcgctgctgc?tgctcgggat?ggtgggcagg 60
gccggccccc?gcccccaggg?tgccactgtg?tccctctggg?agacggtgca?gaaatggcga 120
gaataccgac?gccagtgcca?gcgctccctg?actgaggatc?cacctcctgc?cacagacttg 180
ttctgcaacc?ggaccttcga?tgaatacgcc?tgctggccag?atggggagcc?aggctcgttc 240
gtgaatgtca?gctgcccctg?gtacctgccc?tgggccagca?gtgtgccgca?gggccacgtg 300
taccggttct?gcacagctga?aggcctctgg?ctgcagaagg?acaactccag?cctgccctgg 360
agggacttgt?cggagtgcga?ggagtccaag?cgaggggaga?gaagctcccc?ggaggagcag 420
ctcctgttcc?tctacatcat?ctacacggtg?ggctacgcac?tctccttctc?tgctctggtt 480
atcgcctctg?cgatcctcct?cggcttcaga?cacctgcact?gcaccaggaa?ctacatccac 540
ctgaacctgt?ttgcatcctt?catcctgcga?gcattgtccg?tcttcatcaa?ggacgcagcc 600
ctgaagtgga?tgtatagcac?agccgcccag?cagcaccagt?gggatgggct?cctctcctac 660
caggactctc?tgagctgccg?cctggtgttt?ctgctcatgc?agtactgtgt?ggcggccaat 720
tactactggc?tcttggtgga?gggcgtgtac?ctgtacacac?tgctggcctt?ctcggtctta 780
tctgagcaat?ggatcttcag?gctctacgtg?agcataggct?ggggtgttcc?cctgctgttt 840
gttgtcccct?ggggcattgt?caagtacctc?tatgaggacg?agggctgctg?gaccaggaac 900
tccaacatga?actactggct?cattatccgg?ctgcccattc?tctttgccat?tggggtgaac 960
ttcctcatct?ttgttcgggt?catctgcatc?gtggtatcca?aactgaaggc?caatctcatg 1020
tgcaagacag?acatcaaatg?cagacttgcc?aagtccacgc?tgacactcat?ccccctgctg 1080
gggactcatg?aggtcatctt?tgcctttgtg?atggacgagc?acgcccgggg?gaccctgcgc 1140
ttcatcaagc?tgtttacaga?gctctccttc?acctccttcc?aggggctgat?ggtggccata 1200
ttatactgct?ttgtcaacaa?tgaggtccag?ctggaatttc?ggaagagctg?ggagcgctgg 1260
cggcttgagc?acttgcacat?ccagagggac?agcagcatga?agcccctcaa?gtgtcccacc 1320
agcagcctga?gcagtggagc?cacggcgggc?agcagcatgt?acacagccac?ttgccaggcc 1380
tcctgcagct?ga 1392
<210> SEQ?ID?NO:?2
<211> 464
< 212>amino acid
<213> GLP-1R
<400>?
2
Met?Ala?Gly?Ala?Pro?Gly?Pro?Leu?Arg?Leu?Ala?Leu?Leu?Leu?Leu
5 10 15
Gly?Met?Val?Gly?Arg?Ala?Gly?Pro?Arg?Pro?Gln?Gly?Ala?Thr?Val
20 25 30
Ser?Leu?Trp?Glu?Thr?Val?Gln?Lys?Trp?Arg?Glu?Tyr?Arg?Arg?Gln
35 40 45
Cys?Gln?Arg?Ser?Leu?Thr?Glu?Asp?Pro?Pro?Pro?Ala?Thr?Asp?Leu
50 55 60
Phe?Cys?Asn?Arg?Thr?Phe?Asp?Glu?Tyr?Ala?Cys?Trp?Pro?Asp?Gly
65 70 75
Glu?Pro?Gly?Ser?Phe?Val?Asn?Val?Ser?Cys?Pro?Trp?Tyr?Leu?Pro
80 85 90
Trp?Ala?Ser?Ser?Val?Pro?Gln?Gly?His?Val?Tyr?Arg?Phe?Cys?Thr
95 100 105
Ala?Glu?Gly?Leu?Trp?Leu?Gln?Lys?Asp?Asn?Ser?Ser?Leu?Pro?Trp
110 115 120
Arg?Asp?Leu?Ser?Glu?Cys?Glu?Glu?Ser?Lys?Arg?Gly?Glu?Arg?Ser
125 130 135
Ser?Pro?Glu?Glu?Gln?Leu?Leu?Phe?Leu?Tyr?Ile?Ile?Tyr?Thr?Val
140 145 150
Gly?Tyr?Ala?Leu?Ser?Phe?Ser?Ala?Leu?Val?Ile?Ala?Ser?Ala?Ile
155 160 165
Leu?Leu?Gly?Phe?Arg?His?Leu?His?Cys?Thr?Arg?Asn?Tyr?Ile?His
170 175 180
Leu?Asn?Leu?Phe?Ala?Ser?Phe?Ile?Leu?Arg?Ala?Leu?Ser?Val?Phe
185 190 195
Ile?Lys?Asp?Ala?Ala?Leu?Lys?Trp?Met?Tyr?Ser?Thr?Ala?Ala?Gln
200 205 210
Gln?His?Gln?Trp?Asp?Gly?Leu?Leu?Ser?Tyr?Gln?Asp?Ser?Leu?Ser
215 220 225
Cys?Arg?Leu?Val?Phe?Leu?Leu?Met?Gln?Tyr?Cys?Val?Ala?Ala?Asn
230 235 240
Tyr?Tyr?Trp?Leu?Leu?Val?Glu?Gly?Val?Tyr?Leu?Tyr?Thr?Leu?Leu
245 250 255
Ala?Phe?Ser?Val?Leu?Ser?Glu?Gln?Trp?Ile?Phe?Arg?Leu?Tyr?Val
