CN1318587C - Recombination preparation method of amidating Exendin-4 polypeptide - Google Patents
Recombination preparation method of amidating Exendin-4 polypeptide Download PDFInfo
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Abstract
The present invention discloses a recombination preparation method of amidation Exendin-4 polypeptide. Encoding the DNA sequence of the Exendin-4 polypeptide through manual composition and cloning into the expression carrier to construct recombinant plasmid, then transferring it to the host cells to obtain genetic engineering bacteria integrated by expression Exendin-4 polypeptide, fermenting and training engineering bacteria to obtain the amidation Exendin-4 polypeptide through the treatment technology after expression. Compared with the chemical synthesis method, this preparation method has the characteristics of reducing production cost and meeting large-scale production.
Description
Technical field
The present invention relates to utilize genetic engineering technique to prepare polypeptide or method of protein, relate in particular to and utilize genetic engineering technique to prepare the method for amidating Exendin-4-4 polypeptide, also relate to the amidation technology of polypeptide protein.
Background technology
Exendin-4 is from Monster Gila monster, a kind of 39 amino acid whose polypeptide of separating in the oral secretion of Heloderma horridum or Heloderma suspectum, its amino acid and GLUCAGON LIKE PEPTIDE-1 (Glucagon like peptide-1) has 50% homology, and shows in several animal models and the similarly effect of treatment type ii diabetes of GLUCAGON LIKE PEPTIDE-1.
United States Patent (USP) 5,424,286 disclose the function of Exendin-4 aminoacid sequence and treatment diabetes thereof, and wherein disclosed Exendin-4 polypeptide total length is: hgegtftsdl skqmeeeavrlfiewlkngg pssgappps.
Because Exendin-4 has many advantages on treatment type ii diabetes (NIDDM):
1. when surpassing physiological concentration, blood sugar concentration just promotes secretion of insulin by GLUCAGON LIKE PEPTIDE-1 acceptor, thereby blood sugar regulation concentration, when blood sugar concentration was lower than physiological concentration, then its blood sugar reducing function weakened, and the effect of its blood sugar regulation has the blood sugar concentration dependency; The secretion of glucagon suppression.
2. the inhibition intestines peristalsis reduces the food absorption in vivo, thereby produces the effect that reduces weight in patients, weight increase or obesity and the relevant complication of avoiding patient's life-time service Regular Insulin and oral diabetes medicament to be caused.
3. promote the differentiation of pancreas islet precursor cell, improve the islet secretion function.
In view of the superiority of Exendin-4 on treatment type ii diabetes (NIDDM), preparing Exendin-4 in a large number is to clinical essential condition with this product application.Utilize the gene recombination technology preparation significant with the product that has been used for clinical study abroad.
Summary of the invention
The purpose of this invention is to provide the preparation method of amidating Exendin-4-4 polypeptide, it comprises the steps:
1. design the also dna sequence dna of synthetic coding Exendin-4 aminoacid sequence;
2. make up the expression vector of the recombinant plasmid that contains the Exendin-4 polypeptid coding sequence;
3. change recombinant plasmid over to host cell, obtain the genetic engineering bacterium of amalgamation and expression or non-fusion expression Exendin-4;
4. fermentation culture engineering bacteria;
5. express aftertreatment, obtain amidating Exendin-4-4 polypeptide.
In above-mentioned preparation method, the dna sequence dna of step described in 1. preferably contains the codon of terminal the 40th Gly of coding C-.
In above-mentioned preparation method, the expression vector of step described in 2. be prokaryotic expression carrier preferably.
In above-mentioned preparation method, described prokaryotic expression carrier is the intestinal bacteria fusion expression vector preferably.
In above-mentioned preparation method, described intestinal bacteria fusion expression vector is pThioHis or pGex4T-1 preferably.
In above-mentioned preparation method, described coli expression carrier pGex4T-1 is cloned into the dna sequence dna of coding Exendin-4Gly, forms recombinant plasmid pGSTE4G, with Exendin-4 encoding gene and GST amalgamation and expression.
In above-mentioned preparation method, described intestinal bacteria are preferably BL21.
In above-mentioned preparation method, the expression aftertreatment of step described in 5. preferably includes following step: purified fusion protein, use the Enterkinase cleavage of fusion proteins, separation and purification Exendin-4Gly, with α-amidating enzyme Exendin-4Gly is converted into amidation exendin-4, separation and purification amidation exendin-4.
Embodiment
Used molecular biology experiment operation removes and specifies all with reference to " molecular cloning " and " molecular biology experiment guide " in the following example of the present invention.
DNA operates used reagent all available from the biological Dalian of treasured company, and experiment condition carries out with reference to the condition that manufacturer is recommended.
The structure of embodiment 1 genetic engineering bacterium pGSTE4G
1.Exendin-4Gly obtaining of encoding gene
The Exendin-4Gly encoding gene is according to intestinal bacteria password preference principle, is translated into dna sequence dna by its aminoacid sequence counter-rotating.
The aminoacid sequence of Exendin-4Gly is: hgegtftsdl skqmeeeavr lfiewlknggpssgapppsg,
According to multiple clone site behind the expression vector and restricted protein endoenzyme enzyme recognition site, design following coding gene sequence:
5’-gaattcgatg acgatgacaa gcacggtgaa ggtaccttca cctccgacct gtccaaacag 60
EcoRI
atggaagaag aagctgttcg tctgttcatc gaatggctga aaaacggtgg tccgtcctcc 120
ggtgctccgc cgccgtccgg ttaataagtc gac -3’ 153
SalI
The gained sequence clone is gone among the pUC18 to preserve and is obtained pUCE4G, extract the pUCE4G plasmid DNA with the Promega plasmid extraction kit, small segment with EcoRI/SalI cutting-out 120bp separates small segment with agarose gel electrophoresis, and it is standby to reclaim test kit extraction small segment with gel DNA.
