CN107385015A - Applications of the GLUT 1 in people recombinates the detection of FGF21 protein actives - Google Patents

Applications of the GLUT 1 in people recombinates the detection of FGF21 protein actives Download PDF

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CN107385015A
CN107385015A CN201710443872.9A CN201710443872A CN107385015A CN 107385015 A CN107385015 A CN 107385015A CN 201710443872 A CN201710443872 A CN 201710443872A CN 107385015 A CN107385015 A CN 107385015A
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glut
people
fgf21
recombinates
detection
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陈镝
赵玉峰
苏兴利
梁向艳
魏兰兰
赵妍妍
谢荣
刘英光
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Xian Medical University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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Abstract

Application of the Glucose transporter-1 (GLUT 1) disclosed by the invention in people recombinates the detection of FGF21 protein actives, using the adipocyte of induction differentiation as object, using the elevated-levels of Quantitative Reverse Transcription PCR (qRT PCR) detection Glucose transporter-1 gene expression dose in adipocyte after the people of various concentrations recombinates the processing of FGF21 albumen, and then determine that people recombinates the activity of FGF21 albumen.Present invention discover that good dose-effect relationship be present between Glucose transporter-1 gene expression dose and institute's employment restructuring FGF21 protein concentrations, so that it is determined that Glucose transporter-1 gene expression dose turns into the New Set that people recombinates the detection of FGF21 protein actives in adipocyte, this method operating process is simple, requirement to environment is low, there is good practical value.

