CN101319012A - Calcitonin-gene-related peptide and trout calcitonin amalgamation polypeptide - Google Patents
Calcitonin-gene-related peptide and trout calcitonin amalgamation polypeptide Download PDFInfo
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- CN101319012A CN101319012A CNA2008100649613A CN200810064961A CN101319012A CN 101319012 A CN101319012 A CN 101319012A CN A2008100649613 A CNA2008100649613 A CN A2008100649613A CN 200810064961 A CN200810064961 A CN 200810064961A CN 101319012 A CN101319012 A CN 101319012A
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- calcitonin
- gene
- related peptide
- fusion polypeptide
- salmon
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Abstract
The invention provides calcitonin gene-related peptide and salmon calcitonin fusion polypeptide, relating to fusion polypeptide. In order to generate a product containing both calcitonin and the calcitonin gene-related peptide, the invention provides the calcitonin gene-related peptide and the salmon calcitonin fusion polypeptide. The amino acid sequence of the calcitonin gene-related peptide and the salmon calcitonin fusion polypeptide is shown in SEQ ID No:1; moreover, C-terminal amidation is carried out to the calcitonin gene-related peptide and the salmon calcitonin fusion polypeptide. Experiments verify that the calcitonin gene-related peptide and the salmon calcitonin fusion polypeptide preserve respective activities of the calcitonin gene-related peptide and salmon calcitonin.
Description
Technical field
The present invention relates to a kind of fusion polypeptide.
Background technology
(calcitonin CT) can directly suppress osteoclast (osteoclasts, OC) propagation to thyrocalcitonin, stop the bone dissolving, promoting bone forming, and can alleviate the waist that causes because of osteoporosis rapidly and bore disease pain or morbidity, is one of choice drug of treatment osteoporosis.It can also effectively regulate the balance between bone calcium and the blood calcium, and analgesic activity is arranged; The skeleton pain (as similar rheumatism, tumour) that treatment hypercalcinemia, scleromalacia, hyperthyroidism and a variety of causes are caused etc. has very obvious effects, and in the treatment of itai-itai disease, the analgesic effect of thyrocalcitonin can reach 30～50 times of morphine.Because thyrocalcitonin can be secreted by gastric acid inhibitory, it can be used for treating acute gastric ulcer; Because thyrocalcitonin can suppress the release of pancreatic hormone secretion and inhibitory enzyme, it can be used for treating acute pancreatitis; Thyrocalcitonin also has therapeutic action to hyperparathyroidism.Thyrocalcitonin is also having certain potentiality aspect the treatment of chronic polyarthritis and cardiovascular disorder.(salmon calcitonin is active the highest a kind of in the natural calcitonin sCT) to salmon calcitonin see calcimar, and its activity is 40 times of human calcitonin approximately, has homology with human calcitonin.Cell experiment proof salmon calcitonin see calcimar acts on the human calcitonin receptor, and the amount of the second messenger cAMP that is produced is far longer than human calcitonin, so its usage quantity accounts for clinical in nearly 50% of thyrocalcitonin.
