CN102964453B - Osteogenic growth peptide-calcitonin gene related peptide fusion protein and encoding gene thereof - Google Patents
Osteogenic growth peptide-calcitonin gene related peptide fusion protein and encoding gene thereof Download PDFInfo
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- CN102964453B CN102964453B CN201210534877.XA CN201210534877A CN102964453B CN 102964453 B CN102964453 B CN 102964453B CN 201210534877 A CN201210534877 A CN 201210534877A CN 102964453 B CN102964453 B CN 102964453B
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Abstract
The invention discloses an osteogenic growth peptide-calcitonin gene related peptide fusion protein and an encoding gene thereof and relates to the fusion protein and the encoding gene. The invention aims at providing the osteogenic growth peptide-calcitonin gene related peptide fusion protein and the encoding gene thereof and further getting the fusion protein which simultaneously has the double effects of treating osteoporosis and myocardial ischemia, wherein the two proteins can form complementary action and synergy. The amino acid sequence of the fusion protein is as shown in SEQ ID NO: 2. The nucleotide sequence of the encoding gene is as shown in SEQ ID NO: 1. The osteogenic growth peptide-calcitonin gene related peptide fusion protein disclosed by the invention can fuse an osteogenic growth peptide and a calcitonin gene related peptide, and enable the two, namely the osteogenic growth peptide and the calcitonin gene related peptide to form the complementary action and the synergy; and experiments prove that the fusion protein has great effects of treating the osteoporosis and the myocardial ischemia. The fusion protein disclosed by the invention can be used for preparing medicaments for preventing and treating the osteoporosis and the myocardial ischemia.
Description
Technical field
The present invention relates to a kind of fusion rotein and encoding gene thereof.
Background technology
Current, China has entered aging society, and sufferers of osteoporosis face case increases year by year, and fracture is the most multiple the most common disease in traumatology.Myocardial ischemia is that the hemoperfusion of heart reduces, and causes the oxygen supply of heart to reduce, and energy metabolism of myocardial is undesired, can not support a kind of pathological state of heart normal operation.Myocardial ischemia serious harm the elderly's health, in recent years along with growth in the living standard, coronary atherosclerosis presents the trend of rejuvenation, and also there is the performance of myocardial ischemia in the youngster in some 20-30 years.
SOGP (OGP) has the function such as skeletonization and hemopoietic in the external osteoblastic proliferation of promotion, body, can promote union of fracture, preventing osteoporosis.By 14 aminoacid small peptides, formed, and the C-terminal of its aminoacid sequence and histone H 4 is in full accord, with mouse T cell receptor β chain V district portion homologous, healing for fracture, osteoporosis, the prevention of acute and chronic anemia and treatment and in the effect of tumour Radiotherapy chemotherapy process moderate stimulation medullary cell hematopoiesis, obtained the attention of height and certainly.
Calcitonin-gene-related peptide (Calcitonin Gene Related peptide, CGRP) be first active polypeptide of being found by the first Application DNA gene recombination such as Rosenfeld and molecular biotechnology research in nineteen eighty-three, by 37 seed amino acids, formed, its molecular weight is about 3800 dalton, it and calcitonin gene homology, wherein contain five introns and six exons, for endogenous expands blood vessel peptide matters, can treat myocardial ischemia.
Yet 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 sOGP and calcitonin-gene-related peptide fusion rotein and encoding gene thereof, to obtaining a kind of fusion rotein can simultaneously with treatment osteoporosis and the two kind effects of myocardial ischemia, action compensating and synergy between two kinds of albumen.
The aminoacid sequence of sOGP of the present invention and calcitonin-gene-related peptide fusion rotein is as shown in SEQ ID NO:2.
The nucleotide sequence of above-mentioned sOGP and calcitonin-gene-related peptide 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, sOGP and calcitonin-gene-related peptide are merged, action compensating and synergy between the two, gained fusion rotein confirms to have the effect of good treatment osteoporosis and myocardial ischemia through experiment.Fusion rotein of the present invention can be due to the medicine of preparation prevention and treatment osteoporosis and myocardial ischemia disease, for the research of prevention and treatment osteoporosis and myocardial ischemia 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 sOGP and calcitonin-gene-related peptide fusion rotein is as shown in SEQ ID NO:2.
The preparation method of present embodiment sOGP and calcitonin-gene-related peptide fusion rotein, carries out according to the following steps:
One, fusion rotein OGP/CGRP gene is synthetic: a Kpn I restriction enzyme site of design in OGP and CGRP fusion gene, fusion rotein OGP/CGRP gene by Shanghai Sheng Gong company synthesizing ribonucleotide sequence as shown in SEQ ID NO:1, and at fusion rotein OGP/CGRP gene two ends, add respectively BamHI and EcoR I restriction enzyme site, then be cloned on pUC57 carrier, obtain pUC(OGP/CGRP) carrier;
Two, the structure of recombinant expression vector: the pUC(OGP/CGRP that step 1 is obtained) BamHI and EcoR I double digestion for carrier, through BamHI, be connected with the expression vector pGEX-6P-1 of EcoR I double digestion again with equally, obtain fusion rotein OGP/CGRP DNA recombinant expression vector pGEX-OGP/CGRP;
Three, the structure of engineering strain: then vector pGEX-OGP/CGRP is transformed in e. coli bl21 (DE3), random 37 ℃ 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 that sequencing result is correct is positive recombinant bacterium BL21(DE3-OGP/CGRP); Wherein e. coli bl21 (DE3) obtains for buying;
Four, the abduction delivering of fusion rotein and purifying: by recombinant bacterium BL21(DE3-OGP/CGRP) be placed in 28 ℃ of LB substratum and cultivate 15h, then adopt GST tag fusion protein method to carry out the separation and purification of albumen, the purity of present embodiment sOGP and calcitonin-gene-related peptide fusion rotein is 98%, and fusion protein expression is 39%.