2. make up plasmid pGSTE4G
Extract pGEX 4T-1 plasmid DNA with the Promega plasmid extraction kit,, use the EcoRI/SalI double digestion, and with CIAP with big fragment dephosphorylation.Ready small segment of preceding step is connected the back with the T4 dna ligase transform the e. coli bl21 competent cell of using the preparation of CaCl2 method.With the LB plate screening transformant that contains 100ug/ml Amp.With the transformant picking in the flat board in the LB nutrient solution that contains 100ug/ml Amp, 37 ℃ of shaking culture, with 1mM IPTG abduction delivering, occur 31kd the expression band be correct recon.Gained recon called after pGSTE4G.
Embodiment 2 separation and purification Exendin-4Gly
1. inoculating pGSTE4G contains in the LB nutrient solution of 100ug/ml Amp to 1000ml, 37 ℃ of shaking culture 16 hours are inoculated into fermentation culture in 20 liters of TB substratum with culture, and OD600 reaches at 8 o'clock and adds 1mM IPTG abduction delivering, continue to cultivate after 4 hours centrifugal collection thalline.
2. the gained thalline is with 50mM Tris.Cl pH8.0, and 150mM NaCl suspends, and with the broken thalline of APV high-pressure homogenization, the centrifugal 20min of 14000g collects supernatant liquor.
3. use 50mM Tris.Cl pH8.0 on the gained supernatant liquor, 150mM NaCl equilibrated GST affinity column, with 50mM Tris.Cl pH8.0,150mM NaCl washes foreign protein, use 50mMTris.Cl pH8.0,150mM NaCl, 2mM CaCl2,2u/ml Enterkinase circulated 2 hours in being combined with the GST affinity column of GSTE4G fusion rotein, collected to penetrate liquid.
4. penetrate and use 50mM Tris.Cl pH8.0 on the liquid, 150mM NaCl equilibrated Q-SepharoseFast Flow washes baseline with sample-loading buffer, with 50mM Tris.Cl 300mM NaCl wash-out Exendin-4Gly.
Embodiment 3 Exendin-4Gly are converted into amidating Exendin-4-4 (Exendin-4-NH2)
This step utilizes recombinant alpha-AE (α-amidating enzyme) that Exendin-4Gly is converted into amidating Exendin-4-4 (Exendin-4-NH2).
This step is implemented as follows:
Reaction system: 30mM MES pH6.0,0.0015% (v/v) Triton X-100,11ug/mlcatalase, 5mM KI, 1.5mM sodium ascorbate, 1% (v/v) ethanol, 25000u/ml α-AE, 0.2mM ambient 02,1mM Exendin-4Gly (about 4mg/ml).
Be reflected at 37 ℃ and carried out 16 hours, adjust pH to 8.0, add halfcystine, and prepare purifying amidating Exendin-4-4 (Exendin-4-NH2) to the 5mM termination reaction with 2N NaOH.
Embodiment 4 purifying amidating Exendin-4s-4 (Exendin-4-NH2)
With 50mM Tris.Cl pH8.0,150mM NaCl equilibrated Q-Sepharose Fast Flow washes baseline with sample-loading buffer on the above-mentioned reaction mixture, with 50mM Tris.Cl 300mMNaCl wash-out Exendin-4-NH2; Collected Exendin-4-NH2 goes up the 15 RPC reversed phase chromatography posts with 0.1%TFA equilibrated Source, 0.1%TFA acetonitrile gradient wash-out with 0% to 100%, collect Exendin-4-NH2, transfer pH to 8 with NaOH, last with 50mM Tris.Cl pH8.0,150mM NaCl equilibrated Q-Sepharose Fast Flow removes TFA and acetonitrile, obtains pure product Exendin-4-NH2.
Embodiment 5 reorganization Exendin-4 single vein and subcutaneous injection toxicity tests
1. trial-product
Title: reorganization Exendin-4 (E-4)
Labelled amount: 80 μ g/ml
Solvent: N.F,USP MANNITOL etc.
Preservation condition: 2-8 ℃ preservation.
2. research system
2.1 animal species: Kunming mouse
2.2 sex and quantity: 60 (female 30, male 30)
2.3 animal age: 3 ~ 4 ages in week
2.4 the weight of animals during on-test: female 18.68 ± 0.62, male 19.55 ± 0.84.
2.5 animal-origin: Military Medical Science Institute's Experimental Animal Center
2.6 animal conformity certification number: XCXK (capital) 2002-001
2.7 Experimental Establishment conformity certification: the moving word of doctor 01-2049 number
2.8 reason is selected by the research system: mouse is raised convenient, and is more stable to drug reaction, is one of laboratory animal of relatively generally acknowledging in the world at present.
2.9 the mark identification of research system: picric acid mark.
2.10 quarantine time and discovery: find active, smooth, the no abnormality seen of defecating of animal when on July 31st, 2003 quarantined by hair.
2.11 feeding and management condition: support in plastics are raised box, male and female are divided foster, and 5 in every box is fed with standard feed, freely takes the photograph water, and 12 hours light and shades of illumination alternately.18 ~ 24 ℃ of room temperatures, humidity 40-60%.
