CN104861076A - Fused polypeptide and application of fused polypeptide in accelerating osteogenesis and vascularization - Google Patents
Fused polypeptide and application of fused polypeptide in accelerating osteogenesis and vascularization Download PDFInfo
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- CN104861076A CN104861076A CN201510317325.7A CN201510317325A CN104861076A CN 104861076 A CN104861076 A CN 104861076A CN 201510317325 A CN201510317325 A CN 201510317325A CN 104861076 A CN104861076 A CN 104861076A
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Abstract
The invention discloses a fused polypeptide and application of the fused polypeptide in accelerating osteogenesis and vascularization and belongs to the technical field of biochemistry and molecular biology. The fused polypeptide is formed by fusing polypeptide which plays a role of accelerating bone development and polypeptide which has a vascularization effect. The amino acid sequence of the fused polypeptide is shown in SEQ ID NO: 3. The fused polypeptide disclosed by the invention is formed by fusing the polypeptide which plays the role of accelerating bone development and the polypeptide which has the vascularization effect and has the functions of accelerating bone development and accelerating vascularization. The mutated fused polypeptide disclosed by the invention has the relatively strong functions of accelerating bone development and vascularization, thereby providing a basis for research and development of drugs which accelerate bone development and vascularization.
Description
Technical field
The invention belongs to Biochemistry and Molecular Biology technical field, be specifically related to a kind of fusion polypeptide and promoting the application in skeletonization and vascularization.
Background technology
Delicious peptide (BMPs) is that a class can one group of cytokine of peculiar smell skeletonization, find kind more than 20 up to now, wherein, BMP-2 is most important somatomedin in BMP family, the ability that BMP-2 inducing bone mesenchymal stem cell breaks up to skeletonization direction is the strongest, but this protein half-life is short, be difficult to continue to play a role.The step more complicated of its preparation and purifying thereof, is not easy to obtain, therefore, studies the polypeptide segment of its reactive site, adopts chemical synthesis process synthesis, becomes a kind of research tendency.At present, the research of the reactive site of BMP-2 is ripe, but this fragment activity in vivo, compared with native protein, actively reduces more, and is not easy target and fixes.Natural B MP-2 bioactive peptide molecule is easily degraded in vivo, and the activity in body is very low.
Vascularization polypeptide is the polypeptide factor that a class can promote vasculogenesis, organizational project, it is crucial that tissue regeneration medium vessels is formed, current technology forms deficiency, do not have blood for any regeneration all can not be successful, it is little that native blood vessels forms peptide molecule, and very easily degrade in vivo, the activity in body is very low.
Summary of the invention
The object of the present invention is to provide one can promote osteogenesis and angiopoietic fusion polypeptide simultaneously.
The present invention also aims to provide the above-mentioned preparation method and the application thereof that promote osteogenesis and angiopoietic fusion polypeptide simultaneously.
One has promotion osteogenesis and angiopoietic fusion polypeptide, promotes with having, the polypeptide of osteogenesis effect promotes that the peptide fusion of angiogenic action forms by having; The described aminoacid sequence with the polypeptide promoting osteogenesis effect is as shown in sequence table SEQ ID NO:1; Described have promote that the polypeptide of angiogenic action is as shown in sequence table SEQ ID NO:2.
Described have promote that the polypeptide of osteogenesis effect has the polypeptide promoting osteogenesis effect through the replacement of 1 to 3 amino-acid residue, disappearance or interpolation for the amino acid residue sequence shown in sequence table SEQ ID NO:1.
Described have promote that the polypeptide of angiogenic action has through the replacement of 1 to 3 amino-acid residue, disappearance or interpolation a polypeptide promoting angiogenic action for the amino acid residue sequence shown in sequence table SEQ ID NO:2.
Above-mentioned fusion polypeptide promotes the application in osteogenesis and angiopoietic medicine in preparation.
A kind of pharmaceutical composition comprising above-mentioned fusion polypeptide.