Description

Applications of the GLUT-1 in people recombinates the detection of FGF21 protein actives
Technical field
The invention belongs to glucose transporter applied technical field, and in particular to a kind of GLUT-1 recombinates FGF21 eggs in people Application in white Activity determination.
Background technology
Fat and diabetes B has become the principal disease for threatening human health, and relative new drug development is always It is the focus of Field of Drug Discovery.Wherein, the exploitation of protein medicaments shows good prospect.Fibroblast growth factor 21 (FGF21) are the activated proteins of the secretions such as body liver cell and adipocyte, and FGF21 recombinant proteins can reduce blood fat, drop Hypoglycemia, the illness and its complication for losing weight, significantly mitigating diabetes B.Therefore, FGF21 turns into more research and development companies The drug candidate competitively developed.In medicament research and development, the effect of drugs detection architecture for establishing a stability and high efficiency is evaluation The key of drug candidate, therefore, establishing the detection architecture of an objective evaluation FGF21 recombinant proteins treatment diabetes effect is also An important step in FGF21 research and development.
Adipocyte is the function such as FGF21 target cell, the differentiation and metabolism of FGF21 regulation and control adipocytes.Adipocyte Glucose uptake measure be the FGF21 activity test methods that generally use at present：This method adds in adipocyte culture liquid Enter the analog 1,5-anhydroglucitol of glucose, after 1,5-anhydroglucitol intake cell by hexokinase phosphoric acid turn to 6- phosphoric acid- 1,5-anhydroglucitol, 2-deoxyglucose 6-phosphate is assembled because cannot be introduced into follow-up metabolism in cell, by detecting 6- phosphorus Acid -1,5-anhydroglucitol can reflect that the glucose intake of cell is horizontal.One is to use radioactive substance in this method detection process 1,5-anhydroglucitol is marked, it is higher for experiment protection and environmental protection requirement；In addition, fluoroscopic examination side is used in detection method Method determines the level of 2-deoxyglucose 6-phosphate, and this method is expensive, is unfavorable for high flux screening use, while the inspection Survey is also influenceed by hexokinase activity, can not reflect that glucose uptake ability becomes well when enzymatic activity changes Change.Therefore, the deficiency of glucose uptake measure can not only be made up by researching and developing the system of new FGF21 Function detections, and be expected to obtain More easy reliable detection method is obtained, it is significant that this recombinates FGF21 albumen effects for objective evaluation people.
The content of the invention
It is an object of the invention to provide a kind of applications of GLUT-1 in people recombinates the detection of FGF21 protein actives, to judge People recombinates FGF21 protein actives and provides new thinking, solves that existing detection method is costly, high to environmental requirement asks Topic.
The technical solution adopted in the present invention is applications of the GLUT-1 in people recombinates the detection of FGF21 protein actives.
It is a feature of the present invention that
The addition concentration relationship that GLUT-1 and people recombinate FGF21 albumen is：GLUT-1 gene expression doses recombinate with people The increase of the addition concentration of FGF21 albumen and increase.
GLUT-1 gene expression dose is detected by quantifying PCR method.
In quantifying PCR method detection process, GLUT-1 primer sequence is：
Sense primer：5’-GCC CCC AGA AGG TTA TTG A-3’；
Anti-sense primer：5’-CG TGG TGA GTG TGG TGG AT-3’.
Another technical scheme of the present invention, for judging that people recombinates the GLUT-1 of FGF21 protein actives, GLUT-1 gene expression doses recombinate the up-regulation of FGF21 protein concentrations by people, and are raised in dose dependent.
It is of the invention to be further characterized in that,
GLUT-1 is the GLUT-1 in adipocyte.
The present invention application beneficial effect be：The present invention recombinates FGF21 albumen processing fat carefully by the people of various concentrations Born of the same parents, using the glucose transporter-1 (GLUT- in Quantitative Reverse Transcription PCR (qRT-PCR) detection adipocyte 1) gene expression dose, so that it is determined that glucose transporter-1 expression turns into people's restructuring FGF21 albumen work in adipocyte Property detection New Set, operating process is simple, and the requirement to environment is low, there is good practical value.
Brief description of the drawings
Fig. 1 is the displaing micro picture that cells switch of the present invention is mature fat cell；
Fig. 2 is the amplification curve diagram of glucose transporter-1 and beta-actin genes in quantifying PCR method of the present invention；
Fig. 3 is the solubility curve of PCR primer in quantifying PCR method of the present invention；
Fig. 4 is that the present inventor recombinates the influence to glucose transporter-1 gene expression after FGF21 albumen processing cell 12h Block diagram；
Fig. 5 is the dose-effect relationship that the present inventor recombinates FGF21 protein concentrations and glucose transporter-1 gene expression dose Figure；
Fig. 6 is that people recombinates the depression effect figure that FGF21 acceptor inhibitors recombinate the effect of FGF21 albumen to people.
Embodiment
Glucose transporter-1 (GLUT-1) gene expression dose recombinates FGF21 in people in adipocyte disclosed by the invention Application in protein active detection, the present invention is described in detail with reference to the accompanying drawings and detailed description.
First, cell culture and processing
Mouse Preadipocyte In Vitro cell seeding is in 12 well culture plates, in the incubator culture that temperature is 37 DEG C, training used Nutrient solution is that Nostoc commune Vanch liquid is DMEM nutrient solutions, wherein adding the NBCS of volume fraction 10%, changes fresh training within every 2 days Nutrient solution；After cell growth to contact inhibition 2 days, to change in induction broth, induction broth is DMEM nutrient solutions, wherein Add NBCS, 0.3IU/ml insulin, 20 μm of ol/L dexamethasone and the 20 μm of ol/L Rogers row of volume fraction 10% Ketone, after handling 2 days；Change in insulin nutrient solution, insulin nutrient solution is DMEM nutrient solutions, wherein addition volume fraction is 10% NBCS and 0.3IU/ml insulin, after continuing culture 2 days；Change in Nostoc commune Vanch liquid and continue to cultivate, carefully Fat drips deposit born of the same parents in visible cell after induction, to adipocyte transforming, change fresh medium within every 2 days, cell is used for after 6 days People fills restructuring FGF21 albumen processing.As shown in figure 1, cell fat drips deposition all occurs after induction is broken up, it is changed into maturation Adipocyte.
