CN103031294A - Calcitonin-gene-related peptide and AcAP5 fusion protein and encoding gene thereof - Google Patents
Calcitonin-gene-related peptide and AcAP5 fusion protein and encoding gene thereof Download PDFInfo
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Abstract
The invention discloses a calcitonin-gene-related peptide and AcAP5 (ancylostoma caninum anticoagulant peptide 5) fusion protein and an encoding gene thereof, and relates to a fusion protein and an encoding gene thereof. The invention aims at providing the calcitonin-gene-related peptide and AcAP5 fusion protein and the encoding gene thereof. The fusion protein has double functions of treating hypertension and preventing thrombi, and the two proteins have complementary actions and synergetic effects. The amino acid sequence of the fusion protein is shown as SEQ ID NO: 2. The nucleotide sequence of the encoding gene of the fusion protein is shown as SEQ ID NO: 1. The fusion protein fuses calcitonin-gene-related peptide and AcAP5 which have the complementary actions and synergetic effects. Experiments prove that the obtained fusion protein has good effects of treating the hypertension and preventing the thrombi. The fusion protein can be used for preparing drugs for preventing the thrombi and preventing and treating the hypertension.
Description
Technical field
The present invention relates to a kind of fusion rotein and encoding gene thereof.
Background technology
At present, China has hyperpietic people more than 200,000,000 now, annual increase by 1,000 ten thousand people.Hypertension is the primary Hazard Factor of cardiovascular and cerebrovascular diseases, and hypertensive common cardiovascular and cerebrovascular complication (cerebral apoplexy, heart trouble) accounts for 40% of the total death of resident.In addition, thrombus disease is fallen ill also higher in the elderly, and show according to domestic and international statistical medical information: the lethality rate of cerebral thrombosis almost accounts for first of all diseases.
Calcitonin-gene-related peptide (CGRP) is a kind of biologically active peptides that Rosenfeld equals the nineteen eighty-three discovery, it and thyrocalcitonin (CT) all derive from and are positioned at chromosomal CT/CGRP gene No. 11, owing to the RNA coding that the CT/CGRP gene is different is translated into CGRP and CT, CGRP is a kind of biologically active polypeptides that is comprised of 37 amino acid of answering DNA gene recombination and molecular biotechnology research to find, that the strongest endogenous of finding so far expands the blood vessel peptide matters, to nerve, cardiovascular, breathe, digestion, skeletal muscle, uropoiesis, the systems such as reproduction and immunity have vital role.The effect of the diastole coronary vasodilator of CGRP far is better than Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, and atrial natriuretic peptide and norepinephrine (NE) are stronger about 10000 times than vagusstoff (Ach), serotonin (5-HT) etc., than the strong 10-100 of Racemic isoproterenol doubly.
Ancylostoma caninum anticoagulant hemepeptide (anticoagulant peptide, AcAPs) be that the dog hookworm is when sucking blood, a kind of material with anticoagulant active of its cephalic gland secretion, this material essence is a kind of proteolytic ferment, has the plasma prothrombin time of prolongation, suppresses blood coagulation and promote Fibrinolytic effect.AcAPs uses at present AcAP5, AcAP6, three kinds of recombinant proteins of AcAPc2.Wherein rAcAP5 is comprised of 77 amino acid, contains 10 halfcystines, forms 5 pairs of disulfide linkage (C6-C50, C15-C42, C21-C37, C25-C69, C50-C63), mainly determines its secondary structure by disulfide linkage, thereby determines its biological activity.It is the efficient specific inhibitor of Xa factor (thrombin), and the common pathway of blocking-up blood coagulation reaches blood coagulation resisting function.The anticoagulation of AcAPs, anti thrombotic action make it become clinically a kind of new antithrombotics and antithrombotic reagent.
Yet present these two kinds of albumen independent roles, effect is single.
Summary of the invention
The object of the present invention is to provide calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein and encoding gene thereof, in the hope of obtaining a kind of fusion rotein that can have simultaneously treatment hypertension and antithrombotic dual function, action compensating and synergy between two kinds of albumen.
The aminoacid sequence of calcitonin-gene-related peptide of the present invention and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein is shown in SEQ ID NO:2.
The nucleotide sequence of above-mentioned calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein encoding gene is shown in SEQ ID NO:1.
Beneficial effect of the present invention: the present invention carries out appropriate design, calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 are merged, between the two action compensating and synergy, the gained fusion rotein has good antithrombotic and the hypertensive effect for the treatment of through experiment confirm.Fusion rotein of the present invention can be owing to the medicine of preparation antithrombotic and prevention and treatment high blood pressure disease, for the research of anticoagulation and prevention and treatment high blood pressure disease lays the first stone.
