CN103031294B - 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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- CN103031294B CN103031294B CN201210585415.0A CN201210585415A CN103031294B CN 103031294 B CN103031294 B CN 103031294B CN 201210585415 A CN201210585415 A CN 201210585415A CN 103031294 B CN103031294 B CN 103031294B
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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, the existing hyperpietic of China more than 200,000,000, increases by 1,000 ten thousand people every year.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, shows: the lethality rate of cerebral thrombosis almost accounts for first of all diseases according to domestic and international statistical medical information.
Calcitonin-gene-related peptide (CGRP) is a kind of biologically active peptides that Rosenfeld equals nineteen eighty-three discovery, it and thyrocalcitonin (CT) all derive from and are positioned at chromosomal CT/CGRP gene No. 11, because the RNA coding that CT/CGRP gene is different is translated into CGRP and CT, CGRP is a kind of biologically active polypeptides being made up of 37 amino acid of answering DNA gene recombination and molecular biotechnology research to find, that the strongest endogenous of finding so far expands 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 is far better than Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, and atrial natriuretic peptide and norepinephrine (NE), than strong 10000 times of left and right such as vagusstoff (Ach), serotonins (5-HT), than the strong 10-100 of Racemic isoproterenol doubly.
Ancylostoma caninum anticoagulant hemepeptide (anticoagulant peptide, AcAPs) be that dog hookworm is while 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.What AcAPs applied at present has AcAP5, AcAP6, tri-kinds of recombinant proteins of AcAPc2.Wherein rAcAP5 is made up of 77 amino acid, containing 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.
But this two kinds of albumen independent roles at present, 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, to obtaining a kind of fusion rotein can simultaneously with 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 as 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 as 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, action compensating and synergy between the two, gained fusion rotein confirms to have good antithrombotic and the hypertensive effect for the treatment of through experiment.Fusion rotein of the present invention can be owing to preparing the medicine of 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 as shown in SEQ ID NO:2.
The preparation method of present embodiment calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein, carries out according to the following steps:
One, fusion rotein CGRP/AcAP5 gene is synthetic: a Kpn I restriction enzyme site of design in CGRP and AcAP5 fusion gene, fusion rotein CGRP/AcAP5 gene by Shanghai Sheng Gong company synthesizing ribonucleotide sequence as shown in SEQ ID NO:1, and add respectively BamHI and EcoR I restriction enzyme site at fusion rotein CGRP/AcAP5 gene two ends, then be cloned on pUC57 carrier, obtain pUC(CGRP/AcAP5) carrier;
Two, the structure of recombinant expression vector: the pUC(CGRP/AcAP5 that step 1 is obtained) BamHI and EcoR I double digestion for carrier, connect with the same expression vector pGEX-6P-1 through BamHI and EcoR I double digestion again, obtain 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 e. coli bl21 (DE3), random 37 DEG C of incubated overnight of picking transformed bacteria, adopt plasmid extraction kit (buying from Ai Delai bio tech ltd, Beijing) to extract plasmid DNA, with Kpn I single endonuclease digestion, and with corresponding blank plasmid pGEX-6P-1 in contrast, the plasmid that contains Kpn I restriction enzyme site is carried out to 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: positive recombinant bacterium is placed in to 28 DEG C of LB substratum and cultivates 15h, 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 step 2) carrier is as follows by 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 DEG C of water-baths, 10h.
In step 2, the system of expression vector pGEX-6P-1 double digestion is as follows:
| Composition | Consumption |
| pGEX-6P-1 | 20μL |
| 10×M buffer | 6μL |
| BamHI | 3μL |
| EcoRⅠ | 3μL |
| ddH 2O | 28μL |
Endonuclease reaction condition: 37 DEG C of water-baths, 10h.
In step 2, ligation system is as follows:
| Composition | Consumption |
| Goal gene CGRP/AcAP5 | 13μL |
| Enzyme is cut rear pGEX-6P-1 carrier | 3μL |
| T4 DNA ligase | 2μL |
| 10 × T4 DNA ligase buffe | 2μL |
Ligation condition: 16 DEG C of water-baths, 8～12h.Described T4 DNA ligase, buys from TaKaRa company.By bio-engineering corporation by fusion rotein CGRP/AcAP5 gene clone to pUC57 carrier.