260 265 270
Ser?Ile?Gly?Trp?Gly?Val?Pro?Leu?Leu?Phe?Val?Val?Pro?Trp?Gly
275 280 285
Ile?Val?Lys?Tyr?Leu?Tyr?Glu?Asp?Glu?Gly?Cys?Trp?Thr?Arg?Asn
290 295 300
Ser?Asn?Met?Asn?Tyr?Trp?Leu?Ile?Ile?Arg?Leu?Pro?Ile?Leu?Phe
305 310 315
Ala?Ile?Gly?Val?Asn?Phe?Leu?Ile?Phe?Val?Arg?Val?Ile?Cys?Ile
320 325 330
Val?Val?Ser?Lys?Leu?Lys?Ala?Asn?Leu?Met?Cys?Lys?Thr?Asp?Ile
335 340 345
Lys?Cys?Arg?Leu?Ala?Lys?Ser?Thr?Leu?Thr?Leu?Ile?Pro?Leu?Leu
350 355 360
Gly?Thr?His?Glu?Val?Ile?Phe?Ala?Phe?Val?Met?Asp?Glu?His?Ala
365 370 375
Arg?Gly?Thr?Leu?Arg?Phe?Ile?Lys?Leu?Phe?Thr?Glu?Leu?Ser?Phe
380 385 390
Thr?Ser?Phe?Gln?Gly?Leu?Met?Val?Ala?Ile?Leu?Tyr?Cys?Phe?Val
395 400 405
Asn?Asn?Glu?Val?Gln?Leu?Glu?Phe?Arg?Lys?Ser?Trp?Glu?Arg?Trp
410 415 420
Arg?Leu?Glu?His?Leu?His?Ile?Gln?Arg?Asp?Ser?Ser?Met?Lys?Pro
425 430 435
Leu?Lys?Cys?Pro?Thr?Ser?Ser?Leu?Ser?Ser?Gly?Ala?Thr?Ala?Gly
440 445 450
Ser?Ser?Met?Tyr?Thr?Ala?Thr?Cys?Gln?Ala?Ser?Cys?Ser?***
455 460 464
<210> SEQ?ID?NO:?3
<211> 25
<212> DNA
<213> GLP-1R1
<400> 3
aaaagcttat?ggccggcgcc?cccgg
<210> SEQ?ID?NO:?4
<211> 26
<212> DNA
<213> GLP-1R2
<400> 4
ttctcgagtc?agctgcagga?ggcctg
<210> SEQ?ID?NO:?5
<211> 5428
<212> DNA
<213> PcDNA3.1（+）
<400> 5
gacggatcgg?gagatctccc?gatcccctat?ggtgcactct?cagtacaatc?tgctctgatg 60
ccgcatagtt?aagccagtat?ctgctccctg?cttgtgtgtt?ggaggtcgct?gagtagtgcg 120
cgagcaaaat?ttaagctaca?acaaggcaag?gcttgaccga?caattgcatg?aagaatctgc 180
ttagggttag?gcgttttgcg?ctgcttcgcg?atgtacgggc?cagatatacg?cgttgacatt 240
gattattgac?tagttattaa?tagtaatcaa?ttacggggtc?attagttcat?agcccatata 300
tggagttccg?cgttacataa?cttacggtaa?atggcccgcc?tggctgaccg?cccaacgacc 360
cccgcccatt?gacgtcaata?atgacgtatg?ttcccatagt?aacgccaata?gggactttcc 420
attgacgtca?atgggtggag?tatttacggt?aaactgccca?cttggcagta?catcaagtgt 480
atcatatgcc?aagtacgccc?cctattgacg?tcaatgacgg?taaatggccc?gcctggcatt 540
atgcccagta?catgacctta?tgggactttc?ctacttggca?gtacatctac?gtattagtca 600
tcgctattac?catggtgatg?cggttttggc?agtacatcaa?tgggcgtgga?tagcggtttg 660
actcacgggg?atttccaagt?ctccacccca?ttgacgtcaa?tgggagtttg?ttttggcacc 720
aaaatcaacg?ggactttcca?aaatgtcgta?acaactccgc?cccattgacg?caaatgggcg 780
gtaggcgtgt?acggtgggag?gtctatataa?gcagagctct?ctggctaact?agagaaccca 840
ctgcttactg?gcttatcgaa?attaatacga?ctcactatag?ggagacccaa?gctggctagc 900
gtttaaactt?aagcttggta?ccgagctcgg?atccactagt?ccagtgtggt?ggaattctgc 960
agatatccag?cacagtggcg?gccgctcgag?tctagagggc?ccgtttaaac?ccgctgatca 1020
gcctcgactg?tgccttctag?ttgccagcca?tctgttgttt?gcccctcccc?cgtgccttcc 1080
ttgaccctgg?aaggtgccac?tcccactgtc?ctttcctaat?aaaatgagga?aattgcatcg 1140
cattgtctga?gtaggtgtca?ttctattctg?gggggtgggg?tggggcagga?cagcaagggg 1200
gaggattggg?aagacaatag?caggcatgct?ggggatgcgg?tgggctctat?ggcttctgag 1260
gcggaaagaa?ccagctgggg?ctctaggggg?tatccccacg?cgccctgtag?cggcgcatta 1320
agcgcggcgg?gtgtggtggt?tacgcgcagc?gtgaccgcta?cacttgccag?cgccctagcg 1380
cccgctcctt?tcgctttctt?cccttccttt?ctcgccacgt?tcgccggctt?tccccgtcaa 1440