3. experimental design:
3.1 the foundation of test design
3.1.1 mechanism of action and research background:
E-4 is a kind of 39 amino acid whose peptides, have on its structure 53% with GLP-1 (glucagon-like-peptide-1) homology, and be the GLP-1 receptor antagonist.The biologic activity of GLP-1 comprises: stimulate secretion of insulin, promote the biosynthesizing of Regular Insulin; The secretion of glucagon suppression and the emptying of stomach; Reduce the absorption of food.GLP-1 keeps the active time shorter in vivo, and it can be decomposed by serine protease.
E-4 has and is better than the pharmacological action of GLP-1.Natural E-4 is the material that extracts from the saliva liquid of Gila Heloderma suspectum Yi, and U.S. Amylin pharmaceutical factory synthetic E-4 has finished preclinical test, is carrying out large-scale III clinical trial phase at present.Animal test results proves: E-4 can stimulate secretion of insulin when hyperglycemia, but does not have this effect when hypoglycemia; The emptying of E-4 adjustable gastric, the absorption of the nutritive substance that slows down; For obese animal, E-4 can reduce appetite, reduces body weight; Especially E-4 can make glucose level reduce to normal level.In addition, the effect of E-4 treatment diabetes is that it can stimulate duplicating of beta Cell of islet and regenerates.
3.1.2 clinical plan scheme:
Principal indication: treatment diabetes B
Route of administration: subcutaneous injection
Clinical plan dosage: 5-10 μ g/ people, every day 2 times.
Intend using the course of treatment: long-term prescription.
3.1.3 the relevant regulations of China's drug safety evaluation.
3.2 dosage design
Subcutaneous injection: give mouse with original liquid 0.2ml/10g body weight, then dosage is 1600 μ g/kg.Intravenous injection: give mouse with original liquid 0.2ml/10g body weight, then dosage is 1600 μ g/kg.
3.3 medicine preparation:
Use original liquid.
3.4 medication: single administration.
3.5 route of administration: subcutaneous injection and intravenous injection
3.5 administration capacity: 0.2ml/10g body weight
3.6 administration time: the morning 8:30 ~ 11:30
4. experimental technique
Get 60 of Kunming mouses (male and female half and half), body weight is 18-20g, is divided into 3 groups at random, and 1 group is the normal control group, and 1 group is the subcutaneous injection group, and 1 group is the intravenous injection group.The control group subcutaneous injection gives physiological saline, subcutaneous injection group and intravenous injection group by the dosage of above-mentioned design once or intravenous injection give mouse.Observe performance and the death condition of animal after the administration immediately.
5. observe and record
5.1 appetite: claim animal appetite every day.
5.2 body weight: the 7th day and the 14th day claim the weight of animals before the administration, after the administration, and with compare.
5.3 observed 2 hours continuously after the administration, later morning and afternoon every day each observed and recorded once, carefully write down the poisoning manifestations that animal occurs, observed continuously 14 days.If animal occurs dead, should in time cut open inspection, if there is the visible pathology of naked eyes should do pathologic finding.
6. test-results method for expressing:
After being tried the thing peak concentration and giving animal, observed continuously 14 days, if do not see animal dead, then can be expressed as MTD is this dosage.
The result:
Each animal behavior performance shows no obvious abnormalities behind the subcutaneous and intravenous injection E-4 of mouse, and viewing duration does not have animal dead.The weight of animals rises appreciably during testing, and appetite is normal.
Conclusion:
Under this test conditions, the MTD that the E-4 single intravenous injection gives mouse is 1600 μ g/kg.The MTD that single subcutaneous injection gives mouse is 1600 μ g/kg.
The experimental study of embodiment 6 Exendin-4 hypoglycemic activities
1 material
1.1 laboratory animal
The Wistar rat, male and female half and half, body weight 200 ± 20g.Provide animal conformity certification number by animal testing center, Southeast China University fourth man bridge school district: SYXK (Soviet Union) 2002-0012.
The Db/db mouse, male and female half and half, body weight 50-60g.Provide animal conformity certification number by Shanghai west pul-Bi Kai laboratory animal company limited: 02-20-9 number.
1.2 medicine and reagent
Exendin-4 is provided by Chengdu Zhitian Bioengineering Co., Ltd., lot number 20030618.The glucose assays test kit is Shanghai Rongsheng Bioisystech Co., Ltd's product.The insulin assay test kit is provided by Institute for Atomic Research, Chinese Academy of Sciences Beijing.Streptozotocin is a Sigma company product.Regular iletin is Xuzhou ten thousand nation's biochemical-pharmaceutical factory products.Cholesterol, triglyceride level, glycolated hemoglobin are measured test kit and are built up bio-engineering research by Nanjing and provided.
2, method
2.1 normal rat test
Normal Wistar rats, male and female half and half (200 ± 20) grams is divided into 5 groups at random, and 10 every group, i.e. normal control group, positive controls (Regular Insulin 0.5iu/kg sc), high, medium and low 3 the dosed administration groups of Exendin-4 (0.3,1.0,3.0 μ g/kg).Each administration group subcutaneous injection Exendin-4, normal control group subcutaneous injection equivalent phosphoric acid buffer.4 weeks of successive administration.Measure 0,1,4,8 hour fasting plasma glucose after the first administration, observe the medicine acute effect; 0th, 2,4 weeks were measured serum glucose, insulin concentration on an empty stomach; Carry out the glucose tolerance experiment during the 3rd week; Get blood in 1 hour after the 4th all last administrations, survey cholesterol, triglyceride level.