Described pharmaceutical composition, except containing except fusion polypeptide, also comprises the carrier of pharmaceutical acceptable, vehicle or adjuvant.
The carrier of described pharmaceutical acceptable is keyhole limpet hemocyanin, bovine serum albumin, ovalbumin or oxyphorase.
Described vehicle is gum arabic, lanolin, cocounut oil, potato starch, sweet potato starch, sodium starch glycolate, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, one or more in gas-producing disintegrant.
Described adjuvant is Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, CBP, lipopolysaccharides, cytokine.
Beneficial effect of the present invention: by having, fusion polypeptide of the present invention promotes that osteogenesis and angiopoietic peptide fusion form, have concurrently and promote osteogenesis and angiopoietic effect, mutant fusion polypeptides of the present invention has promotion osteogenesis and angiopoietic effect more by force, for promoting that the research and development of osteogenesis and angiopoietic medicine provide basis.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.
Embodiment 1
1, the preparation of fusogenic peptide
The fusion polypeptide of the present embodiment is obtained by FMOC/tBu solid phase polypeptide synthesis as known in the art:
Skeletonization polypeptide SEQ ID NO:1 (KIPKASSVPTELSAISTLYL);
Angiogenesis polypeptide SEQ ID NO:2 (KLTWQELYQLKYKGI);
Fusion polypeptide SEQ ID NO:3 (KIPKASSVPTELSAISTLYLKLTWQELYQLKYKGI).
2, Osteogenic Cells experiment is become
Test associated materials:
SD MSC mono-strain (RASMX-01001), source: Cyagen
Cell state: cell state is good, and form is homogeneous.
Cell Sterility testing result: negative
Test related reagent:
Human embryonic stem cell medium mTeSR1 (Canadian Stemcell company), 1 × PBS (PBS-10001-100),
Human mesenchymal stem cell Osteoinductive differentiation culture medium C P1202 (Wei Tong biotech firm of Shenzhen), 0.25%Trypsin-0.0,4%EDTA (TEDTA-10001-100), BMP-2 (Beijing Bioisystech Co., Ltd of Yi Qiao divine boat)
Induced osteogenesis polypeptide alkaline phosphatase assay test kit (Science and Technology Ltd. is built up in Nanjing) of the present invention.
Testing sequence:
I, cell prepare: when SD MSC cytogamy reaches 80%, gone down to posterity by cell, are inoculated in through 0.1% gelatin bag by 24 orifice plates crossed, for osteogenic induction.
A. cell dissociation, inoculation:
A. supernatant discarded, cleans cell 2 times with 1 × PBS, adds 1mlPBS and 1ml 0.25%Trypsin-0.04%EDTA peptic cell;
B. digest 1-2 minute, under microscope, visible cell gap increases, cell rounding, claps the wall of culture vessel with have gentle hands, and the SDMSC perfect medium adding 2mL immediately stops digestion.
C. use suction pipe imbitition, blow and beat culture vessel surface gently, 3-5 time repeatedly, make cell thoroughly depart from a bottle ware diapire, cell is moved in centrifuge tube, then in culturing bottle, add 1 × PBS wash 1-2 time, and washing lotion is transferred in centrifuge tube in the lump.
D.1100rpm centrifugal 4 minutes are carried out;
E. supernatant discarded adds complete culture solution, fully mixes, and is on average inoculated in 3 bags and is crossed in 12 orifice plates of gelatin, shake up, be positioned in the CO2gas incubator of 37 DEG C and cultivate.
The preparation of II, induced osteogenesis polypeptide: weighed by induced osteogenesis polypeptide and be distributed into 3 parts (1mg/ parts), get a copy of it and dissolve, other are stored in-20 DEG C of refrigerators for for subsequent use.Add the aseptic water dissolution 1mg osteogenin of 1ml in centrifuge tube, filter, be sub-packed in EP pipe, be stored in-80 DEG C of refrigerator-freezers for use.