gctctaaatc?gggggctccc?tttagggttc?cgatttagtg?ctttacggca?cctcgacccc 1500
aaaaaacttg?attagggtga?tggttcacgt?agtgggccat?cgccctgata?gacggttttt 1560
cgccctttga?cgttggagtc?cacgttcttt?aatagtggac?tcttgttcca?aactggaaca 1620
acactcaacc?ctatctcggt?ctattctttt?gatttataag?ggattttgcc?gatttcggcc 1680
tattggttaa?aaaatgagct?gatttaacaa?aaatttaacg?cgaattaatt?ctgtggaatg 1740
tgtgtcagtt?agggtgtgga?aagtccccag?gctccccagc?aggcagaagt?atgcaaagca 1800
tgcatctcaa?ttagtcagca?accaggtgtg?gaaagtcccc?aggctcccca?gcaggcagaa 1860
gtatgcaaag?catgcatctc?aattagtcag?caaccatagt?cccgccccta?actccgccca 1920
tcccgcccct?aactccgccc?agttccgccc?attctccgcc?ccatggctga?ctaatttttt 1980
ttatttatgc?agaggccgag?gccgcctctg?cctctgagct?attccagaag?tagtgaggag 2040
gcttttttgg?aggcctaggc?ttttgcaaaa?agctcccggg?agcttgtata?tccattttcg 2100
gatctgatca?agagacagga?tgaggatcgt?ttcgcatgat?tgaacaagat?ggattgcacg 2160
caggttctcc?ggccgcttgg?gtggagaggc?tattcggcta?tgactgggca?caacagacaa 2220
tcggctgctc?tgatgccgcc?gtgttccggc?tgtcagcgca?ggggcgcccg?gttctttttg 2280
tcaagaccga?cctgtccggt?gccctgaatg?aactgcagga?cgaggcagcg?cggctatcgt 2340
ggctggccac?gacgggcgtt?ccttgcgcag?ctgtgctcga?cgttgtcact?gaagcgggaa 2400
gggactggct?gctattgggc?gaagtgccgg?ggcaggatct?cctgtcatct?caccttgctc 2460
ctgccgagaa?agtatccatc?atggctgatg?caatgcggcg?gctgcatacg?cttgatccgg 2520
ctacctgccc?attcgaccac?caagcgaaac?atcgcatcga?gcgagcacgt?actcggatgg 2580
aagccggtct?tgtcgatcag?gatgatctgg?acgaagagca?tcaggggctc?gcgccagccg 2640
aactgttcgc?caggctcaag?gcgcgcatgc?ccgacggcga?ggatctcgtc?gtgacccatg 2700
gcgatgcctg?cttgccgaat?atcatggtgg?aaaatggccg?cttttctgga?ttcatcgact 2760
gtggccggct?gggtgtggcg?gaccgctatc?aggacatagc?gttggctacc?cgtgatattg 2820
ctgaagagct?tggcggcgaa?tgggctgacc?gcttcctcgt?gctttacggt?atcgccgctc 2880
ccgattcgca?gcgcatcgcc?ttctatcgcc?ttcttgacga?gttcttctga?gcgggactct 2940
ggggttcgaa?atgaccgacc?aagcgacgcc?caacctgcca?tcacgagatt?tcgattccac 3000
cgccgccttc?tatgaaaggt?tgggcttcgg?aatcgttttc?cgggacgccg?gctggatgat 3060
cctccagcgc?ggggatctca?tgctggagtt?cttcgcccac?cccaacttgt?ttattgcagc 3120
ttataatggt?tacaaataaa?gcaatagcat?cacaaatttc?acaaataaag?catttttttc 3180
actgcattct?agttgtggtt?tgtccaaact?catcaatgta?tcttatcatg?tctgtatacc 3240
gtcgacctct?agctagagct?tggcgtaatc?atggtcatag?ctgtttcctg?tgtgaaattg 3300
ttatccgctc?acaattccac?acaacatacg?agccggaagc?ataaagtgta?aagcctgggg 3360
tgcctaatga?gtgagctaac?tcacattaat?tgcgttgcgc?tcactgcccg?ctttccagtc 3420
gggaaacctg?tcgtgccagc?tgcattaatg?aatcggccaa?cgcgcgggga?gaggcggttt 3480
gcgtattggg?cgctcttccg?cttcctcgct?cactgactcg?ctgcgctcgg?tcgttcggct 3540
gcggcgagcg?gtatcagctc?actcaaaggc?ggtaatacgg?ttatccacag?aatcagggga 3600
taacgcagga?aagaacatgt?gagcaaaagg?ccagcaaaag?gccaggaacc?gtaaaaaggc 3660
cgcgttgctg?gcgtttttcc?ataggctccg?cccccctgac?gagcatcaca?aaaatcgacg 3720
ctcaagtcag?aggtggcgaa?acccgacagg?actataaaga?taccaggcgt?ttccccctgg 3780
aagctccctc?gtgcgctctc?ctgttccgac?cctgccgctt?accggatacc?tgtccgcctt 3840
tctcccttcg?ggaagcgtgg?cgctttctca?tagctcacgc?tgtaggtatc?tcagttcggt 3900