Various concentrations 0ng/ml, 1ng/ml, 3ng/ml, 10ng/ml, 30ng/ are separately added into adipocyte culture liquid Ml, 100ng/ml, 300ng/ml, 1000ng/ml people recombinate FGF21 albumen (U.S.'s Peprotech Products), processing 12 After hour, adipocyte extracts for RNA.
2nd, adipocyte RNA extractions and reverse transcription
Broth out in 12 orifice plates is clean, 350 μ L lysate RL are added per hole, are split with the grinding of cell grinding rod Solution, then all solution are transferred in Filter column CS with 12000rpm centrifugation 2min, collect filtrate；Add again into filtrate Enter the ethanol of 350 μ L 70%, mix and be transferred in adsorption column CR3 with 12000rpm centrifugation 1min, outwell in collecting pipe Waste liquid, adsorption column CR3 is put back in collecting pipe；350 μ L protein liquid removal RW1 are added into adsorption column CR3, with 12000rpm speed Degree centrifugation 1min, outwells the waste liquid in collecting pipe, adsorption column CR3 is put back in collecting pipe；80 μ L are added into adsorption column CR3 DNase I working solutions, room temperature place 15min；350 μ L protein liquid removal RW1 are added into adsorption column CR3, with 12000rpm speed Degree centrifugation 1min, outwells the waste liquid in collecting pipe, adsorption column CR3 is put back in collecting pipe；500 μ L are added into adsorption column CR3 Rinsing liquid RW, is stored at room temperature 2min, with 12000rpm centrifugation 1min, outwells the waste liquid in collecting pipe, by adsorption column CR3 Put back in collecting pipe；Repetition adds 500 μ L rinsing liquid RW into adsorption column CR3,2min is stored at room temperature, with 12000rpm speed 2min is centrifuged, outwells the waste liquid in collecting pipe, adsorption column CR3 is placed in into room temperature places 5min thoroughly to dry remnants rinsing Liquid；Adsorption column CR3 is transferred in RNase-Free centrifuge tubes, adds 50 μ L Rnase-Free ddH2O, room temperature places 2min, With 12000rpm centrifugation 2min, RNA solution is obtained.
ELIASA detects RNA concentration and purity, agarose gel electrophoresis detection RNA mass.2 are sequentially added in eight connecting legs μ L 5x gDNA Eraser Buffer, then 1 μ L gDNA Eraser, 1 μ g Total RNA are sequentially added thereto, finally use RNase Free dH2O complement to 10 μ L, mix, and react at room temperature 5min.Following reagent, 4 μ L 5x are separately added into each pipe PrimeScript Buffer, 4 μ L RNase Free dH2O, 1 μ L PrimeScript RT Enzyme Mix I, 1 μ L RT Primer Mix, overall reaction system are 20 μ L, are mixed.Reaction condition is 37 DEG C, 15min, 85 DEG C, 5s.Will after the completion of reaction CDNA be stored in 4 DEG C it is standby, it is long-term to be transferred to -20 DEG C when preserving.
3rd, the detection of glucose transporter-1 (GLUT-1) gene expression dose
Glucose transporter-1 gene expression dose is detected using quantifying PCR method, with the expression of beta-actin genes As reference gene.6 μ L dH2O, 2 μ L templates cDNA, the primer of 2 μ L glucoses transporter -1,10 μ are sequentially added in eight connecting legs L SYBR Premix Ex TaqII (2x), overall reaction system are 20 μ L, are mixed.
Reaction condition is：The first step is 94 DEG C, 5min；Second step is 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min circulations Carry out 40 times；3rd step is heating dissolving amplified production, obtains solubility curve.
Glucose transporter-1 primer sequence is：
Sense primer：5’-GCC CCC AGA AGG TTA TTG A-3’；
Anti-sense primer：5’-CG TGG TGA GTG TGG TGG AT-3’.
Beta-actin primer sequence is：
Sense primer：5’-CGT TGG CAT CCA CGA AAC TA-3’；
Anti-sense primer：5’-GGT GCT GGG AGG TAC AGG G-3’.
Using beta-actin gene expressions as reference, expression analysis is carried out to experimental result：As shown in Fig. 2 with The copy number of the increase of amplification number, glucose transporter-1 and beta-actin genes exponentially increases after necessarily amplification number Add, finally reach plateau；As shown in figure 3, solubility curve performance is unimodal, illustrate the high specificity of amplified production；Such as Fig. 4 institutes Show, people, which recombinates FGF21 (U.S.'s Peprotech Products, concentration 100ng/ml) processing cells, can dramatically increase fat in 12 hours (* * represent statistical analysis P for the expression of fat intracellular glucose transport body -1<0.01, N=6)；As shown in figure 5, glucose turns The fortune gene expression dose of body -1 recombinates the increase of FGF21 addition concentration with people and increased, and shows as good dose-effect relationship；Such as figure Shown in 6,4 groups of experiments are carried out, one group is that control group is blank group, and two groups of addition people recombinate FGF21 albumen, three groups of addition people weights Group FGF21 protein inhibitors AZD4547, four groups add people and recombinate FGF21 albumen and inhibitor AZD4547 simultaneously, the results showed that, People significantly inhibits FGF21 after recombinating FGF21 acceptor inhibitors AZD4547 (U.S.'s MCE Products, concentration 10nmol/L) processing To effect (the * * expression statistical analysis P of GLUT-1 expression<0.01, N=6).
4th, the identification of specific amplification
Quantitative pcr amplification product is carried out DNA sequencing, glucose transporter-1 sequencing result is as follows：“ccc cca gaa ggt tat tga gga gtt cta caa tca aac atg gaa cca ccg cta cgg aga gcc cat ccc Atc cac cac act cac cac g ", amplified fragments size is 85bp, is reported with American National Biotechnology Information center GLUT-1mRNA (sequence numbers：NM 011400.3) 315-399 base sequences it is completely the same.
Found according to above experimental result, glucose transporter-1 (GLUT-1) gene expression dose recombinates FGF21 by people The up-regulation of addition concentration, and raised in dose dependent, glucose transporter-1 gene expression dose turns into people in adipocyte Recombinate the New Set of FGF21 protein actives detection.