III, induced osteogenesis:
A. just induction: when cytogamy reaches about 80%, carry out osteogenic induction.Often organize two multiple hole, wherein 2 hole negative controls: use mescenchymal stem cell complete culture solution to cultivate; 2 hole positive controls: use interstital stem cell Osteoinductive differentiation complete culture solution to cultivate; 2 holes are cultivated containing the BMP-2 mescenchymal stem cell complete culture solution of 10ug/ml; 2 holes are containing 30ug/ml's
BMP-2 mescenchymal stem cell complete culture solution is cultivated; 2 holes are cultivated containing the induced osteogenesis polypeptide mescenchymal stem cell complete culture solution of the present invention of 10ug/ml; 2 holes are cultivated containing the induced osteogenesis polypeptide mescenchymal stem cell complete culture solution of the present invention of 30ug/ml.2 plates repeat.
B. within every 3 days, carry out induction and change liquid.
IV. alkaline phosphatase detects:
Get different detection time and put cell, measure test kit (Nanjing is built up) according to alkaline phosphatase (AKP) and each group of AKP content is detected.Concrete operations are as follows:
A. digest each porocyte, after centrifugal, carry out cell counting, adjustment cell quantity, ensure that 50ul cell re-suspension liquid contains and be greater than 5X10
5cell.With 0.05ml damping fluid re-suspended cell, add corresponding solution successively: measure in pipe and add 0.05mL cell suspension, 0.05mL damping fluid, 0.05mL matrix liquid; 0.05mL 0.1mg/ml phenol Standard Applying Solution is added, 0.05mL damping fluid, 0.05mL matrix liquid in standard pipe; 0.05mL distilled water is added, 0.05mL damping fluid, 0.05mL matrix liquid in blank tube.Abundant mixing 37 DEG C of water-baths 15 minutes, add 1.5mL nitrite ion, mixing, 520nm, 0.5cm or 1cm optical path colorimetric, and blank tube returns to zero, and surveys each pipe absorbancy.
V. experimental result
Two weeks alkaline phosphatase assay:
As can be seen from the above results, the fusion polypeptide of the present embodiment and the result of positive control sample are contrasted, can find that fusion polypeptide of the present invention completely can induced cell growth.And be better than the effect of single skeletonization polypeptide.And the result of the sample of fusion polypeptide of the present invention and BMP-2 is contrasted, can find that fusion polypeptide of the present invention can realize the effect of the induced cell growth suitable with BMP-2, and fusion polypeptide of the present invention and BMP-2 compound use better effects if.
The gene order of fusion polypeptide of the present invention and BMP-2 bioactive peptide is proceeded to animal expression vector by engineered method, proceed in Mice Body, obtain the cartilaginous tissue of mouse, crack protein matter, detected the amount of fusion polypeptide and the BMP-2 bioactive peptide of expressing by WesternBlot, find fusion polypeptide, the degradation speed of cell is slow in vivo, improve more than 10 times than natural BMP-2 bioactive peptide stability, active duration is long.
2, vasculogenesis experiment
Vasculogenesis process be complicated process, angiogenesis Therapy is carried out with the ishemic part of mouse lower limb ischemia model, perform the operation after 1 day, in the below at ligation position, i.e. gastrocnemius muscle position, carry out the injection of angiogenesis polypeptide and fusion polypeptide respectively, the polypeptide 0.1mL of annotation 0.1mg/mL, injection every day once, 8 days after surgery, 16 days, 32 days, the mouse left and right leg blood flow velocity ratio of Post operation angiogenesis polypeptide treatment in 8 days is 0.22, the mouse left and right leg velocity ratio of fusion polypeptide treatment is 0.30, the mouse left and right leg velocity ratio for the treatment of of control group is 0.10, the mouse left and right leg blood flow velocity of Post operation angiogenesis polypeptide treatment in 32 days is 0.39 than being the mouse left and right leg velocity ratio that 0.28 fusion polypeptide is treated, the mouse left and right leg velocity ratio for the treatment of of control group is 0.11.