2.2 streptozotocin (STZ) hyperglycemic rat test
The Wistar rat, male (200 ± 20) gram is except that the normal control group, fasting is ip 50mg/kg STZ (be dissolved in the 0.1mol/L PH4.5 citrate buffer, 2% concentration is faced and used preceding preparation) after 12 hours, measure blood sugar after 72 hours, the selected experiment of＞13.1mmol/L.Be divided into 5 groups at random, 10 every group, i.e. model control group, positive controls (Regular Insulin 0.5iu/kg sc), high, medium and low 3 the dosed administration groups of Exendin-4 (0.3,1.0,3.0 μ g/kg).Each administration group subcutaneous injection Exendin-4, normal control group subcutaneous injection equivalent phosphoric acid buffer.4 weeks of successive administration.Measure 0,1,4,8 hour fasting plasma glucose after the first administration, observe the medicine acute effect; 0th, 2,4 weeks were measured serum glucose, insulin concentration on an empty stomach; Carry out the glucose tolerance experiment during the 3rd week; Get blood in 1 hour after the 4th all last administrations, survey glycolated hemoglobin, cholesterol, triglyceride level.
2.3 spontaneous diabetes db/db mouse test
4 the week age db/db mouse, be divided into 5 groups at random, 10 every group, male and female half and half.Be model control group, positive drug group (Regular Insulin 1iu/kg sc), high, medium and low 3 the dosed administration groups of Exendin-4 (0.6,2.0,6.0 μ g/kg).Other establishes the normal control group is nondiabeticlittermate.Each administration group subcutaneous injection Exendin-4, model control group and normal control group subcutaneous injection equivalent phosphoric acid buffer in continuous 8 weeks, detect following index: measure 0,1,4,8 hour fasting plasma glucose concentration during first administration, observe the acute effect of medicine.Get blood when experiment finishes, measure blood sugar, Regular Insulin, glycolated hemoglobin, cholesterol, triglyceride level.
3, result
3.1 Exendin-4 is to the acute effect of normal rat blood sugar
As seen from Table 1, Exendin-4 does not have acute hypoglycemic activity to normal rat
Table 1 Exendin-4 to the acute effect of normal rat blood sugar (mmol/L) (X ± S, n=10)
| Group | 0h | 1h | 4h | 8h |
| Normal control group Regular Insulin group 3.0 μ g/kgEX-4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 4.63±0.52 4.94±0.62 4.78±0.84 4.80±0.96 4.72±0.88 | 4.28±0.61 3.42±0.36* 4.47±0.46 4.42±0.92 4.66±0.98 | 4.86±0.79 2.88±0.38** 4.33±1.03 4.56±0.61 4.86±0.47 | 4.55±0.82 3.15±0.52** 4.62±0.92 4.25±1.16 4.68±0.82 |
Compare * P＜0.05 * * P＜0.01 with the normal control group
3.2 the Exendin-4 successive administration influence to normal rat fasting blood-glucose, Regular Insulin in 1 month
As seen from Table 2, with normal group relatively, Exendin-4 successive administration 1 month does not have obvious influence to normal rat fasting blood-glucose, Regular Insulin.
The influence to normal rat fasting blood-glucose, Regular Insulin in 1 month of table 2 Exendin-4 successive administration (X ± S, n=10)
| Group | Glucose (mmol.L-1 -1) | Regular Insulin (mU.L -1) | ||||
| 0 week | 2 weeks | 4 weeks | 0 week | 2 weeks | 4 weeks | |
| Normal control group Regular Insulin group 3.0 μ g/kgEX-4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 4.63±0.52 4.94±0.62 4.78±0.84 4.80±0.96 4.72±0.88 | 4.76±0.81 3.89±0.54* 4.59±0.62 4.48±1.04 4.81±0.95 | 4.58±0.73 3.76±0.47* 4.72±0.77 4.62±0.48 4.64±0.72 | 16.2±4.5 18.5±5.2 20.1±6.9 19.4±4.7 18.2±4.5 | 17.5±5.3 26.5±4.2** 16.8±4.1 18.7±3.8 19.7±8.1 | 18.2±4.1 30.1±6.8** 17.3±5.8 19.2±3.0 18.7±6.9 |
Compare * P＜0.05 * * P＜0.01 with the normal control group
3.3 Exendin-4 is to the influence of normal rat sugar tolerance
As seen from Table 3, Exendin-4 can obviously reduce the rising of blood sugar behind the abdominal injection glucose, improves the sugar tolerance of normal rat.
Table 3 Exendin-4 to the influence of normal rat sugar tolerance (X ± S, n=10)
| Group | 0h | 0.5h | 1h | 2h |
| Normal control group Regular Insulin group 3.0 μ g/kgEX 4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 4.35±0.63 4.52±0.74 4.54±0.62 4.75±0.71 4.66±0.74 | 12.18±1.83 9.02±1.49** 7.83±1.82** 8.92±1.69** 9.74±1.85* | 9.55±1.72 7.03±1.55* 6.25±1.71** 6.88±1.43* 8.42±1.67 | 7.12±1.44 6.08±1.67 5.37±1.49* 5.78±1.24* 6.73±1.28 |
Compare * P＜0.05 * * P＜0.01 with the normal control group
3.4 Exendin-4 is to the influence of normal rat serum ester
As seen from Table 4, Exendin-4 does not have obvious influence to normal rat cholesterol, triglyceride level.