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 Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein is shown in SEQ ID NO:2.
The preparation method of present embodiment calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein, carry out according to the following steps:
One, fusion rotein CGRP/AcAP5 gene is synthetic: Kpn I restriction enzyme site of design in CGRP and the AcAP5 fusion gene, give birth to the fusion rotein CGRP/AcAP5 gene of worker's company's synthesizing ribonucleotide sequence shown in SEQ ID NO:1 by Shanghai, and add respectively BamHI and EcoR I restriction enzyme site at fusion rotein CGRP/AcAP5 gene two ends, then be cloned on the pUC57 carrier, obtain pUC(CGRP/AcAP5) carrier;
Two, the structure of recombinant expression vector: with the pUC(CGRP/AcAP5 of step 1 acquisition) carrier BamHI and EcoR I double digestion, again with equally be connected expression vector pGEX-6P-1 with EcoR I double digestion through BamHI and be connected acquisition fusion rotein CGRP/AcAP5 dna recombinant expression vector pGEX-CGRP/AcAP5;
Three, the structure of engineering strain: then vector pGEX-CGRP/AcAP5 is transformed in the e. coli bl21 (DE3), 37 ℃ of incubated overnight of picking transformed bacteria at random, adopt plasmid extraction kit (buying from Beijing Ai Delai bio tech ltd) to extract plasmid DNA, with Kpn I single endonuclease digestion, and with corresponding blank plasmid pGEX-6P-1 in contrast, the plasmid that will contain Kpn I restriction enzyme site carries out gene sequencing, and what sequencing result was correct is positive recombinant bacterium; Wherein e. coli bl21 (DE3) obtains for buying;
Four, the abduction delivering of fusion rotein and purifying: place 28 ℃ of LB substratum to cultivate 15h positive recombinant bacterium, then adopt GST tag fusion protein method to carry out the separation and purification of albumen, the purity of present embodiment calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein is 98%, and fusion protein expression is 38.1%.
PUC(CGRP/AcAP5 in the step 2) carrier is as follows with the system of BamHI and EcoR I double digestion:
| Composition | Consumption |
| PUC(CGRP/AcAP5) carrier | 20μL |
| 10×M buffer | 6μL |
| BamHI | 3μL |
| EcoRⅠ | 3μL |
| ddH 2O | 28μL |
Endonuclease reaction condition: 37 ℃ of water-baths, 10h.
The system of expression vector pGEX-6P-1 double digestion is as follows in the step 2:
| Composition | Consumption |
| pGEX-6P-1 | 20μL |
| 10×M buffer | 6μL |
| BamHI | 3μL |
| EcoRⅠ | 3μL |
| ddH 2O | 28μL |
Endonuclease reaction condition: 37 ℃ of water-baths, 10h.
The ligation system is as follows in the step 2:
| Composition | Consumption |
| Goal gene CGRP/AcAP5 | 13μL |
| Enzyme is cut rear pGEX-6P-1 carrier | 3μL |
| The T4 dna ligase | 2μL |
| 10 * T4 dna ligase buffe | 2μL |
Ligation condition: 16 ℃ of water-baths, 8～12h.Described T4 dna ligase is bought from TaKaRa company.By bio-engineering corporation with fusion rotein CGRP/AcAP5 gene clone to the pUC57 carrier.
