CN103293315B

CN103293315B - RGA method detects insulin secretion accelerating peptide fusion BA

Info

Publication number: CN103293315B
Application number: CN201210195635.2A
Authority: CN
Inventors: 王军志; 饶春明; 于雷; 王兰; 高凯
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2012-06-14
Filing date: 2012-06-14
Publication date: 2016-05-25
Anticipated expiration: 2032-06-14
Also published as: CN103293315A

Abstract

A kind of new insulin secretion accelerating peptide fusion (Exendin-4-HSA, Byetalog) biological activity determination method. Does is its general principle: by GLP-1R and cAMP response element (cAMP? response? element, CRE) Reporter gene vector cotransfection CHO-K1 cell, pressurization screening separates with monoclonal to cultivate and obtains stablizing monoclonal cell strain CHOglplr/crec14. After GLP-1R and ligand binding, through a series of signal transduction, the expression of activating ELK 1 reporter gene luciferase, stimulates the variation of rear luciferase expression to carry out quantitatively Byetalog activity by detecting Byetalog. Concrete detecting step is: 6000/hole of CHOglplr/crec14 cell spreads 96 orifice plates, cultivates 16-18 hour, adds variable concentrations Byetalog to stimulate 6 hours, detects luciferase activity. The method is simple to operate, is quick on the draw, and variability is little, significant for quality control and the clinical practice of Byetalog.

Description

RGA method detects insulin secretion accelerating peptide fusion BA

Technical field:

The present invention relates to the active detection field of bio-pharmaceutical, for insulin secretion accelerating peptide fusion (Byetalog)Determination of biological activity set up one more fast and accurately reporter gene quantitative approach (reportgeneassay,RGA)。

Background technology:

The research application of insulin secretion accelerating peptide (Exendin-4) becomes the study hotspot in treating diabetes field in recent yearsIt-. Exendin-4 can effectively solve the multiple challenges of current diabetes B treatment: effectively reducing blood sugar does not increase low simultaneouslyBlood sugar risk; Improve β cell function; Lose weight; Reduce cardiovascular risk. Insulin secretion accelerating peptide fusion(Exendin-4-HSA, Byetalog) is the long-acting protein medicament of the treatment diabetes B of China's research and development, commonly uses at presentActivity determination method is cAMP enzyme linked immunosorbent assay, needs loaded down with trivial details ELISA detecting step and expensive kit. Exendin-4 is pancreasGlycemic plain sample peptide-1 (GLP-1) analog, the performance of its biological effect is by being combined with GLP-1R, activates adenylate cyclaseEnzyme (AC), produces cAMP, and then activates the transcription factor that cAMP relies on, the latter and cAMP response element (cAMPresponseElement, CRE) in conjunction with activating transcribing of downstream gene. Reporter gene method (reportgeneassay, RGA) is a kind of fastFast, easy detection method, utilizes the variation of reporter gene expression after drug effect to reflect the activity of medicine. In conjunction withThe concrete mechanism of action of Exendin-4 and the advantage of RGA, we are CRE reporter gene and GLP-1R cotransfection CHO-K1 cell,Set up one easier, cheaply detection method.

Summary of the invention:

1. goal of the invention:

Set up one quicker, easy, accurately objective Byetalog determination of activity reporter gene method, promotes this productResearch and development, quality control and clinical practice.

2. technical scheme:

The present invention is by reporting base by GLP-1R carrier and cAMP response element (cAMPresponseelement, CRE)Because of carrier cotransfection CHO-K1 cell, and pressurize to screen to separate and turn out monoclonal cell strain, then set up corresponding detection methodByetalog activity is carried out quantitatively. Its techniqueflow is: foundation (GLP1R and the report base of CHOGLP1R/CRE cell lineBecause of the strain of carrier monoclonal cell) → RGA assay method is set up and methodology is verified.

3. beneficial effect:

The foundation of this detection method is conducive to the research and development of Byetalog medicine, and quality control and clinical practice, haveHigher using value.

By with existing method comparison, have the following advantages:

(1) cost is lower, does not need expensive reagent box;

(2) simple to operate, do not need loaded down with trivial details ELISA step;

(3) cycle short, can in 24 hours, complete all experiments were;

(4) result is objective reliable, and the degree of accuracy is high, makes a variation little.

Brief description of the drawings:

Fig. 1-1westernblot detects GLP-1R and expresses

Reactivity comparison to Byetalog before and after Fig. 1-2 CHO-K1 transient transfection GLP-1R/CRE

The reactivity comparison of Fig. 2-1 monoclonal cell strain

The comparison of the different cell densities in Fig. 3-1

The experimental result that Fig. 3-2Byetalog action time is different

The pre-diluted concentration in Fig. 3-3 and detection range

The preliminary methodology checking in Fig. 4-1

Detailed description of the invention:

1. materials and methods:

1.1 research objects: insulin secretion accelerating peptide fusion

1.2GLP-1R plasmid: buy the gene Co., Ltd in Aureal Dongyuan County.

PGL4.26 carrier: buy in promega.

CHO-K1 cell: buy in ATCC.