gtaggtcgtt?cgctccaagc?tgggctgtgt?gcacgaaccc?cccgttcagc?ccgaccgctg 3960
cgccttatcc?ggtaactatc?gtcttgagtc?caacccggta?agacacgact?tatcgccact 4020
ggcagcagcc?actggtaaca?ggattagcag?agcgaggtat?gtaggcggtg?ctacagagtt 4080
cttgaagtgg?tggcctaact?acggctacac?tagaagaaca?gtatttggta?tctgcgctct 4140
gctgaagcca?gttaccttcg?gaaaaagagt?tggtagctct?tgatccggca?aacaaaccac 4200
cgctggtagc?ggtttttttg?tttgcaagca?gcagattacg?cgcagaaaaa?aaggatctca 4260
agaagatcct?ttgatctttt?ctacggggtc?tgacgctcag?tggaacgaaa?actcacgtta 4320
agggattttg?gtcatgagat?tatcaaaaag?gatcttcacc?tagatccttt?taaattaaaa 4380
atgaagtttt?aaatcaatct?aaagtatata?tgagtaaact?tggtctgaca?gttaccaatg 4440
cttaatcagt?gaggcaccta?tctcagcgat?ctgtctattt?cgttcatcca?tagttgcctg 4500
actccccgtc?gtgtagataa?ctacgatacg?ggagggctta?ccatctggcc?ccagtgctgc 4560
aatgataccg?cgagacccac?gctcaccggc?tccagattta?tcagcaataa?accagccagc 4620
cggaagggcc?gagcgcagaa?gtggtcctgc?aactttatcc?gcctccatcc?agtctattaa 4680
ttgttgccgg?gaagctagag?taagtagttc?gccagttaat?agtttgcgca?acgttgttgc 4740
cattgctaca?ggcatcgtgg?tgtcacgctc?gtcgtttggt?atggcttcat?tcagctccgg 4800
ttcccaacga?tcaaggcgag?ttacatgatc?ccccatgttg?tgcaaaaaag?cggttagctc 4860
cttcggtcct?ccgatcgttg?tcagaagtaa?gttggccgca?gtgttatcac?tcatggttat 4920
ggcagcactg?cataattctc?ttactgtcat?gccatccgta?agatgctttt?ctgtgactgg 4980
tgagtactca?accaagtcat?tctgagaata?gtgtatgcgg?cgaccgagtt?gctcttgccc 5040
ggcgtcaata?cgggataata?ccgcgccaca?tagcagaact?ttaaaagtgc?tcatcattgg 5100
aaaacgttct?tcggggcgaa?aactctcaag?gatcttaccg?ctgttgagat?ccagttcgat 5160
gtaacccact?cgtgcaccca?actgatcttc?agcatctttt?actttcacca?gcgtttctgg 5220
gtgagcaaaa?acaggaaggc?aaaatgccgc?aaaaaaggga?ataagggcga?cacggaaatg 5280
ttgaatactc?atactcttcc?tttttcaata?ttattgaagc?atttatcagg?gttattgtct 5340
catgagcgga?tacatatttg?aatgtattta?gaaaaataaa?caaatagggg?ttccgcgcac 5400
atttccccga?aaagtgccac?ctgacgtc 5428
<210> SEQ?ID?NO:?6
<211> 6752
<212> DNA
<213> GLP-1R/PcDNA3.1(+)
<400> 6
gacggatcgg?gagatctccc?gatcccctat?ggtgcactct?cagtacaatc?tgctctgatg 60
ccgcatagtt?aagccagtat?ctgctccctg?cttgtgtgtt?ggaggtcgct?gagtagtgcg 120
cgagcaaaat?ttaagctaca?acaaggcaag?gcttgaccga?caattgcatg?aagaatctgc 180
ttagggttag?gcgttttgcg?ctgcttcgcg?atgtacgggc?cagatatacg?cgttgacatt 240
gattattgac?tagttattaa?tagtaatcaa?ttacggggtc?attagttcat?agcccatata 300
tggagttccg?cgttacataa?cttacggtaa?atggcccgcc?tggctgaccg?cccaacgacc 360
cccgcccatt?gacgtcaata?atgacgtatg?ttcccatagt?aacgccaata?gggactttcc 420
attgacgtca?atgggtggag?tatttacggt?aaactgccca?cttggcagta?catcaagtgt 480
atcatatgcc?aagtacgccc?cctattgacg?tcaatgacgg?taaatggccc?gcctggcatt 540
atgcccagta?catgacctta?tgggactttc?ctacttggca?gtacatctac?gtattagtca 600
tcgctattac?catggtgatg?cggttttggc?agtacatcaa?tgggcgtgga?tagcggtttg 660
actcacgggg?atttccaagt?ctccacccca?ttgacgtcaa?tgggagtttg?ttttggcacc 720
aaaatcaacg?ggactttcca?aaatgtcgta?acaactccgc?cccattgacg?caaatgggcg 780
gtaggcgtgt?acggtgggag?gtctatataa?gcagagctct?ctggctaact?agagaaccca 840
ctgcttactg?gcttatcgaa?attaatacga?ctcactatag?ggagacccaa?gctggctagc 900