Claims (6)

  1. Applications of the 1.GLUT-1 in people recombinates the detection of FGF21 protein actives.
  2. 2. application according to claim 1, it is characterised in that described GLUT-1 recombinates the addition of FGF21 albumen with people Concentration relationship is：GLUT-1 gene expression doses recombinate the increase of the addition concentration of FGF21 albumen with people and increased.
  3. 3. application according to claim 1, it is characterised in that described GLUT-1 gene expression dose passes through quantitative PCR method detects.
  4. 4. application according to claim 3, it is characterised in that in described quantifying PCR method detection process, GLUT-1's Primer sequence is：
    Sense primer：5’-GCC CCC AGA AGG TTA TTG A-3’；
    Anti-sense primer：5’-CG TGG TGA GTG TGG TGG AT-3’.
  5. 5. for judging that people recombinates the GLUT-1 of FGF21 protein actives, it is characterised in that described GLUT-1 gene expression doses The up-regulation of FGF21 protein concentrations is recombinated by people, and raised in dose dependent.
  6. 6. the GLUT-1 according to claim 5 for being used to judge that people recombinates FGF21 protein actives, it is characterised in that described GLUT-1 be adipocyte in GLUT-1.
CN201710443872.9A 2017-06-13 2017-06-13 Applications of the GLUT 1 in people recombinates the detection of FGF21 protein actives Pending CN107385015A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102296110A (en) * 2011-07-18 2011-12-28 西北农林科技大学 Method for detecting mononucleotide polymorphism of FGF 21 gene of yellow cattle

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102296110A (en) * 2011-07-18 2011-12-28 西北农林科技大学 Method for detecting mononucleotide polymorphism of FGF 21 gene of yellow cattle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JULIE S. MOYERS ET.AL: "Molecular Determinants of FGF-21 Activity—Synergy and Cross-Talk With PPARg Signaling", 《JOURNAL OF CELLULAR PHYSIOLOGY》 *
陈妍等: "成纤维细胞生长因子FGF21的生物学活性检测方法综述", 《科技经济导刊》 *

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Application publication date: 20171124