Table 4 Exendin-4 to the influence of normal rat cholesterol, triglyceride level (X ± S, n=10)
| Group | Cholesterol (mmol/L) | Triglyceride level (mmol/L) |
| Normal control group Regular Insulin group 3.0 μ g/kgEX-4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 1.66±0.18 1.72±0.17 1.58±0.16 1.69±0.19 1.52±0.16 | 1.95±0.10 1.86±0.09 1.92±0.08 1.90±0.12 2.01±0.14 |
3.5 Exendin-4 is to the acute effect of STZ rat fasting blood-glucose
As seen from Table 5, but EXendin-4 dose-dependently ground reduces the STZ rat fasting blood-glucose, and 4 hours blood sugar drops to lower-most point after the subcutaneous injection, partly recovers in the time of 8 hours.
Table 5 Exendin-4 to the acute effect of STZ rat fasting blood-glucose (X ± S, n=10)
| Group | 0h | 1h | 4h | 8h |
| Normal control group model group Regular Insulin group 3.0 μ g/kgEX-4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 5.14±0.75 19.48±2.81*** 19.35±2.78 19.54±2.65 20.11±3.02 19.18±2.37 | 5.27±0.48 19.74±3.12*** 15.19±2.37# 15.27±2.52# 17.29±2.86 19.21±2.58 | 5.43±0.58 20.25±2.75*** 11.75±2.06## 12.04±2.17## 14.73±2.25## 18.39±3.14 | 4.98±0.92 19.33±3.07*** 13.38±2.71# 14.89±3.15# 17.58±3.75 18.94±2.73 |
Compare * * * P＜0.001 with the normal control group
Compare #P＜0.05 ##P＜0.01 with model group
3.6 the Exendin-4 successive administration influence to STZ rat fasting blood-glucose, Regular Insulin in 1 month
Table 6 is the result show, compares with model group, and the Exendin-4 successive administration is in the time of 1 month, and the 2nd week beginning fasting plasma glucose concentration obviously reduces, and serum insulin level raises.
The influence to STZ rat fasting blood-glucose, Regular Insulin in 1 month of table 6 Exendin-4 successive administration (X ± S, n=10)
| Group | Glucose (mmol.L 1) | Regular Insulin (mU.L 1) | ||||
| 0 week | 2 weeks | 4 weeks | 0 week | 2 weeks | 4 weeks | |
| Normal control group model group Regular Insulin group 3.0 μ g/kgEX-4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 5.14±0.75 19.48±2.81 19.35±2.78 19.54±2.65 20.11±3.02 19.18±2.37 | 4.92±0.86 21.18±3.17** 14.27±2.28## 13.29±2.17## 15.22±3.04## 17.95±2.17# | 5.22±0.65 22.45±3.56** 12.10±2.45## 11.05±3.35## 12.67±3.01## 16.37±2.84# | 19.2±3.7 12.4±2.8** 11.8±3.1 13.5±2.8 12.4±3.5 13.2±4.3 | 19.5±5.3 13.6±3.0** 22.1±5.7## 21.2±4.6## 18.4±3.9## 17.6±4.0## | 18.2±4.2 13.8±3.7** 4.2±4.5## 20.5±5.1## 20.1±6.2## 17.3±5.3## |
Compare * * P＜0.01 with the normal control group
Compare ##P＜0.01 with model group
3.7 Exendin-4 is to the influence of STZ rat sugar tolerance
As seen table 7 result compares with normal group, and behind the model group rats by intraperitoneal injection glucose, 0.5,1 hour blood sugar increasing value obviously increases.And give obviously to suppress behind the Exendin-4 rising of blood sugar continuously.
Table 7 Exendin-4 to the shadow of each time point change of blood sugar value (mmol/L) of STZ rat to (X ± S, n=10)
| Group | 0.5h | 1h | 2h |
| Normal control group model group Regular Insulin group 3.0 μ g/kgEX-4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 3.68±0.66 6.75±0.81** 3.16±0.79## 3.83±0.72## 3.92±0.85## 5.74±0.70 | 3.89±0.70 8.04±0.61** 4.07±0.65## 3.25±0.81## 3.95±0.83## 4.32±0.57## | 2.86±1.07 3.06±0.78 2.08±1.47 2.25±0.89 2.78±0.50 2.13±0.59 |
Compare * * P＜0.01 with the normal control group
Compare ##P＜0.01 with model group
3.8 Exendin-4 is to the influence of STZ rat fat and glycolated hemoglobin
Table 8 result shows Exendin-4 successive administration 1 month, and each dosage can reduce the concentration of glycolated hemoglobin very significantly, and high dosage also can reduce serum cholesterol level.And triglyceride level is not had obvious effect.
Table 8 Exendin-4 to the influence of STZ rat fat and glycolated hemoglobin (X ± S, n=10)
| Group | Cholesterol (mmol/L) | Triglyceride level (mmol/L) | Glycolated hemoglobin/% |
| Normal control group model group Regular Insulin group 3.0 μ g/kgEX-4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 1.68±0.16 3.01±0.14** 2.11±0.07# 1.95±0.12# 2.98±0.16 3.15±0.21 | 1.82±0.07 1.91±0.12 2.03±0.18 2.14±0.23 1.95±0.09 2.11±0.24 | 5.27±0.62 17.25±2.03** 10.34±1.85## 9.75±1.14## 11.82±1.35## 12.14±1.73## |
Compare * * P＜0.01 with the normal control group
Compare ##P＜0.05 ##P＜0.01 with model group
3.9 Exendin-4 is to the acute effects of db/db mouse blood sugar
As seen from Table 9, but Exendin-4 dose-dependently ground reduces db/db mouse fasting plasma glucose, and 4 hours blood sugar drops to lower-most point after the subcutaneous injection, and amplitude reaches 30%, 8 hour part and recovers.