Be the effect of checking present embodiment, carry out following experiment:
The experiment of CGRP-AcAP5 fusion rotein antithrombotic:
Get the SD rat, be divided at random 11 groups, 8 every group, every group of dosage is as follows: negative control group gives the physiological saline with volume; Positive controls is selected heparin sodium injection, and dosage is 1650 U/kg; CGRP low dose group administration 40 μ g/kg; Dosage group administration 200 μ g/kg among the CGRP; CGRP high dose group administration 1mg/kg; AcAP5 low dose group administration 40 μ g/kg; Dosage group administration 200 μ g/kg among the AcAP5; AcAP5 high dose group administration 1mg/kg; CGRP-AcAP5 fusion rotein low dose group administration 40 μ g/kg; Dosage group administration 200 μ g/kg in the CGRP-AcAP5 fusion rotein; CGRP-AcAP5 fusion rotein high dose group administration 1mg/kg.All by intravenous administration.Behind intravenous administration, the carotid artery with separator well places in the detection joint of YLS-14B instrument for generating thrombus in small animal immediately, starts the thrombus instrument for generating, with constant current galvanic current stimulation (1 mA).The record probe is measured the circulation of blood and is conversed blood vessel blockage degree (timed interval of measurement is 4 s, is expressed as a percentage data, and all processes continues 5 min) by infrared scan.The result is as shown in table 1:
The effect of table 1 CGRP-AcAP5 fusion rotein Chinese People's Anti-Japanese Military and Political College Rat common carotid thrombus
| Group | n | Dosage | Carotid chocking-up degree (%) |
| Negative control group | 8 | 0μg/kg | 100.00±0.00 |
| Positive controls | 8 | 1650U/kg | 49.26±41.98 |
| The CGRP low dose group | 8 | 40μg/kg | 50.86±33.76 * |
| Dosage group among the CGRP | 8 | 200μg/kg | 35.81±23.96 * |
| The CGRP high dose group | 8 | 1mg/kg | 25.87±21.86 * |
| The AcAP5 low dose group | 8 | 40μg/kg | 48.31±36.74 * |
| Dosage group among the AcAP5 | 8 | 200μg/kg | 32.64±65.56 * |
| The AcAP5 high dose group | 8 | 1mg/kg | 22.74±23.79 ** |
| CGRP-AcAP5 fusion rotein low dose group | 8 | 40μg/kg | 40.26±41.31 * |
| Dosage group in the CGRP-AcAP5 fusion rotein | 8 | 200μg/kg | 26.39±42.63 * |
| CGRP-AcAP5 fusion rotein high dose group | 8 | 1mg/kg | 10.69±14.56 ** |
Compare with control group: * P＜0.05, * * P＜0.01
The result shows: although injection AcAP5 protein solution and CGRP-AcAP5 fusion rotein solution have antithrombotic effect, but the antithrombotic effect of CGRP-AcAP5 fusion rotein is best, and be significantly increased than the effect of injecting separately the AcAP5 protein solution, illustrate that CGRP and AcAP5 merge the effect that can strengthen Ancylostoma caninum anticoagulant peptide AcAP5.
By hypertension experiment and antithrombotic experimental result as can be known, calcitonin-gene-related peptide (CGRP) and Ancylostoma caninum anticoagulant peptide (AcAP5) fusion can promote mutually synergy.
The experiment of CGRP-AcAP5 fusion rotein treatment myocardial ischemia in rats:
Get 30 of SD rats, cause myocardial infarction and ischemia model; Rat is divided into three groups (control group, experiment A group and experiment B groups) at random, and experiment A group is pressed 2.5mg/kgb.w dosage injection CGRP albumen; Experiment B group is pressed 2.5mg/kgb.w dosage injection CGRP-AcAP5 fusion rotein; The physiological saline of control group injection Isodose.
Experiment is carried out 4 times, and the ratio that rat heart muscle is carried out hazardous location, necrotic area and necrotic area and hazardous location detects, and detected result is as shown in table 2, wherein AAR(Area at risk) be the hazardous location, IS(Infarct size) be the necrotic area.
Table 2 infarcted region size (± S)
*P<0.05，**P<0.01
The result shows: although experiment A group is compared the effect that the treatment myocardial ischemia is arranged with experiment B group with control group, but the result for the treatment of of CGRP-AcAP5 fusion rotein is best, and be significantly increased than the effect of injecting separately CGRP albumen, illustrate that AcAP5 and CGRP merge the effect that can strengthen calcitonin-gene-related peptide (CGRP).
By osteoporosis experiment and myocardial ischemia experimental result as can be known, Ancylostoma caninum anticoagulant hemepeptide (AcAP5) and calcitonin-gene-related peptide (CGRP) fusion can promote mutually synergy.
CGRP-AcAP5 fusion rotein toxicity test:
Harbin Medical University's Experimental Animal Center provides SPF level kunming mice.Get 60 6 week age, male and female half and half, weights be that the mouse of 18 ± 2g is as experimental subjects.Mouse is divided into 2 groups at random: experimental group and control group, adopt maximum tolerance administration (according to clinical Interferon, rabbit consumption 50 μ g/kg).Experimental group injection CGRP-AcAP5 fusion rotein, total dose 1mg/kg, once daily 0.02mL.Control group mice injection Isodose physiological saline.
Administration process small mouse is acted normally, feed, drinking-water is normal, the urine excrement is normal, hair color along white and glossy, activity freely, between reactive good, the mouse without mutually baiting phenomenon.Raised 14 days and 30 days continuously, mouse does not all occur dead.