(calcitonin gene-related peptide is one of the important neurotransmitter of regulating cardiovascular activity CGRP) to calcitonin-gene-related peptide, is the strongest vasodilator material of human body known to so far.It is stronger 240 times than pannonit, Sodium Nitroprusside that calcitonin-gene-related peptide expands the coronary artery effect, and it is by a kind of biologically active polypeptides of the sensory nerve ending release of capsaicin sensitive, is distributed widely in nerve and cardiovascular systems.In cardiovascular systems, the calcitonin-gene-related peptide biological activity has diversity, as powerful vasodilator effect, inhibition smooth muscle cell proliferation and endothelial cell apoptosis, also can promote cytodifferentiation propagation, participates in angiogenesis.The effect of the diastole coronary vasodilator of CGRP far be better than the P material (substance P, SP), atrial natriuretic peptide and norepinephrine (norepinephrine, NE); Than vagusstoff (acetylcholine, Ach), serotonin (5-hydroxytryptamine, 5-HT) etc. strong about 10000 times, stronger 10～100 times than Racemic isoproterenol.The agent that CGRP vasorelaxation reaction presents is in various degree imitated---dependency, and increased response when promptly suitably strengthening the implantation dosage of CGRP, concentration is low, and then effect is slow, but longer duration.(Shen YT such as Shen, Pittman TJ, Buie PS.Functional role of alpha-calcitonin gene-related peptide in theregulation of the cardiovascular system[J] .Pharmacol Exp Ther, 2001,298 (3): although 551-558.) think that ectogenic α-CGRP can cause the diastole of different sites vescular bed, the endogenous α-CGRP under standard state does not play an important role to regulation of blood vessels.Can suppress vascular smooth muscle cell proliferation, protection endotheliocyte and angiogenesis inhibiting through experimental verification calcitonin-gene-related peptide repeatedly, prevent and treat hypertension, prevent congestive heart failure, prevent and treat stenocardia, prevent and treat myocardial infarction and treatment shock.And have the function of modulating the pain sensation in spinal levels, can regulate the osteoclastic bone absorption and promote the scleroblast division.
Have only the thyrocalcitonin product in the market, and calcitonin-gene-related peptide only rests on the laboratory study stage.
Summary of the invention
The objective of the invention is in order to obtain a kind of product that had not only contained thyrocalcitonin but also contained calcitonin-gene-related peptide, and a kind of calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide are provided.
The aminoacid sequence of calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide is shown in SEQ ID NO:1; And calcitonin-gene-related peptide and the amidation of salmon calcitonin see calcimar fusion polypeptide C end.
Calcitonin-gene-related peptide and salmon calcitonin see calcimar enzymic activity have separately been kept through experimental verification calcitonin-gene-related peptide of the present invention and salmon calcitonin see calcimar fusion polypeptide; And the linked reaction speed that produces nicotinamide adenine dinucleotide reduced (NADH) is to add simultaneously more than the twice of reaction system speed of response of two kinds of single (calcitonin-gene-related peptide and salmon calcitonin see calcimar) polypeptide, transition state time while (transienttime) shortens greatly, foreshortens to about 1/4 of the former transition state time.
Thereby the present invention transforms its amino-acid residue the phenomenon of conformation interdependence according to the msCT biological activity in order to improve the msCT biological activity, and the Xie Ansuan of the 8th of msCT is become glycine, the 16th leucine with the L-Ala displacement, delete 22 tyrosine (msCT[Gly again
8, Ala
16, del-Tyr
22]), the biological activity of msCT is brought up to 8600IU/mg by 4500IU/mg.
(it is 4500IU/mg that C holds amidated calcitonin-gene-related peptide of the present invention and salmon calcitonin see calcimar fusion polypeptide biologic activity to calcitonin-gene-related peptide of the present invention and salmon calcitonin see calcimar fusion polypeptide C end amidation can raising thyrocalcitonin active about 30 times, and non-amidated calcitonin-gene-related peptide and the salmon calcitonin see calcimar fusion polypeptide biologic activity of C end is 100～200IU/mg).
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the aminoacid sequence of present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide is as follows:
AlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSe rGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPhe
GlyThrCysSerAsnLeuSerThrCysGlyLeuGlyLysLeuSerGlnGluAlaHisLysLe uGlnThrProArgThrAsnThrGlySerGlyThrPro; And calcitonin-gene-related peptide and the amidation of salmon calcitonin see calcimar fusion polypeptide C end.
Present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion gene are synthetic by Nanjing base day limited liability company, and fusion polypeptide is that this laboratory structure bacterial strain changes tomato over to after affinitive layer purification obtains.