gtttaaactt?aagcttatgg?ccggcgcccc?cggcccgctg?cgccttgcgc?tgctgctgct 960
cgggatggtg?ggcagggccg?gcccccgccc?ccagggtgcc?actgtgtccc?tctgggagac 1020
ggtgcagaaa?tggcgagaat?accgacgcca?gtgccagcgc?tccctgactg?aggatccacc 1080
tcctgccaca?gacttgttct?gcaaccggac?cttcgatgaa?tacgcctgct?ggccagatgg 1140
ggagccaggc?tcgttcgtga?atgtcagctg?cccctggtac?ctgccctggg?ccagcagtgt 1200
gccgcagggc?cacgtgtacc?ggttctgcac?agctgaaggc?ctctggctgc?agaaggacaa 1260
ctccagcctg?ccctggaggg?acttgtcgga?gtgcgaggag?tccaagcgag?gggagagaag 1320
ctccccggag?gagcagctcc?tgttcctcta?catcatctac?acggtgggct?acgcactctc 1380
cttctctgct?ctggttatcg?cctctgcgat?cctcctcggc?ttcagacacc?tgcactgcac 1440
caggaactac?atccacctga?acctgtttgc?atccttcatc?ctgcgagcat?tgtccgtctt 1500
catcaaggac?gcagccctga?agtggatgta?tagcacagcc?gcccagcagc?accagtggga 1560
tgggctcctc?tcctaccagg?actctctgag?ctgccgcctg?gtgtttctgc?tcatgcagta 1620
ctgtgtggcg?gccaattact?actggctctt?ggtggagggc?gtgtacctgt?acacactgct 1680
ggccttctcg?gtcttatctg?agcaatggat?cttcaggctc?tacgtgagca?taggctgggg 1740
tgttcccctg?ctgtttgttg?tcccctgggg?cattgtcaag?tacctctatg?aggacgaggg 1800
ctgctggacc?aggaactcca?acatgaacta?ctggctcatt?atccggctgc?ccattctctt 1860
tgccattggg?gtgaacttcc?tcatctttgt?tcgggtcatc?tgcatcgtgg?tatccaaact 1920
gaaggccaat?ctcatgtgca?agacagacat?caaatgcaga?cttgccaagt?ccacgctgac 1980
actcatcccc?ctgctgggga?ctcatgaggt?catctttgcc?tttgtgatgg?acgagcacgc 2040
ccgggggacc?ctgcgcttca?tcaagctgtt?tacagagctc?tccttcacct?ccttccaggg 2100
gctgatggtg?gccatattat?actgctttgt?caacaatgag?gtccagctgg?aatttcggaa 2160
gagctgggag?cgctggcggc?ttgagcactt?gcacatccag?agggacagca?gcatgaagcc 2220
cctcaagtgt?cccaccagca?gcctgagcag?tggagccacg?gcgggcagca?gcatgtacac 2280
agccacttgc?caggcctcct?gcagctgact?cgagtctaga?gggcccgttt?aaacccgctg 2340
atcagcctcg?actgtgcctt?ctagttgcca?gccatctgtt?gtttgcccct?cccccgtgcc 2400
ttccttgacc?ctggaaggtg?ccactcccac?tgtcctttcc?taataaaatg?aggaaattgc 2460
atcgcattgt?ctgagtaggt?gtcattctat?tctggggggt?ggggtggggc?aggacagcaa 2520
gggggaggat?tgggaagaca?atagcaggca?tgctggggat?gcggtgggct?ctatggcttc 2580
tgaggcggaa?agaaccagct?ggggctctag?ggggtatccc?cacgcgccct?gtagcggcgc 2640
attaagcgcg?gcgggtgtgg?tggttacgcg?cagcgtgacc?gctacacttg?ccagcgccct 2700
agcgcccgct?cctttcgctt?tcttcccttc?ctttctcgcc?acgttcgccg?gctttccccg 2760
tcaagctcta?aatcgggggc?tccctttagg?gttccgattt?agtgctttac?ggcacctcga 2820
ccccaaaaaa?cttgattagg?gtgatggttc?acgtagtggg?ccatcgccct?gatagacggt 2880
ttttcgccct?ttgacgttgg?agtccacgtt?ctttaatagt?ggactcttgt?tccaaactgg 2940
aacaacactc?aaccctatct?cggtctattc?ttttgattta?taagggattt?tgccgatttc 3000
ggcctattgg?ttaaaaaatg?agctgattta?acaaaaattt?aacgcgaatt?aattctgtgg 3060
aatgtgtgtc?agttagggtg?tggaaagtcc?ccaggctccc?cagcaggcag?aagtatgcaa 3120
agcatgcatc?tcaattagtc?agcaaccagg?tgtggaaagt?ccccaggctc?cccagcaggc 3180
agaagtatgc?aaagcatgca?tctcaattag?tcagcaacca?tagtcccgcc?cctaactccg 3240
cccatcccgc?ccctaactcc?gcccagttcc?gcccattctc?cgccccatgg?ctgactaatt 3300
ttttttattt?atgcagaggc?cgaggccgcc?tctgcctctg?agctattcca?gaagtagtga 3360