For the effect of checking present embodiment, carry out following experiment:
The experiment of CGRP-AcAP5 fusion rotein antithrombotic:
Get 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 of same 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 in CGRP; CGRP high dose group administration 1mg/kg; AcAP5 low dose group administration 40 μ g/kg; Dosage group administration 200 μ g/kg in 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 CGRP-AcAP5 fusion rotein; CGRP-AcAP5 fusion rotein high dose group administration 1mg/kg.All by intravenous administration.After intravenous administration, immediately the carotid artery of separator well is placed in the detection joint of YLS-14B instrument for generating thrombus in small animal, start thrombus instrument for generating, with constant current galvanic current stimulation (1 mA).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.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 |
| CGRP low dose group | 8 | 40μg/kg | 50.86±33.76 * |
| Dosage group in CGRP | 8 | 200μg/kg | 35.81±23.96 * |
| CGRP high dose group | 8 | 1mg/kg | 25.87±21.86 * |
| AcAP5 low dose group | 8 | 40μg/kg | 48.31±36.74 * |
| Dosage group in AcAP5 | 8 | 200μg/kg | 32.64±65.56 * |
| 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 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 ** |
With control group comparison: * P<0.05, * * P<0.01
Result shows: although injection AcAP5 protein solution and CGRP-AcAP5 fusion rotein solution have antithrombotic effect, but antithrombotic effect the best of CGRP-AcAP5 fusion rotein, and be significantly increased than the effect of injecting separately AcAP5 protein solution, illustrate that CGRP and AcAP5 merge the effect that can strengthen Ancylostoma caninum anticoagulant peptide AcAP5.
From hypertension experiment and antithrombotic experimental result, calcitonin-gene-related peptide (CGRP) and Ancylostoma caninum anticoagulant peptide (AcAP5) merge and 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 to 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 hazardous location, IS(Infarct size) be necrotic area.
Table 2 infarcted region size (± S)
*P<0.05，**P<0.01
Result shows: although experiment A group has the effect for the treatment of myocardial ischemia with experiment B group compared with control group, but result for the treatment of the best of CGRP-AcAP5 fusion rotein, 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).
From osteoporosis experiment and myocardial ischemia experimental result, Ancylostoma caninum anticoagulant hemepeptide (AcAP5) and calcitonin-gene-related peptide (CGRP) merge and can promote mutually, synergy.
CGRP-AcAP5 fusion rotein toxicity test:
Harbin Medical University's Experimental Animal Center provides SPF level kunming mice.Get mouse that 60 6 week age, male and female half and half, weights are 18 ± 2g 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, urine excrement is normal, hair color along white and glossy, activity freely, between reactive good, mouse without mutually baiting phenomenon.Raise 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, use Beckman automatic clinical chemistry analyzer to detect Main Biochemical: 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 statistics sample, in every group, randomly draw 7 mouse, core, liver, spleen, lung, kidney HE dyeing does 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 | Number of days | 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: respectively organize the heart, liver, spleen, lung, kidney and the marrow of mouse all without abnormal changes such as oedema, sex change, necrosis.
Pathological section and the bone marrow smear of this experiment to the heart, spleen, lung, kidney observed, and is showed no obvious damaging change.The organ coefficient statistical analysis of 5 kinds of internal organs is shown to each group difference is without significance.All illustrate that CGRP-AcAP5 fusion rotein and meta-bolites thereof do not produce organic lesion to internal organs.The each group difference of Biochemistry test is without significantly illustrating that CGRP-AcAP5 fusion rotein is to liver, the infringement of kidney non-functional.Show that CGRP-AcAP5 fusion rotein has good 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, change 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 the expression amount of CGRP in intestinal bacteria.
Present embodiment fusion rotein can carry out administration by injection or oral mode, does not have first pass effect.
The secondary structure that has changed CGRP and Ancylostoma caninum anticoagulant hemepeptide AcAP5 while building 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, utilize present embodiment e. coli bl21 (DE3-CGRP/AcAP5) fermentation CGRP-AcAP5 fusion rotein to there is separation and purification and be easy to feature.
Medicine, reagent, enzyme, competent cell and the plasmids etc. that use in present embodiment are all bought acquisition, if without particular requirement concentration be product annotation concentration.
Embodiment two: described in embodiment one, the nucleotide sequence of calcitonin-gene-related peptide and Ancylostoma caninum anticoagulant peptide AcAP5 fusion rotein encoding gene is as 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 as 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 as shown in SEQ ID NO:1.
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| cappello m et al，.GenBank:AAC47318.《GenBank》.1996, * |
| 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》.2009, * |
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