Table 9 Exendin-4 to the acute shadow of db/db mouse blood sugar to (X ± S, n=10)
| Group | 0h | 1h | 4h | 8h |
| Model group Regular Insulin group 6.0 μ g/kgEX-4 2.0 μ g/kgEX-4 0.6 μ g/kgEX-4 | 15.37±3.12 14.65±4.28 14.72±4.05 15.16±3.87 15.71±3.54 | 16.04±3.28 10.48±3.17** 9.26±2.15** 12.02±2.61* 12.52±2.94* | 15.69±3.44 9.49±2.13** 10.58±2.86** 11.57±3.45* 13.88±3.22 | 16.48±4.11 13.75±5.02 12.16±3.19* 14.28±4.33 15.32±3.29 |
Compare * P＜0.05 * * P＜0.01 with model group
3.10 the Exendin-4 successive administration influence to db/db mouse blood sugar, Regular Insulin in 2 months
Table 10 result shows that the Exendin-4 successive administration can obviously reduce db/db mice serum blood sugar, insulin level in 2 months.
Table 10 Exendin-4 multiple dosing to the influence of db/db mouse blood sugar and Regular Insulin (X ± S, n=10)
| Group | Blood sugar/mmolL 1 | Regular Insulin/mUL |
| Normal control group model group Regular Insulin group 3.0 μ g/kgEX-4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 5.48±0.67 16.85±3.18** 8.12±2.11## 7.16±1.29## 10.25±2.26## 12.32±2.18# | 17.24±2.87 36.14±4.64** 24.38±4.17## 22.11±3.85## 25.05±3.34## 26.49±3.12## |
Compare * * P＜0.01 with the normal control group
Compare #P＜0.05 ##P＜0.01 with model group
3.11 Exendin-4 is to the influence of db/db mouse glycolated hemoglobin, blood fat
Table 11 result shows that the Exendin-4 successive administration can obviously reduce the level of db/db mouse cholesterol, triglyceride level and glycolated hemoglobin in 2 months.
Table 11 Exendin-4 to the influence of db/db mouse glycolated hemoglobin, blood fat (X ± S, n=10)
| Group | Cholesterol (mmol/L) | Triglyceride level (mmol/L) | Glycolated hemoglobin/% |
| Normal control group model group Regular Insulin group 3.0 μ g/kgEX-4 1.0 μ g/kgEX-4 0.3 μ g/kgEX-4 | 1.47±0.12 4.55±0.18** 1.82±0.15## 1.74±0.19## 2.16±0.22## 3.20±0.12# | 2.15±0.14 5.82±0.22** 3.03±0.12# 3.54±0.21# 3.92±0.15# 3.23±0.20# | 5.27±0.62 21.27±2.15** 8.28±1.43## 7.15±1.37## 10.56±1.61## 13.94±2.71## |
Compare * * P＜0.01 with the normal control group
Compare #P＜0.05 ##P＜0.01 with model group
Embodiment 7 reorganization Exendin-4 general pharmacologies are learned the experimental study report
1. materials and methods:
1.1 title: heavy Exendin-4.
1.1.1 labelled amount: 80ug/ml
1.1.2 proterties and physico-chemical property: this product is white lyophilized powder, and is soluble in water
1.1.3 preservation condition: 2-8 ℃ preservation.
1.2 research system:
1.2.1 animal species: Kunming mouse.
1.2.2 quantity and sex: 120 of mouse, male and female half and half.
1.2.3 the weight of animals: mouse body weight 18-22g.
1.2.4 animal-origin: mouse is purchased the Experimental Animal Center in Chinese Military Medical Science Institute.
1.2.5 the reason that the research system is selected: mouse genetic background is clear, and more stable to the reaction of medicine, individual difference is less, it is convenient to raise, and the bodily form is less, is convenient to test operation, saves for the reagent thing.
1.2.7 the mark identification of research system: mouse is adopted the picric acid mark.
1.2.8 the quarantine time: on March 25,16 days to 2004 December in 2003
1.2.9 quarantine result: each animal health is active, and dorsal body setae is smooth.Diet and activity there is no unusual performance.
1.2.10 feeding and management condition: 5 in the every cage of mouse, feed with standard feed, freely take the photograph water, room temperature 20-25 ℃, humidity 40-70%, 12 hours light and shades of illumination are alternately.
1.3 laboratory apparatus and reagent
1.3.1GJ-8502 type computer controlled automatic mouse autonomic activities experiment instrument, institute of Materia Medica,Chinese Academy of Medical Sciences production.
1.3.2 0.3% Sodital sodium solution
1.3.3 wire netting etc.
1.4 dosage design and foundation:
1.4.1 the foundation of test design:
1.4.1.1 mechanism of action and research background:
Exendin-4 (E-4) is a kind of 39 amino acid whose peptides, have on its structure 53% with the GLP-1 homology, and be the GLP-1 receptor antagonist.The biologic activity of GLP-1 comprises: stimulate secretion of insulin, promote the biosynthesizing of Regular Insulin; The secretion of glucagon suppression and the emptying of stomach; Reduce the absorption of food.GLP-1 keeps the active time shorter in vivo, and it can be decomposed by serine protease.
E-4 has and is better than the pharmacological action of GLP-1.Natural E-4 is the material that extracts from the saliva liquid of Gila Heloderma suspectum Yi, and U.S. Amylin pharmaceutical factory synthetic E-4 has finished preclinical test, is carrying out large-scale III clinical trial phase at present.Animal test results proves: E-4 can stimulate secretion of insulin when hyperglycemia, but does not have this effect when hypoglycemia; The emptying of E-4 adjustable gastric, the absorption of the nutritive substance that slows down; For obese animal, E-4 can reduce appetite, reduces body weight; Especially E-4 can make glucose level reduce to normal level.In addition, the effect of E-4 treatment diabetes is that it can stimulate duplicating of beta Cell of islet and regenerates.