ggaggctttt?ttggaggcct?aggcttttgc?aaaaagctcc?cgggagcttg?tatatccatt 3420
ttcggatctg?atcaagagac?aggatgagga?tcgtttcgca?tgattgaaca?agatggattg 3480
cacgcaggtt?ctccggccgc?ttgggtggag?aggctattcg?gctatgactg?ggcacaacag 3540
acaatcggct?gctctgatgc?cgccgtgttc?cggctgtcag?cgcaggggcg?cccggttctt 3600
tttgtcaaga?ccgacctgtc?cggtgccctg?aatgaactgc?aggacgaggc?agcgcggcta 3660
tcgtggctgg?ccacgacggg?cgttccttgc?gcagctgtgc?tcgacgttgt?cactgaagcg 3720
ggaagggact?ggctgctatt?gggcgaagtg?ccggggcagg?atctcctgtc?atctcacctt 3780
gctcctgccg?agaaagtatc?catcatggct?gatgcaatgc?ggcggctgca?tacgcttgat 3840
ccggctacct?gcccattcga?ccaccaagcg?aaacatcgca?tcgagcgagc?acgtactcgg 3900
atggaagccg?gtcttgtcga?tcaggatgat?ctggacgaag?agcatcaggg?gctcgcgcca 3960
gccgaactgt?tcgccaggct?caaggcgcgc?atgcccgacg?gcgaggatct?cgtcgtgacc 4020
catggcgatg?cctgcttgcc?gaatatcatg?gtggaaaatg?gccgcttttc?tggattcatc 4080
gactgtggcc?ggctgggtgt?ggcggaccgc?tatcaggaca?tagcgttggc?tacccgtgat 4140
attgctgaag?agcttggcgg?cgaatgggct?gaccgcttcc?tcgtgcttta?cggtatcgcc 4200
gctcccgatt?cgcagcgcat?cgccttctat?cgccttcttg?acgagttctt?ctgagcggga 4260
ctctggggtt?cgaaatgacc?gaccaagcga?cgcccaacct?gccatcacga?gatttcgatt 4320
ccaccgccgc?cttctatgaa?aggttgggct?tcggaatcgt?tttccgggac?gccggctgga 4380
tgatcctcca?gcgcggggat?ctcatgctgg?agttcttcgc?ccaccccaac?ttgtttattg 4440
cagcttataa?tggttacaaa?taaagcaata?gcatcacaaa?tttcacaaat?aaagcatttt 4500
tttcactgca?ttctagttgt?ggtttgtcca?aactcatcaa?tgtatcttat?catgtctgta 4560
taccgtcgac?ctctagctag?agcttggcgt?aatcatggtc?atagctgttt?cctgtgtgaa 4620
attgttatcc?gctcacaatt?ccacacaaca?tacgagccgg?aagcataaag?tgtaaagcct 4680
ggggtgccta?atgagtgagc?taactcacat?taattgcgtt?gcgctcactg?cccgctttcc 4740
agtcgggaaa?cctgtcgtgc?cagctgcatt?aatgaatcgg?ccaacgcgcg?gggagaggcg 4800
gtttgcgtat?tgggcgctct?tccgcttcct?cgctcactga?ctcgctgcgc?tcggtcgttc 4860
ggctgcggcg?agcggtatca?gctcactcaa?aggcggtaat?acggttatcc?acagaatcag 4920
gggataacgc?aggaaagaac?atgtgagcaa?aaggccagca?aaaggccagg?aaccgtaaaa 4980
aggccgcgtt?gctggcgttt?ttccataggc?tccgcccccc?tgacgagcat?cacaaaaatc 5040
gacgctcaag?tcagaggtgg?cgaaacccga?caggactata?aagataccag?gcgtttcccc 5100
ctggaagctc?cctcgtgcgc?tctcctgttc?cgaccctgcc?gcttaccgga?tacctgtccg 5160
cctttctccc?ttcgggaagc?gtggcgcttt?ctcatagctc?acgctgtagg?tatctcagtt 5220
cggtgtaggt?cgttcgctcc?aagctgggct?gtgtgcacga?accccccgtt?cagcccgacc 5280
gctgcgcctt?atccggtaac?tatcgtcttg?agtccaaccc?ggtaagacac?gacttatcgc 5340
cactggcagc?agccactggt?aacaggatta?gcagagcgag?gtatgtaggc?ggtgctacag 5400
agttcttgaa?gtggtggcct?aactacggct?acactagaag?aacagtattt?ggtatctgcg 5460
ctctgctgaa?gccagttacc?ttcggaaaaa?gagttggtag?ctcttgatcc?ggcaaacaaa 5520
ccaccgctgg?tagcggtttt?tttgtttgca?agcagcagat?tacgcgcaga?aaaaaaggat 5580
ctcaagaaga?tcctttgatc?ttttctacgg?ggtctgacgc?tcagtggaac?gaaaactcac 5640
gttaagggat?tttggtcatg?agattatcaa?aaaggatctt?cacctagatc?cttttaaatt 5700
aaaaatgaag?ttttaaatca?atctaaagta?tatatgagta?aacttggtct?gacagttacc 5760
aatgcttaat?cagtgaggca?cctatctcag?cgatctgtct?atttcgttca?tccatagttg 5820
