CN102964453A - 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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- CN102964453A CN102964453A CN201210534877XA CN201210534877A CN102964453A CN 102964453 A CN102964453 A CN 102964453A CN 201210534877X A CN201210534877X A CN 201210534877XA CN 201210534877 A CN201210534877 A CN 201210534877A CN 102964453 A CN102964453 A CN 102964453A
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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 the sufferers of osteoporosis face case increases year by year, and fracture is the most multiple the most common disease in the 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 the performance of myocardial ischemia also appears 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, the body, can promote union of fracture, preventing osteoporosis.Formed by 14 aminoacid small peptides, 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 have 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, formed by 37 seed amino acids, its molecular weight is about 3800 dalton, it and calcitonin gene homology, wherein contain five introns and six exons, for endogenous expands the blood vessel peptide matters, can treat myocardial ischemia.
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 sOGP and calcitonin-gene-related peptide fusion rotein and encoding gene thereof, in the hope of obtaining a kind of fusion rotein that can have simultaneously 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 shown in SEQ ID NO:2.
The nucleotide sequence of above-mentioned sOGP and calcitonin-gene-related peptide fusion rotein encoding gene is 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, between the two action compensating and synergy, the gained fusion rotein has the effect of good treatment osteoporosis and myocardial ischemia through experiment confirm.Fusion rotein of the present invention can be owing 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 shown in SEQ ID NO:2.
The preparation method of present embodiment sOGP and calcitonin-gene-related peptide fusion rotein, carry out according to the following steps:
One, fusion rotein OGP/CGRP gene is synthetic: Kpn I restriction enzyme site of design in OGP and the CGRP fusion gene, give birth to the fusion rotein OGP/CGRP 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 OGP/CGRP gene two ends, then be cloned on the pUC57 carrier, obtain pUC(OGP/CGRP) carrier;
Two, the structure of recombinant expression vector: with the pUC(OGP/CGRP 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 OGP/CGRP dna recombinant expression vector pGEX-OGP/CGRP;
Three, the structure of engineering strain: then vector pGEX-OGP/CGRP 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 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: with recombinant bacterium BL21(DE3-OGP/CGRP) place 28 ℃ of LB substratum to 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 the step 2) carrier is as follows with the system of BamHI and EcoR I double digestion:
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:
Endonuclease reaction condition: 37 ℃ of water-baths, 10h.
The ligation system is as follows in the step 2:
Ligation condition: 16 ℃ of water-baths, 8～12h.Described T4 dna ligase is bought from TaKaRa company.
Be 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, extract bilateral ovaries under 40 aseptic conditions, wherein 8 Sham-operated control group excision bilateral one fritter fat.Get 40 of modeling group survival healthy rats after 12 weeks, 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 is respectively got the rat femur head after the execution, immerse in 4% glutaraldehyde fixing, with the 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 are 20kV.
Observations sees Table 1, the result shows, although 1 group of positive controls, experiment with test 2 groups and compare the osteoporotic effect for the treatment of with negative control group, but the result for the treatment of of OGP-CGRP fusion rotein is best, and be significantly increased than the effect of taking separately the OGP protein solution, illustrate that OGP and CGRP merge the effect that can be enhanced to osteogenic growth peptide (OGP).
Long-pending (the X ± SD) of the comparison of table 1 bone trabecula width and surface of bone
Compare with negative control group: * 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 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 OGP-CGRP fusion rotein is best, and be significantly increased than the effect of injecting separately CGRP albumen, illustrate that OGP 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, sOGP (OGP) and calcitonin-gene-related peptide (CGRP) fusion can promote mutually synergy.
(3) OGP-CGRP 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 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, 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 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, 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 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.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 | 3.46±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 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 OGP-CGRP 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 OGP-CGRP fusion rotein is to liver, the infringement of kidney non-functional.Show that the OGP-CGRP fusion rotein has preferably biocompatibility, to mouse without obviously acute toxicity, long term toxicity.
Embodiment two: the nucleotide sequence of embodiment one described sOGP and calcitonin-gene-related peptide fusion rotein encoding gene is 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 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 shown in SEQ ID NO:1.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4212542A4 (en) * | 2020-09-10 | 2024-08-28 | Shaanxi Micot Tech Co Ltd | Bispecific fusion polypeptide compound |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560076A (en) * | 2004-03-09 | 2005-01-05 | 复旦大学 | Derivant with reducing calcium gene related peptide and preparation process thereof |
| CN1857713A (en) * | 2006-03-20 | 2006-11-08 | 吴忠 | Osteoporosis treating medicine |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560076A (en) * | 2004-03-09 | 2005-01-05 | 复旦大学 | Derivant with reducing calcium gene related peptide and preparation process thereof |
| CN1857713A (en) * | 2006-03-20 | 2006-11-08 | 吴忠 | Osteoporosis treating medicine |
Non-Patent Citations (2)
| Title |
|---|
| GENBANK: "GenBank:1005250A", 《NCBI GENBANK》 * |
| 余琼等: "重组人成骨生长肽治疗去卵巢大鼠骨质疏松的实验观察", 《中国地方病学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4212542A4 (en) * | 2020-09-10 | 2024-08-28 | Shaanxi Micot Tech Co Ltd | Bispecific fusion polypeptide compound |
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