There is one section in present embodiment calcitonin-gene-related peptide and the salmon calcitonin see calcimar fusion gene and is called as the encoding sequence of " affine handle " (affinity handle) to be connected, this section " affine handle " can combine specifically with the aglucon on the affinity column, like this expressed fusion protein can with the method for affinity chromatography fast and easily purifying come out, resulting fusion polypeptide albumen is after the cutting of chemical process or enzyme process is come, mix products is gone up same affinity column again, the fusion rotein of useing one section polypeptide of " affine handle " or albumen as and not being cut open as yet is adsorbed on the post, and reorganization purpose product then is in the moving phase.Therefore can obtain the calcitonin-gene-related peptide and the salmon calcitonin see calcimar fusion polypeptide of higher degree.
Present embodiment adds that at the C of calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide end a Gly provides amino donor for the amidation of peptide acyl glycine acidylate monooxygenase.
Present embodiment designs a Kpn I restriction enzyme site (shown in the aminoacid sequence underscore) between CGRP and msCT aminoacid sequence, can be used for verifying positive colony and other genetic manipulation.
Present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide are carried out experimentation on animals:
Experiment 1: the osteoporosis model rat is divided into two groups (treatment group and model group) at random, is the blank group with the normal rat; The treatment group is by body weight 0.5 μ g/kg dosage injection present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide, and model group is injected the physiological saline of Isodose.Experiment was carried out 84 days, rat blood serum acid phosphatase (TRAP), alkaline phosphatase (ALP) and Bone Gla protein (BGP) is detected, and rat has been carried out bone densitometry.Detected result as shown in Table 1 and Table 2.
Table 1 rat blood serum TRAP, ALP, BGP measurement result (X ± S)
| Group | The mouse number | TRAP(U/L) | ALP(U/L) | BGP(μg/L) |
| The blank group | 10 | 18.67±3.31 | 152.84±21.08 | 1.20±0.25 |
| Model group | 10 | 29.96±3.05 | 156.22±23.34 | 1.32±0.23 |
| The treatment group | 10 | 19.25±4.23 ** | 233.70±42.05 ** | 1.97±0.18 ** |
Table 2 rat femur, shin bone, lumbar vertebrae density value (X ± S)
| Group | The mouse number | Femur (g/cm 3) | Shin bone (g/cm 3) | Lumbar vertebrae (g/cm 3) |
| The blank group | 10 | 0.61±0.04 | 0.58±0.03 | 0.62±0.04 |
| Model group | 10 | 0.41±0.12 | 0.42±0.08 | 0.44±0.06 |
| The treatment group | 10 | 0.59±0.07 * | 0.59±0.05 * | 0.58±0.04 * |
Experimental data explanation present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide according to table 1 and table 2 have excellent result of treatment to osteoporosis, prove that present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide have the salmon calcitonin see calcimar activity.
20 of experiment 2:SD rats are caused myocardial infarction and ischemia model, be divided into two groups (treatment group and model group) then at random, the treatment group is by body weight 2.5mg/kg dosage injection present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide, and model group is injected the physiological saline of Isodose.Experiment is carried out 4 times, and the ratio that rat heart muscle is carried out hazardous location (AAR), necrotic area (IS) and necrotic area and hazardous location detects, and detected result is as shown in table 3.
Table 3
| Group | Hazardous location (g) | Necrotic area (g) | IS/AAR(％) |
| Model group treatment group | 0.24±0.03 2.25±0.03 | 0.14±0.03 0.07±0.03 ** | 54±13 28±14 ** |
Test-results shows that calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide are a kind of heart source property myocardial preservation materials.Experimental data according to table 3 illustrates that the myocardial infarction that present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide cause myocardial ischemia has excellent result of treatment, proves that present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide have the calcitonin-gene-related peptide activity.
Experimentation on animals proof present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide have the activity of calcitonin-gene-related peptide and two kinds of peptides of salmon calcitonin see calcimar simultaneously.