cctgactccc?cgtcgtgtag?ataactacga?tacgggaggg?cttaccatct?ggccccagtg 5880
ctgcaatgat?accgcgagac?ccacgctcac?cggctccaga?tttatcagca?ataaaccagc 5940
cagccggaag?ggccgagcgc?agaagtggtc?ctgcaacttt?atccgcctcc?atccagtcta 6000
taattgttg?ccgggaagct?agagtaagta?gttcgccagt?taatagtttg?cgcaacgttg 6060
ttgccattgc?tacaggcatc?gtggtgtcac?gctcgtcgtt?tggtatggct?tcattcagct 6120
ccggttccca?acgatcaagg?cgagttacat?gatcccccat?gttgtgcaaa?aaagcggtta 6180
gctccttcgg?tcctccgatc?gttgtcagaa?gtaagttggc?cgcagtgtta?tcactcatgg 6240
ttatggcagc?actgcataat?tctcttactg?tcatgccatc?cgtaagatgc?ttttctgtga 6300
ctggtgagta?ctcaaccaag?tcattctgag?aatagtgtat?gcggcgaccg?agttgctctt 6360
gcccggcgtc?aatacgggat?aataccgcgc?cacatagcag?aactttaaaa?gtgctcatca 6420
ttggaaaacg?ttcttcgggg?cgaaaactct?caaggatctt?accgctgttg?agatccagtt 6480
cgatgtaacc?cactcgtgca?cccaactgat?cttcagcatc?ttttactttc?accagcgttt 6540
ctgggtgagc?aaaaacagga?aggcaaaatg?ccgcaaaaaa?gggaataagg?gcgacacgga 6600
aatgttgaat?actcatactc?ttcctttttc?aatattattg?aagcatttat?cagggttatt 6660
gtctcatgag?cggatacata?tttgaatgta?tttagaaaaa?taaacaaata?ggggttccgc 6720
gcacatttcc?ccgaaaagtg?ccacctgacg?tc 6752
<210> SEQ?ID?NO:?7
<211> 1002
<212> DNA
<213> GAPDH
<400> 7
atggtgaagg?tcggtgtgaa?cggatttggc?cgtattgggc?gcctggtcac?cagggctgcc 60
atttgcagtg?gcaaagtgga?gattgttgcc?atcaacgacc?ccttcattga?cctcaactac 120
atggtctaca?tgttccagta?tgactccact?cacggcaaat?tcaacggcac?agtcaaggcc 180
gagaatggga?agcttgtcat?caacgggaag?cccatcacca?tcttccagga?gcgagacccc 240
actaacatca?aatggggtga?ggccggtgct?gagtatgtcg?tggagtctac?tggtgtcttc 300
accaccatgg?agaaggccgg?ggcccacttg?aagggtggag?ccaaaagggt?catcatctcc 360
gccccttctg?ccgatgcccc?catgtttgtg?atgggtgtga?accacgagaa?atatgacaac 420
tcactcaaga?ttgtcagcaa?tgcatcctgc?accaccaact?gcttagcccc?cctggccaag 480
gtcatccatg?acaactttgg?cattgtggaa?gggctcatga?ccacagtcca?tgccatcact 540
gccacccaga?agactgtgga?tggcccctct?ggaaagctgt?ggcgtgatgg?ccgtggggct 600
gcccagaaca?tcatccctgc?atccactggt?gctgccaagg?ctgtgggcaa?ggtcatccca 660
gagctgaacg?ggaagctcac?tggcatggcc?ttccgtgttc?ctacccccaa?tgtgtccgtc 720
gtggatctga?cgtgccgcct?ggagaaacct?gccaagtatg?atgacatcaa?gaaggtggtg 780
aagcaggcat?ctgagggccc?actgaagggc?atcttgggct?acactgagga?ccaggttgtc 840
tcctgcgact?tcaacagcaa?ctcccactct?tccaccttcg?atgccggggc?tggcattgct 900
ctcaatgaca?actttgtcaa?gctcatttcc?tggtatgaca?atgaatacgg?ctacagcaac 960
agggtggtgg?acctcatggc?ctacatggcc?tccaaggagt?aa 1002
<210> SEQ?ID?NO:?8
<211> 18
<212> DNA
<213> GAPDH1
<400> 8
5’-TCAACGGCAC AGTCAAGG-3’
<210> SEQ?ID?NO:?9
<211> 18
<212> DNA
<213> GAPDH2
<400> 9
5’-ACCAGTGGAT GCAGGGAT-3’
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| CN103344773A (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) |
| 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) |
| CN103966171A (en) * | 2014-05-29 | 2014-08-06 | 昆明贝尔吉科技有限公司 | A cell line for screening peptides and non-peptides GLP-1 analogs and its preparation method and application |
| US9694053B2 (en) | 2013-12-13 | 2017-07-04 | Sanofi | Dual GLP-1/glucagon receptor agonists |
