CN100445297C - Amolops loloensis insulin release promoting peptide and use in producing medicine - Google Patents
Amolops loloensis insulin release promoting peptide and use in producing medicine Download PDFInfo
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- CN100445297C CN100445297C CNB200610010932XA CN200610010932A CN100445297C CN 100445297 C CN100445297 C CN 100445297C CN B200610010932X A CNB200610010932X A CN B200610010932XA CN 200610010932 A CN200610010932 A CN 200610010932A CN 100445297 C CN100445297 C CN 100445297C
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Abstract
The present invention belongs to the field of biomedicine technology, and is especially Amolops loloensis insulin release promoting peptide and its application in preparing medicine. The present invention obtains insulin release promoting peptide from the skin secretion of Amolops loloensis through a conventional biochemical process, measures its sequence and synthesizes the polypeptide based on the obtained sequence. Amolops loloensis insulin release promoting peptide is one kind of single strand polypeptide of molecular weight of 1627.04 dalton, isoelectri point of 8.75 and total sequence of Phe-Leu-Pro-Ile-Val-Gly-Lys-Leu-Leu-Ser-Gly-Leu-Ser-Gly-Leu- Leu(FLPIVGKLLSGLSGLL-NH2). It has excellent insulin release promoting effect, no hemolytic activity, no plasma clotting activity and other advantages, and may be used in preparing medicine for treating diabetes.
Description
Technical field:
The invention provides a kind of FLPIVGKLLSGLSGLL-NH2 and the application in pharmacy, belong to field of biomedicine technology.
Background technology:
Diabetes be many gene and environmental factors cause with sugar, fat, protein metabolism disorder, and cause multisystem, internal secretion, the metabolic disease of a kind of pilosity that multi-organ function damages.Statistics shows that China's eighties＜1% is raised to 3.6-5% at present, and wherein (about 3,800 ten thousand people) belong to type ii diabetes more than 90%.By 2000, total diabetic subject 1.35 hundred million people in the whole world estimated will be increased to 2.99 hundred million in 2025.Diabetes itself are not fearful, and fearful is complication, and diabetic complication comprises cancer, sleep apnea, depression, myocardial infarction, peripheral vascular disease, hypertension and modal type ii diabetes.Type ii diabetes claims " non insulin dependent diabetes " again, and main pathology shows as insulin resistant and by the insulin secretion relative deficiency due to the islet cell function imbalance, forms persistent hyperglycemia, and can produce multiple mortality complication.Its clinical manifestation symptom mostly with body generation insulin resistance, self excretory Regular Insulin is few or hysteresis etc. is relevant.Up to now, this disease does not still have the way of radical cure, and it not only can increase the weight of patient economy burden, can make also that the patient is disabled, premature death.
At present, treat type ii diabetes in the world, be aided with medicines such as biguanides, a-glycosidase inhibitor based on sulfonylureas.But, because the side effect of sulfonylureas is big, life-time service can cause beta Cell of islet depletion because of the Regular Insulin excessive secretion, takes among the diabetics of this type of medicine, has every year 10% patient to use other class medicines such as Regular Insulin instead because of the treatment inefficacy.And present pharmacological research and methods of treatment still can not contain all related metabolic defectives of this disease.In the finder's of boston, u.s.a hospital general in 1984 enteron aisle, exist a class to promote since the polypeptide (general designation GLP-1) of insulin secretion, for treatment of diabetes provides new direction.With present Remedies for diabetes, especially sulfonylureas is compared, and the GLP-1 analogue has some new action target spots, as delay stomach emptying, glucagon suppression produces, and the biosynthesizing of increase Regular Insulin and β cell quantity etc. have become the new highlight for the treatment of type ii diabetes.Believe in the near future,, will become the new tool of type ii diabetes treatment undoubtedly along with definite the further perfect of administering mode that reach of GLP-1 dosage.
The main method of present stage generation lead compound still depends on carries out random screening to a large amount of compounds.And the vast many species natural biological resource of China is the important source of structure diversity small molecules organic compound, and the some of them biologically active substance is put down in writing by the traditional Chinese medical science already and adopted.A lot of amphibian animals belong to traditional Chinese medicine and national medicine and widespread use in China, as Chinese toad (Bufo gargarizans), Bombina maxima (Bombinamaxima), Rana nigromaculata (pelophylaxnigromaculata), Rana guentheri (Hylarana guentheri) and rana limnocharis (Euphlyctis limnocharis) etc.The skin of these amphibian animals and internal organ have pharmacologically active and clinical efficacy widely.
There is long history in China to the application of batrachians medicine, and except having a series of organic medicinal small molecules such as alkaloid, batrachians skin also contains a large amount of bioactive peptide materials, and this is the important source that new drug is found.
The contriver searches for FLPIVGKLLSGLSGLL-NH2 complete sequence structure of the present invention and relatively finds no any identical polypeptide through albumen database.