To produce the speed of NADH be to add simultaneously more than the twice of reaction system speed of response of two kinds of single (calcitonin-gene-related peptide or salmon calcitonin see calcimar) polypeptide for present embodiment calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide linked reaction after tested, and the transition state time foreshortens to former about 1/4 simultaneously.
Sequence table
＜110〉Heilongjiang University
＜120〉calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide
<160>1
<210>1
<211>70
<212>PRT
＜213〉artificial sequence
<220>
＜223〉calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide.
<400>1
Ala Cys Asp Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu
1 5 10 15
Ser Arg Ser Gly Gly Val Val Lys Asn Asn Phe Val Pro Thr Asn Val
20 25 30
Gly Ser Lys Ala Phe Gly Thr Cys Ser Asn Leu Ser Thr Cys Gly Leu
35 40 45
Gly Lys Leu Ser Gln Glu Ala His Lys Leu Gln Thr Pro Arg Thr Asn
50 55 60
Thr Gly Ser Gly Thr Pro
65 70
Claims (1)
1, calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion gene polypeptide expressed is characterized in that the aminoacid sequence of calcitonin-gene-related peptide and salmon calcitonin see calcimar fusion polypeptide is as follows:
AlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSe rGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheG lyThrCysSerAsnLeuSerThrCysGlyLeuGlyLysLeuSerGlnGluAlaHis LysLeuGlnThrProArgThrAsnThrGlySerGlyThrPro; And calcitonin-gene-related peptide and the amidation of salmon calcitonin see calcimar fusion polypeptide C end.
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| CN2008100649613A CN101319012B (en) | 2008-07-21 | 2008-07-21 | Calcitonin-gene-related peptide and trout calcitonin amalgamation polypeptide |
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| CN101319012A true CN101319012A (en) | 2008-12-10 |
| CN101319012B CN101319012B (en) | 2010-08-11 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102964451A (en) * | 2012-12-12 | 2013-03-13 | 黑龙江大学 | msCT-CTx fusion protein for treating osteoporosis and reliving pain and nucleic acid encoding fusion protein |
| CN102965327A (en) * | 2012-12-12 | 2013-03-13 | 黑龙江大学 | msCT-rhLeptin fusion protein transgenic engineering strain |
| CN102964452A (en) * | 2012-12-12 | 2013-03-13 | 黑龙江大学 | msCT-rhLeptin fusion protein for treating osteoporosis and obesity and nucleic acid encoding fusion protein |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031293B (en) * | 2012-12-31 | 2014-05-14 | 黑龙江大学 | Calcitonin-gene-related peptide and AcAPc2 fusion protein and encoding gene thereof |
-
2008
- 2008-07-21 CN CN2008100649613A patent/CN101319012B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102964451A (en) * | 2012-12-12 | 2013-03-13 | 黑龙江大学 | msCT-CTx fusion protein for treating osteoporosis and reliving pain and nucleic acid encoding fusion protein |
| CN102965327A (en) * | 2012-12-12 | 2013-03-13 | 黑龙江大学 | msCT-rhLeptin fusion protein transgenic engineering strain |
| CN102964452A (en) * | 2012-12-12 | 2013-03-13 | 黑龙江大学 | msCT-rhLeptin fusion protein for treating osteoporosis and obesity and nucleic acid encoding fusion protein |
| CN102964451B (en) * | 2012-12-12 | 2014-06-11 | 黑龙江大学 | msCT-CTx fusion protein for treating osteoporosis and reliving pain and nucleic acid encoding fusion protein |
| CN102965327B (en) * | 2012-12-12 | 2014-06-11 | 黑龙江大学 | msCT-rhLeptin fusion protein transgenic engineering strain |
| CN102964452B (en) * | 2012-12-12 | 2015-09-09 | 黑龙江大学 | For the msCT-rhLeptin fusion rotein of curing osteoporosis with obesity and the nucleic acid of this fusion rotein of encoding |
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| CN101319012B (en) | 2010-08-11 |
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