| US9745360B2 (en) | 2012-12-21 | 2017-08-29 | Sanofi | Dual GLP1/GIP or trigonal GLP1/GIP/glucagon agonists |
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| US9751926B2 (en) | 2013-12-13 | 2017-09-05 | Sanofi | Dual GLP-1/GIP receptor agonists |
| US9758561B2 (en) | 2014-04-07 | 2017-09-12 | Sanofi | Dual GLP-1/glucagon receptor agonists derived from exendin-4 |
| 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 |
| US9789165B2 (en) | 2013-12-13 | 2017-10-17 | Sanofi | Exendin-4 peptide analogues as dual GLP-1/GIP receptor agonists |
| US9982029B2 (en) | 2015-07-10 | 2018-05-29 | Sanofi | Exendin-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists |
| CN108220383A (en) * | 2018-01-05 | 2018-06-29 | 北京博康健基因科技有限公司 | The detection kit and detection method of a kind of rExendin-4 pharmaceutical activity and application |
| US10758592B2 (en) | 2012-10-09 | 2020-09-01 | Sanofi | Exendin-4 derivatives as dual GLP1/glucagon agonists |
| US10806797B2 (en) | 2015-06-05 | 2020-10-20 | Sanofi | Prodrugs comprising an GLP-1/glucagon dual agonist linker hyaluronic acid conjugate |
| CN112538461A (en) * | 2020-12-19 | 2021-03-23 | 上海精翰生物科技有限公司 | GLP1R reporter gene stable transfer cell strain, construction method and application |
| CN114763562A (en) * | 2021-05-28 | 2022-07-19 | 生物岛实验室 | Glucagon-like peptide-1 receptor stable expression cell strain |
| CN116735894A (en) * | 2023-06-14 | 2023-09-12 | 上海市胸科医院 | Protein markers for identifying subtypes of non-small cell lung cancer and their applications |
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| CN103344773B (en) * | 2013-06-19 | 2015-04-15 | 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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| 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 |
| CN103966171A (en) * | 2014-05-29 | 2014-08-06 | 昆明贝尔吉科技有限公司 | A cell line for screening peptides and non-peptides GLP-1 analogs and its preparation method and application |
| US10806797B2 (en) | 2015-06-05 | 2020-10-20 | Sanofi | Prodrugs comprising an GLP-1/glucagon dual agonist linker hyaluronic acid conjugate |
| US9982029B2 (en) | 2015-07-10 | 2018-05-29 | Sanofi | Exendin-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists |
| CN108220383A (en) * | 2018-01-05 | 2018-06-29 | 北京博康健基因科技有限公司 | The detection kit and detection method of a kind of rExendin-4 pharmaceutical activity and application |
| CN112538461A (en) * | 2020-12-19 | 2021-03-23 | 上海精翰生物科技有限公司 | GLP1R reporter gene stable transfer cell strain, construction method and application |
| CN112538461B (en) * | 2020-12-19 | 2024-02-06 | 上海精翰生物科技有限公司 | GLP1R reporter gene stable transgenic cell strain, construction method and application |
| CN114763562A (en) * | 2021-05-28 | 2022-07-19 | 生物岛实验室 | Glucagon-like peptide-1 receptor stable expression cell strain |
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