1.4.1.2 pharmacodynamics test:
Cultivate in body, the pancreas islet that exsomatizes, under high glucose concn condition, E-4 can stimulate secretion of insulin.
The hypoglycemic activity of E-4 is studied with stimulating insulin secretion to act in a lot of animal models, comprises ob/ob, db/db mouse, the fat Zucker rat of diabetes and diabetes rhesus monkey etc.
Result of study shows that mouse test: hypoglycemic reaches at 35% o'clock, and the most dosage of GLP-1 1h behind medicine eliminates, and the E-4 time length surpasses 4h.Db/db mouse dosage is 0.001,0.01,0.1,1,10 100 μ g/100 μ L/, and ED50 is 0.059 ± 0.15 μ g/kg; Ob/ob mouse dosage is 0.001,0.01,0.1,1,10 100 μ g/100 μ L/, and ED50 is 0.136 μ g/kg.The rhesus monkey test: E-4 can reduce diabetes rhesus monkey blood sugar concentration, and dose-dependence is arranged, and dosage is 0.1,1,10,100 μ g/kg, and the ED50 of hypoglycemic 37% is 0.25 ± 0.09 μ g.
1.4.1.3 pharmacokinetic parameter:
The type ii diabetes patient accepts 3 to 4 E-4 in 48 hours, dosage reaches 0.1～0.4 μ g/kg, and drug plasma can detect at 15 hours at least as a result.Another studies show that, the E-4 of 4 dosage 0.2,0.4,0.6,0.8 μ g/kg of diabetes B patient continuous subcutaneous acceptance in 23 hours, and steady state plasma concentration kept 4 hours at least.
1.4.1.4 safety evaluation test:
Do not see E-4 clinical before the report of safety evaluation test, in the E-4 clinical trial, dosage is 0.1 μ g/kg, does not have tangible safety issue, slight headache is only arranged, feels sick, vomiting and slight hypoglycemia symptom.
1.4.1.5 clinical plan scheme:
Principal indication: type ii diabetes
Route of administration: subcutaneous injection
Clinical plan dosage: 5-10 μ g/ people, every day 2 times
1.4.1.6 relevant laws and regulations:
Annex three in " medicine registration management way " (the trying) of the promulgation of China drug surveilance office: biological products registration classification and declaration material project demand, and new drug pharmacology (toxicity) preclinical study governing principle.
1.4.2 test dose:
Mouse test Exendin-4 dosage is 1.0,3.0,10.0 μ g/kg, and this dosage is greater than the pharmacodynamics dose,equivalent (conversion of mouse dose,equivalent is 1.30-2.60 μ g/kg) of mouse
1.5 experimental technique:
120 of mouse (body weight 18～22g, male and female half and half) are divided into 4 groups of control groups, low dose group, middle dosage group, high dose group at random by sex, body weight,
Medication: subcutaneous injection, capacity 0.2ml/10g.
Observed content:
A. general behavior is observed: 40 mouse, and male and female half and half, by above-mentioned grouping administration, the performance of animal general behavior, posture, gait after the direct viewing administration, hydrostomia, amyostasia and pupil change.
B. to the influence of normal mouse autonomic activities: 40 mouse, male and female half and half are by above-mentioned grouping administration, whenever, take turns every group of 1 animals administer, gave next round in 12 minutes at interval, 1h tests respectively and respectively organizes mouse autonomic activities number in the 10min after administration, the every wheel measured 4, surveys 10 altogether and takes turns, result between comparative group.
C. to the influence of vetanarcol hypnosis sub-threshold dose effect: 40 mouse, male and female half and half, by above-mentioned grouping administration, 1h difference abdominal injection vetanarcol (30mg/kg) after the administration, observe sleep quality in the 30min, if any animal righting reflex loss appears, the latent period that needs record number of animals to occur and occur sleeping.
D. climb the net ability: the influence of normal mouse being climbed the net ability.Wire netting is 45 ° places on the experiment table, 40 mouse, male and female half and half by after the above-mentioned grouping medication each two of experiment mice being put in and letting alone climbing on the net, are observed in 10 minutes each and are organized the mouse number of elements that falls.
2. test-results:
2.1 influence to the mouse general behavior:
After subcutaneous injection gave Exendin-4 1.0,3.0,10.0 μ g/kg, each was organized mouse and shows no obvious abnormalities variation, no abnormality seen posture, gait, and phenomenons such as no hydrostomia, amyostasia, tic, muscle paralysis take place, and pupil does not have change.
2.2 influence to the mouse autonomic activities:
After subcutaneous injection gave Exendin-4 1.0,3.0,10.0 μ g/kg, the no abnormal change of each treated animal autonomic activities was compared with the physiological saline control group, and no difference of science of statistics sees table 12 for details.
Table 12 Exendin-4 to the influence of mouse autonomic activities (
N=10)
| Group | Dosage (μ g/kg) | Autonomic activities (number of times/10 minute) |
| Dosage group high dose group in the control group low dose group | 0 1.0 3.0 10.0 | 271.3±102.4 270.8±106.4 278.3±63.5 259.3±80.3 |
Compare p＞0.05 with control group
2.3 influence to the effect of vetanarcol hypnosis sub-threshold dose:
Subcutaneous injection gives Exendin-4 1.0,3.0,10.0 μ g/kg, 30min difference abdominal injection vetanarcol (30mg/kg) after the administration, observe sleep quality in the 30min, righting reflex loss occurring with animal continues to be as the criterion more than 1 minute, do not see the latent period of respectively organizing the number of animals of appearance sleep and occurring sleeping and see table 13 for details by significant difference.