The administration of CGRP-AcAP5 fusion rotein is after 14 days, 30 days, put to death mouse, pluck eyeball and get blood, centrifugal 5 min of 3000r/min, draw serum, detect Main Biochemical with the Beckman automatic clinical chemistry analyzer: aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), blood urea nitrogen (BUN), uric acid (URCA), total bilirubin (TBIL), TOTAL BILE ACID (TBA).Blood biochemistry index is as shown in table 3.
Table 3 blood biochemistry index
The administration of CGRP-AcAP5 fusion rotein is put to death mouse after 14 days, 30 days, cores, liver, spleen, lung, kidney, observes internal organs color, form, calculates each organ coefficient.Estimate according to the statistics sample, in every group, randomly draw 7 mouse, core, liver, spleen, lung, kidney HE dyeing do histopathologic examination; Get myeloid tissue Wright's staining observation of cell and have or not oedema, sex change, necrosis etc.The pathological observation result of main organs is as shown in table 4.
Table 4 internal organs pathological observation result
| Group | Fate | The heart | Liver | Spleen | Lung | Kidney |
| Control group | 14 | 3.59±0.78 | 30.62±2.63 | 4.64±0.75 | 2.90±0.76 | 7.83±0.72 |
| Experimental group | 14 | 3.58±0.82 | 30.59±2.52 | 4.57±0.84 | 2.91±0.75 | 7.81±0.90 |
| Control group | 30 | 3.46±0.84 | 30.82±2.84 | 4.75±0.94 | 2.91±0.76 | 7.90±0.84 |
| Experimental group | 30 | 3.76±0.80 | 31.47±3.42 | 4.52±0.72 | 2.90±0.80 | 7.87±0.83 |
The HE dyeing of experimental group and the control group main organs heart, liver, spleen, lung, kidney and marrow Wright dyeing paired observation are found: each organizes the heart, liver, spleen, lung, kidney and the marrow of mouse all without abnormal changes such as oedema, sex change, necrosis.
This experiment is observed pathological section and the bone marrow smear of the heart, spleen, lung, kidney, is showed no obvious damaging change.Organ coefficient statistical analysis to 5 kinds of internal organs draws each group difference without significance.Illustrate that all CGRP-AcAP5 fusion rotein and meta-bolites thereof do not produce organic lesion to internal organs.Each group difference of Biochemistry test is without illustrating that significantly the CGRP-AcAP5 fusion rotein is to liver, the infringement of kidney non-functional.Show that the CGRP-AcAP5 fusion rotein has preferably biocompatibility, to mouse without acute toxicity, long term toxicity.
Present embodiment CGRP-AcAP5 fusion rotein inserts GlyThr between calcitonin-gene-related peptide (CGRP) and Ancylostoma caninum anticoagulant hemepeptide, changed the secondary structure of polypeptide, but not only do not make AcAP5 and CGRP loss of biological activity, improved on the contrary its biological activity.The expression amount of the interior CGRP-AcAP5 fusion rotein of present embodiment e. coli bl21 (DE3-CGRP/AcAP5) is also high than single AcAP5 or CGRP Expression in Escherichia coli amount.
The present embodiment fusion rotein can carry out administration by injection or oral mode, does not have first pass effect.
Changed the secondary structure of CGRP and Ancylostoma caninum anticoagulant hemepeptide AcAP5 when making up present embodiment e. coli bl21 (DE3-CGRP/AcAP5), this change does not produce toxicity in vivo, and the fusion rotein that fermentation produces has security; And the change of secondary structure do not affect chromatography and purifying, utilizes present embodiment e. coli bl21 (DE3-CGRP/AcAP5) fermentation CGRP-AcAP5 fusion rotein to have separation and purification and is easy to characteristics.
The medicine that uses in the present embodiment, reagent, enzyme, competent cell and plasmid etc. are all bought acquisition, if without particular requirement then concentration be product annotation concentration.
Embodiment two: the nucleotide sequence of embodiment one described calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein encoding gene is shown in SEQ ID NO:1.
Claims (2)
1. calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein is characterized in that the aminoacid sequence of described fusion rotein is shown in SEQ ID NO:2.
2. the encoding gene of calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein as claimed in claim 1 is characterized in that the nucleotide sequence of gene is shown in SEQ ID NO:1.
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| CN102321588A (en) * | 2011-10-10 | 2012-01-18 | 黑龙江大学 | Chinese hamster ovary genetic engineering cell line for performing high-level secretory expression on AcAPc2 |
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| CAPPELLO M ET AL，: "GenBank:AAC47318", 《GENBANK》 * |
| 余琼等，: "重组鲑鱼降钙素与降钙素基因相关肽融合基因分子设计及大肠杆菌表达载体构建", 《食品工业科技》 * |
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