PUC(OGP/CGRP in step 2) carrier is as follows by the system of BamHI and EcoR I double digestion:
Endonuclease reaction condition: 37 ℃ of water-baths, 10h.
In step 2, the system of expression vector pGEX-6P-1 double digestion is as follows:
Endonuclease reaction condition: 37 ℃ of water-baths, 10h.
In step 2, ligation system is as follows:
Ligation condition: 16 ℃ of water-baths, 8～12h.Described T4 DNA ligase, buys from TaKaRa company.
For the effect of checking present embodiment, carry out following experiment:
(1) OGP-CGRP fusion rotein treatment osteoporosis experiment:
Get 48 of female sd inbred rats, under 40 aseptic conditions, extract bilateral ovaries, wherein 8 Sham-operated control group excision bilateral one fritter fat.After 12 weeks, get 40 of modeling group survival healthy rats, be divided at random 5 groups, 8 every group.Sham-operated control group is got 8, amounts to 5 groups, respectively oral following medicine: Sham-operated control group: 0.5% CMC-Na solution 5ml/kg; Model control group: 0.5% CMC-Na solution 5ml/kg.Positive controls: alendronate sodium (Alen) 5mg/kg; Test 1 group: 0.5% OGP protein solution 5ml/kg; Test 2 groups: 0.5% OGP-CGRP fusion rotein solution 5ml/kg.Successive administration three months, respectively gets rat femur head after execution, immerse in 4% glutaraldehyde fixing, with dentistry diamond saw, femoral head sagittal plane is cut, get its a slice, through cleaning, 10% clorox soaks 6h, ultrasonic cleaning 15min, Gradient elution using ethanol, ether soaks, dry, ion sputtering film coating, SX-40 scanning electron microscopic observation, acceleration voltage is 20kV.
Observations is in Table 1, result shows, although positive controls, test 1 group with test 2 groups compare with negative control group treatment osteoporotic effect, but the result for the treatment of of OGP-CGRP fusion rotein is best, and than the effect of taking separately OGP protein solution, be significantly increased, illustrate that OGP and CGRP merge the effect that can be enhanced to osteogenic growth peptide (OGP).
The comparison of table 1 bone trabecula width and surface of bone amass (X ± SD)
With negative control group comparison: * P<0.05, * * P<0.01
(2) experiment of OGP-CGRP 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 OGP-CGRP 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 compares with experiment B group the effect that has treatment myocardial ischemia with control group, but the result for the treatment of of OGP-CGRP fusion rotein is best, and than the effect of injecting separately CGRP albumen, be significantly increased, illustrate that OGP and CGRP merge the effect that can strengthen calcitonin-gene-related peptide (CGRP).
From osteoporosis experiment and myocardial ischemia experimental result, sOGP (OGP) and calcitonin-gene-related peptide (CGRP) merge and can promote mutually, synergy.
(3) OGP-CGRP 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 50ug/kg).Experimental group injection OGP-CGRP 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 OGP-CGRP 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, with Beckman automatic clinical chemistry analyzer, 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 OGP-CGRP 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.According to statistics sample, estimate, 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 | Number of days | The heart | Liver | Spleen | Lung | Kidney |
| Control group | 14 | 3.46±0.90 | 31.44±2.73 | 4.38±0.78 | 2.91±0.83 | 7.85±0.79 |
| Experimental group | 14 | 3.31±0.81 | 30.43±3.02 | 4.35±0.78 | 2.85±0.86 | 7.81±0.93 |
| Control group | 30 | 3.67±0.61 | 31.93±2.95 | 4.72±0.84 | 2.90±0.77 | 7.96±0.89 |
| Experimental group | 30 | 3.58±0.78 | 30.74±3.71 | 4.58±0.87 | 2.89±0.52 | 7.88±0.84 |
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 heart, liver, spleen, lung, kidney and marrow of organizing mouse is all without abnormal changes such as oedema, sex change, necrosis.
This experiment is observed the pathological section of the heart, spleen, lung, kidney and bone marrow smear, 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 OGP-CGRP fusion rotein and meta-bolites thereof do not produce organic lesion to internal organs.Each group difference of Biochemistry test is without significantly illustrating that OGP-CGRP fusion rotein is to liver, the infringement of kidney non-functional.Show that OGP-CGRP fusion rotein has good biocompatibility, to mouse without obviously acute toxicity, long term toxicity.
Embodiment two: described in embodiment one, the nucleotide sequence of sOGP and calcitonin-gene-related peptide fusion rotein encoding gene is as shown in SEQ ID NO:1.
Claims (2)
1. sOGP and calcitonin-gene-related peptide 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 sOGP and calcitonin-gene-related peptide 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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