Byetalog standard items and sample: Chinese food and pharmaceutical research calibrating institute recombinant technique product chambers are retained.

1.3 reagent:

Growth medium: F12K+10% hyclone (GIBCO, #10099)+1% dual anti-(GIBCO, #15240)

Plating medium: Opti-MEM (GIBCO, #31985)

MegaTran1.0 transfection reagent: origene, TT200003

G418：CalBiochem，#345810

Hygromycin: Cellgro, 30-240-CR

Select culture medium: growth medium+200ug/mlG418+300ug/ml hygromycin

Glolysis：promega，E2661

Luciferase substrate: promega, E2650

GLP-1R antibody: Abcam, ab39072

Chemical luminous substrate: pierce, P1010

TMB：Amersco，J644

1.4 instruments:

ELIASA, SpectraMaxM5

Chemiluminescence imager, LAS-3000

1.5 DAS:

SoftMaxPro software

SigmaPlot11.0 software

1.6 experiment flows:

1.6.1pGL4.26-CRE structure

Chemical synthesising DNA response element CRE, sequence is as follows:

CRE normal chain:

5’-CTAGCGCACCAGACAGTGACGTCAGCTGCCAGATCCCATGGCCGTCATACTGTGACGTCTTTCAGACACCCCATTGACGTCAATGGGAGAACA-3’

CRE minus strand:

3’-GCGTGGTCTGTCACTGCAGTCGACGGTCTAGGGTACCGGCAGTATGACACTGCAGAAAGTCTGTGGGGTAACTGCAGTTACCCTCTTGTCTAG-5’

After positive minus strand annealing, be connected into pGL4.26 carrier, deliver to the order-checking of the prosperous biotechnology of AudioCodes company, entirely true.

1.6.2 transient transfection and detection

1.6.2.1 transfection

(1) shift to an earlier date 18-24 hour, 6 orifice plates paving CHO-K1 cells, 10,000 cells/well;

(2) dilute plasmid with 200ulopti-MEM, after mixing, add transfection reagent TransMeg (plasmid w/ transfection reagentV, 1: 3), mix 8s, leave standstill 10 minutes;

(3) plasmid-transfection reagent mixture is added in 6 orifice plate cells, after 3-4 hour, change growth medium.

(4) harvesting after cultivation 48h.

1.6.2.2westernblot detect the expression of GLP-1R

12%SDS-PAGE, 100V electrophoresis 10 minutes, 150V electrophoresis is to bromophenol blue to bottommost; 7 points of 20V voltage stabilizing transferring filmsClock, 5% skimmed milk power room temperature sealing 1h, within 1: 1000, dilution primary antibodie 4 is spent night, and 1: 2000 dilution two anti-room temperature 1h, adds substrate and keeps awayLight reaction 5 minutes, takes a picture.

1.6.2.3RGA detect the reactivity to Byetalog

(1) digestion counting cells, plating medium is diluted to 10,000 cell/ml, and 100ul/ hole spreads 96 orifice plates, cultivates16-18h；

(2) 100ul/ hole adds the Byetalog sample stimulus 6h of serial dilution;

(3) discard cell conditioned medium, 100ul/ hole adds lysate Glo-lysis, room temperature concussion cracking 15min;

(4) draw 50ul cell lysate and proceed to white light tight ELISA Plate, add luciferase substrate 50ul/ hole, mixedAfter even, go up machine testing immediately.

1.6.3 pressurization screening and monoclonal cell strain separate and cultivate

(1) 48h after transfection, changes G418 and hygromycin and selects culture medium, screens about 3 weeks, within every 3-4 days, changes onceG418 selects culture medium;

(2) after clone grows, digestion counting, is diluted to 1 cell/200ul, and 200ul/ hole spreads to 96 orifice plates;

(3) with selecting medium culture 2 weeks, micro-Microscopic observation, selects monoclonal cell hole, and is transferred to 24 holesPlate, reaches 6 orifice plates again and expands cultivation after covering with.

1.6.4 monoclonal cell strain detects Byetalog is reactive

Separate cultivation and obtain 24 monoclonal cell strains, detect respectively the reactivity to Byetalog, cell density is 10,000/hole, the pre-diluted concentration of Byetalog is 10ug/ml, 10 6 of multiple proportions serial dilutions gradients, 1 blank, totally 8 concentrationPoint, respectively establishes 2 multiple holes. Byetalog drug effect detects uciferase activity for 6 hours.

1.6.5RGA activity determination method is set up:

1.6.5.1 the linear response of more different cell densities

According to result of the test above, the pre-diluted concentration of Byetalog is 10ug/ml, and adjusting doubling dilution multiple is 4 times,Drug treating time is 6 hours, detects cell density and is respectively 2000,4000, and the dosage in 6000,8000 and 10000/hole is anti-Answer curve.

1.6.5.2 the linear response of different pharmaceutical stimulation time

According to result of the test above, cell density is 6000/hole, and adjusting the pre-diluted concentration of Byetalog is 30ug/Ml, doubling dilution multiple is 4 times, detects the dose-effect curve that acts on respectively 4,6 and 8 hours.