Table 13 Exendin-4 to the influence of vetanarcol hypnosis sub-threshold dose effect (x ± s, n=10)
| Group | Dosage (μ g/kg) | Latent period (S) | Righting reflex loss number in the 30min |
| Dosage group high dose group in the control group low dose group | 0 1.0 3.0 10.0 | 351±239 341±36.8 430±325 348±243 | 2 2 2 2 |
Compare p＞0.05 with control group
2.4 mouse is climbed the influence of net ability:
After subcutaneous injection gave Exendin-4 1.0,3.0,10.0 μ g/kg, each treated animal climbs the net ability and the physiological saline control group compares, and no difference of science of statistics sees table 14 for details.
Table 14 Exendin-4 climbs the influence (n=10) of net ability to mouse
| Group | Dosage (μ g/kg) | The mouse number of elements of being caught in the 10min (1h behind the medicine) |
| Dosage group high dose group in the control group low dose group | 0 1.0 3.0 10.0 | 0 0 0 0 |
3. conclusion:
Under this test conditions, Exendin-4 1.0,3.0 and 10.0 μ g/kg subcutaneous administrations, no abnormality seens such as the performance of mouse general behavior, posture, gait, the autonomic activities number of administration animal, to the reaction of vetanarcol hypnosis sub-threshold dose, climb net ability etc. and relatively do not have significant difference with control group.Prompting this product does not all have obvious influence to spirit, nerve.
Sequence table
＜110〉Chengdu Zhitian Bioengineering Co., Ltd.
＜120〉recombination and preparation of amidating Exendin-4-4 polypeptide
<130>MP040485-2
<150>200410038842.2
<151>2004-04-30
<160>1
<210>1
<211>153
<212>DNA
<213>Escherichia coli
<400>1
gaattcgatg acgatgacaa gcacggtgaa ggtaccttca cctccgacct gtccaaacag 60
atggaagaag aagctgttcg tctgttcatc gaatggctga aaaacggtgg tccgtcctcc 120
ggtgctccgc cgccgtccgg ttaataagtc gac 153
Claims (7)
1. the recombination and preparation of amidating Exendin-4-4 polypeptide, it comprises the steps:
1. design the also dna sequence dna of synthetic coding Exendin-4 aminoacid sequence;
2. make up the expression vector of the recombinant plasmid that contains the Exendin-4 polypeptid coding sequence;
3. change recombinant plasmid over to host cell, obtain the genetic engineering bacterium of amalgamation and expression or non-fusion expression Exendin-4;
4. fermentation culture engineering bacteria;
5. express aftertreatment, obtain amidating Exendin-4-4 polypeptide;
It is characterized in that the dna sequence dna of step described in 1. contains the codon of terminal the 40th Gly of coding C-.
2. preparation method as claimed in claim 1 is characterized in that, the expression vector of step described in 2. is prokaryotic expression carrier.
3. preparation method as claimed in claim 2 is characterized in that, described prokaryotic expression carrier is the intestinal bacteria fusion expression vectors.
4. preparation method as claimed in claim 3 is characterized in that, described intestinal bacteria fusion expression vector is pThioHis or pGex4T-1.
5. preparation method as claimed in claim 4 is characterized in that, described intestinal bacteria fusion expression vector pGex4T-1 is cloned into the dna sequence dna of coding Exendin-4Gly, forms recombinant plasmid pGSTE4G, with Exendin-4 encoding gene and GST amalgamation and expression.
6. as the described preparation method of claim 3-5, it is characterized in that described intestinal bacteria are BL21.
7. as the described preparation method of claim 1-5, it is characterized in that, the expression aftertreatment of step described in 5. comprises the steps: purified fusion protein, use the Enterkinase cleavage of fusion proteins, separation and purification Exendin-4Gly, with α-amidating enzyme Exendin-4Gly is converted into amidating Exendin-4-4, separation and purification amidating Exendin-4-4.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5424286A (en) * | 1993-05-24 | 1995-06-13 | Eng; John | Exendin-3 and exendin-4 polypeptides, and pharmaceutical compositions comprising same |
| WO2000069911A1 (en) * | 1999-05-17 | 2000-11-23 | Conjuchem, Inc. | Long lasting insulinotropic peptides |
| CN1376166A (en) * | 1999-07-12 | 2002-10-23 | 西兰医药联合股份有限公司 | Peptide that lower blood glucose levels |
| CN1455001A (en) * | 2002-04-29 | 2003-11-12 | 上海复星生物医药研究院有限公司 | Exendin-4 polypeptide preparation method |
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2004
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5424286A (en) * | 1993-05-24 | 1995-06-13 | Eng; John | Exendin-3 and exendin-4 polypeptides, and pharmaceutical compositions comprising same |
| WO2000069911A1 (en) * | 1999-05-17 | 2000-11-23 | Conjuchem, Inc. | Long lasting insulinotropic peptides |
| CN1376166A (en) * | 1999-07-12 | 2002-10-23 | 西兰医药联合股份有限公司 | Peptide that lower blood glucose levels |
| CN1455001A (en) * | 2002-04-29 | 2003-11-12 | 上海复星生物医药研究院有限公司 | Exendin-4 polypeptide preparation method |
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