Summary of the invention:
The object of the present invention is to provide a kind of new having to promote the FLPIVGKLLSGLSGLL-NH2 of Regular Insulin release and the application in pharmacy.
FLPIVGKLLSGLSGLL-NH2 of the present invention adopts the ordinary method preparation, that is: ether stimulates amolops loloensis, the centrifugal removal precipitation of the skin juice of collecting, lyophilize is after the separation and purification of a series of biological chemistry means obtains this polypeptide, measure the aminoacid sequence structure with automatic Protein Sequencer, according to synthetic this polypeptide of resulting aminoacid sequence.Adopt the time-of-flight mass spectrometry (TOFMS) determining molecular weight, iso-electric point is measured in isoelectrofocusing, and Protein Sequencer is confirmed the aminoacid sequence structure automatically.
FLPIVGKLLSGLSGLL-NH2 of the present invention is for separating a kind of single chain polypeptide that obtains from Chinese amphibian animal amolops loloensis skin juice, molecular weight 1627.04 dalton, iso-electric point 8.75, polypeptide primary structure total order is classified as: Phe-Leu-Pro-Ile-Val-Gly-Lys-Leu-Leu-Ser-Gly-Leu-Ser-Gly-Leu-Leu (FLPIVGKLLSGLSGLL-NH
2)
FLPIVGKLLSGLSGLL-NH2 of the present invention is the product that obtains from the natural biological resource, has the effect that good promotion Regular Insulin discharges, also have advantages such as no hemolytic activity, no clotting of plasma activity simultaneously, can be used as being employed of preparation treatment diabetes medicament.
Description of drawings:
Accompanying drawing shows that FLPIVGKLLSGLSGLL-NH2 of the present invention promotes the effect that Regular Insulin discharges.Among the figure: contrast to containing the balanced salt buffered soln (HBSS) of glucose 16.7mM; Amolopin concentration is 125ug/ul.
Embodiment:
Further specify essentiality content of the present invention with embodiment below, but content of the present invention is not limited thereto.
The preparation of FLPIVGKLLSGLSGLL-NH2:
Live body amolops loloensis water is cleaned up, place Glass Containers with cover, drip anhydrous diethyl ether (1 liter of volumetric capacity adds 1 milliliter of anhydrous diethyl ether), encloses container 3-5 minute, as seen a large amount of foam-like materials is secreted out from amolops loloensis skin, collect secretory product, preserve after centrifugal (10000rpm, 20 minutes), the lyophilize.
The first step, Sephadex G-50 gel filtration chromatography, the starting material amolops loloensis skin juice lyophilized powder 300mg of Huo Deing is dissolved in the 5ml pure water as stated above, and 4 ℃ of centrifugal 10min of 10000rpm remove precipitation.Sephadex G-50 gel-filtration column (long 1000mm, diameter 26mm) is used 0.1M Na
2HPO
4-NaH
2PO
4PH 6.0 damping fluid balances, fully after with on the sample in this post.Use same buffer solution elution, collect active peak.
In second step, anti-phase high pressure liquid chromatography is: the peak of the activeconstituents that Sephadex G-50 gel-filtration obtains is splined on through the abundant equilibrated C of the ultrapure water that contains 1 ‰ trifluoroacetic acids
18Reversed-phase column (long 300mm, the diameter 5mm) gradient elution of the acetonitrile damping fluid from 0% to 80% that contains 1 ‰ trifluoroacetic acids.Collection has the active peak that promotes that Regular Insulin discharges.
The 3rd step, the FLPIVGKLLSGLSGLL-NH2 of purifying is identified its purity with the high-efficient liquid phase chromatogram HPLC method, molecular weight determination adopts time-of-flight mass spectrometry (TOFMS) (Time of flight mass spectrometry, TOF-MS), isoelectric focusing electrophoresis is measured iso-electric point, measures the aminoacid sequence structure with automatic Protein Sequencer.
FLPIVGKLLSGLSGLL-NH2 by method for preparing is to separate a kind of single chain polypeptide that obtains from Chinese amphibian animal amolops loloensis skin juice, molecular weight 1627.04 dalton, iso-electric point 8.75, polypeptide primary structure total order is classified as: Phe-Leu-Pro-Ile-Val-Gly-Lys-Leu-Leu-Ser-Gly-Leu-Ser-Gly-Leu-Leu (COOH-FLPIVGKLLSGLSGLL-NH
2)
FLPIVGKLLSGLSGLL-NH2 promotes the effect that Regular Insulin discharges:
The Regular Insulin of FLPIVGKLLSGLSGLL-NH2 discharges and promotes the active Regular Insulin of glucose induction that adopts to discharge activity test method (GSIS, Glucose-stimulated insulin secretion).