1.6.5.3 pre-diluted concentration and detection range

According to result of the test above, cell density is 6000/hole, and Byetalog doubling dilution multiple is 4 times, medicineAction time is 6 hours, detects the pre-diluted concentration of Byetalog and is respectively 10ug/ml, and the dosage of lug/ml and 0.1ug/ml is anti-Answer curve.

1.6.6 preliminary methodology checking

By newly-built RGA method, 1 batch of Byetalog stoste is carried out to determination of activity, do 50% recovery of standard addition, each dose simultaneouslyMeasure 3 multiple holes, measure 3 times.

2. result and discussion

2.1CHO-K1 cell transfecting GLP-1R plasmid, westernblot detects it and expresses, and the results are shown in Figure 1-1, wild typeCHO-K1 cell does not substantially detect GLP-1R and expresses, and cell overexpression GLP-1R albumen after transfection.

2.2CRE plasmid separately or with GLP-1R plasmid co-transfection CHO-K1 cell, and detect the reaction to ByetalogProperty, the results are shown in Fig. 1-2, the separately cell of transfection CRE, Byetalog does not affect uciferase activity substantially, and CRE andThe cell of GLP-1R cotransfection, along with the increase luciferase activity of Byetalog concentration strengthens, and dose-effect curve relationMeet four parametric equations, linear better (seeing Fig. 1-1).

2.3CRE plasmid and GLP-1R plasmid co-transfection CHO-K1 cell, mould by 200ug/mlG418 and 300ug/ml tideElement pressurization screening, the stable clone obtaining carries out monoclonal and separates cultivation, obtains 24 monoclonal cell strains, and it is right to detect respectivelyByetalog reactivity, the results are shown in Figure 2-1. The pre-diluted concentration of Byetalog is 10ug/ml, 10 6 of doubling dilutions gradients, 1Blank concentration. All responding property of 3,4,6,12,14,16, No. 20 clones, wherein 3,4,14, No. 20 clone's signal values are stronger, noiseRelatively good, No. 14 clones' ED₅₀Be worth minimumly, detection sensitivity is the highest, intends cloning CHOglplr/crec14 with this and sets upByetalog activity test method.

The foundation of 2.4 detection methods

Respectively to CHOglplr/crec14 cell bed board density, drug treating time, the parameters such as drug effect concentration rangeBe optimized, see Fig. 3-1,3-2 and 3-3. By comparing ED₅₀Value and signal to noise ratio, tentatively determined detection scheme: cell bed boardDensity is 6000 cells/well, and the pre-diluted concentration of Byetalog medicine is 1ug/ml, 4 doubling dilutions, and the medicine irritation time is6h. 2.5 use RGA methods are measured 3 times the 1 batch of Byetalog stoste, do 50% rate of recovery simultaneously, wherein one-time detection curve see Fig. 4-1, result gathers in Table 2-1:

Table 2-1RGA method detects Byetalog stoste BA

In table, result shows, the specific activity average that the method detects this batch of stoste is 4.68 × 10⁴U/mg, CV% value is4.14%, the rate of recovery is between 70-130%, and repeatability and accuracy are all higher, are applicable to detect as conventional methodByetalog activity.

Claims

1. application report gene approach detects the bioactive method of insulin secretion accelerating peptide fusion, it is characterized in that:

First, according to the principle of the mechanism of action of GLP-1 acceptor and reporter gene, build the reactive transgenosis cell strain of GLP-1,Concrete grammar is: by GLP-1R carrier and cAMP response element CRE Reporter gene vector cotransfection CHO-K1 cell, it is right to detectThe reactivity of Byetalog, result demonstration, along with the increase luciferase activity of Byetalog concentration strengthens, and dose response songLine relation meets four parametric equations, better linear;

By 200ug/mlG418 and 300ug/ml hygromycin pressurization screening, carry out monoclonal and separate cultivation, obtain stable clone24 monoclonal cell strains, detect respectively the reactivity to Byetalog, obtain clone signal value stronger, signal to noise ratio is good, ED₅₀ValueThe monoclonal cell strain CHOglplr/crec14 minimum, detection sensitivity is the highest, then clone CHOglplr/crec14 for rightThe detection of Byetalog activity;

Secondly, respectively to CHOglplr/crec14 cell bed board density, drug treating time, drug effect concentration range parameterBe optimized, by comparing ED₅₀Value and signal to noise ratio, determine following detected parameters and condition: cell bed board density be 6000 thinBorn of the same parents/hole, the pre-diluted concentration of Byetalog medicine is 1ug/ml, 4 doubling dilutions, the medicine irritation time is 6h;

The 3rd, detecting step and checking: by RGA method, 1 batch of Byetalog stoste is measured 3 times, done 50% rate of recovery, result simultaneouslyShow, the specific activity average that this method detects this batch of stoste is 4.68 × l0⁴U/mg, CV% value is 4.14%, the rate of recovery is at 70-Between 130%, repeatability and accuracy are all higher, are applicable to detect Byetalog activity as conventional method.