One. the separation of islet cells and cultivation (Islet isolation and culturing)
1. get pancreas:
Only get one day SD rat 5-6 of fasting, body weight 200-250g/, male and female are not limit, and behind the excessive etherization, disconnected neck is put to death.The rat of above-mentioned execution is sterilized with 70% alcohol whole body, under aseptic condition on the Bechtop, take out pancreas, place balanced salt solution in HBSS (available from JRH), the rinsing bloodstain, and cut off excess tissue and fat.
2. digestion:
With pancreas 37 ℃ of concussion digestion 30 minutes in 20ml collagenase IV (available from sigma) Digestive system (concentration is 0.167%), be digested the fine sand shape to tissue block, 4 ℃ or ice-cold HBSS balanced salt solution stop digestion.Centrifugal 5 minutes of 1000r/min removes the collagenase in the supernatant liquor, washs 2 times with quadrat method again.
3. purifying:
With Ficoll400 (molecular weight 40,000 is available from Pharmacia) discontinuous density gradient centrifuging purifying islet cells.Add 25%Ficoll4002ml with postdigestive islet cells mixing at the 15ml centrifuge tube, add 23%Ficoll4002ml thereon gently, 20%Ficoll4001ml, 11%Ficoll4001ml, centrifugal 20 minutes of 3000r/min, sucking-off 11%-20%, the cell of 20%-23% two bed interfaces is with RPMI1640 nutrient solution (available from HYCLONE) washed twice.The islet cells suspension that takes a morsel is with dithizone (DTZ, dithizone) dyeing, and taking pictures.
4. manual selecting:
Pancreas islet after purifying and the washing is placed culture dish, the manual pancreas islet of selecting under anatomical lens.Inoculate 96 porocyte culture plates, 10 pancreas islet of every hole inoculation, add 200ul RPMI1640 nutrient solution and (contain 10% foetal calf serum, penicillin, 100 units/ml Streptomycin sulphate), at 37 ℃, cultivate more than 12 hours under the 5%CO2 condition, make isolating pancreas islet recover normal physiological status, detect so that Regular Insulin discharges.
Two. the Regular Insulin of glucose induction discharges active detect (GSIS)
1. on Bechtop, with the cell culture fluid sucking-off in the 96 porocyte culture plates.
2. add HBSS balanced salt solution (glucose concn is the high sugared condition of the 16.7mM) 200ul that is mixed with PBS (negative control) or biologically active peptides sample (experimental group),, continued to hatch under the 5%CO2 condition 1 hour at 37 ℃.Each sample is all done 3 repetitions.
3. get supernatant liquor, centrifugal 5 minutes of 1000r/min ,-20 ℃ are frozen.Insulin content is put the chamber of exempting from by Nanjing Military Command's hospital general's clinical laboratory and is detected.The results are shown in Figure of description.
By Figure of description as seen, FLPIVGKLLSGLSGLL-NH2 has the effect that significant promotion Regular Insulin discharges, and the Regular Insulin that islet cells discharged presents the trend of rising along with the increase of FLPIVGKLLSGLSGLL-NH2 concentration.
Above-mentioned test shows: FLPIVGKLLSGLSGLL-NH2 can be used as being employed of preparation treatment diabetes medicament.
Claims (1)
1. FLPIVGKLLSGLSGLL-NH2, it is characterized in that this FLPIVGKLLSGLSGLL-NH2 is to separate a kind of single chain polypeptide that obtains from amolops loloensis, molecular weight 1627.04 dalton, iso-electric point 8.75, polypeptide primary structure total order is classified as: Phe-Leu-Pro-Ile-Val-Gly-Lys-Leu-Leu-Ser-Gly-Leu-Ser-Gly-Leu-Leu (FLPIVGKLLSGLSGLL).
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200610010932XA CN100445297C (en) | 2006-05-30 | 2006-05-30 | Amolops loloensis insulin release promoting peptide and use in producing medicine |
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| CNB200610010932XA CN100445297C (en) | 2006-05-30 | 2006-05-30 | Amolops loloensis insulin release promoting peptide and use in producing medicine |
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| CN100445297C true CN100445297C (en) | 2008-12-24 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106478803B (en) * | 2016-10-20 | 2019-12-20 | 南方医科大学 | Peptide for promoting insulin release of hypsizygus marmoreus, gene thereof and application thereof in pharmacy |
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Non-Patent Citations (4)
| Title |
|---|
| 云南省两栖动物新纪录-棕点湍蛙. 胡健生等.四川动物，第22卷第3期. 2003 |
| 云南省两栖动物新纪录-棕点湍蛙. 胡健生等.四川动物，第22卷第3期. 2003 * |
| 基于12S和16S rDNA序列的湍蛙属部分物种的系统关系. 金义文等.四川省动物学会第八次会员代表大会暨第九次学术年会论文集. 2004 |
| 基于12S和16S rDNA序列的湍蛙属部分物种的系统关系. 金义文等.四川省动物学会第八次会员代表大会暨第九次学术年会论文集. 2004 * |
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