Vessel wall generates could promote blood flow velocity, above explanation, and angiogenesis polypeptide and fusion polypeptide can play stimulates capillary blood vessel to be formed, and improves the ability of blood flow velocity.And fusion polypeptide effect is stronger.
During injection, the keyhole limpet hemocyanin 0.01ml of 0.1mg/mL is added in fusion polypeptide, the mouse left and right leg blood flow velocity ratio of Post operation angiogenesis polypeptide treatment in 8 days is 0.25, the mouse left and right leg velocity ratio of fusion polypeptide treatment is 0.35, the mouse left and right leg velocity ratio for the treatment of of control group is 0.10, the mouse left and right leg blood flow velocity of Post operation angiogenesis polypeptide treatment in 32 days is 0.43 than being the mouse left and right leg velocity ratio that 0.33 fusion polypeptide is treated, and the mouse left and right leg velocity ratio for the treatment of of control group is 0.11.Prove that this keyhole limpet hemocyanin facilitates fusion polypeptide and plays effect.
During injection, 0.001mg sweet potato starch is added in fusion polypeptide, the mouse left and right leg blood flow velocity ratio of Post operation angiogenesis polypeptide treatment in 8 days is 0.24, the mouse left and right leg velocity ratio of fusion polypeptide treatment is 0.34, the mouse left and right leg velocity ratio for the treatment of of control group is 0.10, the mouse left and right leg blood flow velocity of Post operation angiogenesis polypeptide treatment in 32 days is 0.41 than being the mouse left and right leg velocity ratio that 0.32 fusion polypeptide is treated, and the mouse left and right leg velocity ratio for the treatment of of control group is 0.11.Prove that this sweet potato starch facilitates fusion polypeptide and plays effect.
During injection, 0.001mg cocounut oil is added in fusion polypeptide, the mouse left and right leg blood flow velocity ratio of Post operation angiogenesis polypeptide treatment in 8 days is 0.24, the mouse left and right leg velocity ratio of fusion polypeptide treatment is 0.34, the mouse left and right leg velocity ratio for the treatment of of control group is 0.10, the mouse left and right leg blood flow velocity of Post operation angiogenesis polypeptide treatment in 32 days is 0.41 than being the mouse left and right leg velocity ratio that 0.32 fusion polypeptide is treated, and the mouse left and right leg velocity ratio for the treatment of of control group is 0.11.Prove that this cocounut oil facilitates fusion polypeptide and plays effect.
During injection, 0.0001mg cross-linked polyvinylpyrrolidone is added in fusion polypeptide, the mouse left and right leg blood flow velocity ratio of Post operation angiogenesis polypeptide treatment in 8 days is 0.24, the mouse left and right leg velocity ratio of fusion polypeptide treatment is 0.34, the mouse left and right leg velocity ratio for the treatment of of control group is 0.10, the mouse left and right leg blood flow velocity of Post operation angiogenesis polypeptide treatment in 32 days is 0.41 than being the mouse left and right leg velocity ratio that 0.32 fusion polypeptide is treated, and the mouse left and right leg velocity ratio for the treatment of of control group is 0.11.Prove that cross-linked polyvinylpyrrolidone oil facilitates fusion polypeptide and plays effect.
The gene order of fusion polypeptide of the present invention and vascularization polypeptide is proceeded to animal expression vector by engineered method, proceed in Mice Body, obtain the cartilaginous tissue of mouse, crack protein matter, detected the amount of fusion polypeptide and the BMP-2 bioactive peptide of expressing by Western Blot, the fusion polypeptide of the present embodiment, the degradation speed of cell is slow in vivo, improve more than 10 times than natural vascularization polypeptide stability, active duration is long.
Claims (6)
1. have and promote osteogenesis and an angiopoietic fusion polypeptide, it is characterized in that, promote with having, the polypeptide of osteogenesis effect promotes that the peptide fusion of angiogenic action forms by having, its aminoacid sequence is as shown in sequence table SEQ ID NO:3; The described aminoacid sequence with the polypeptide promoting osteogenesis effect is as shown in sequence table SEQ ID NO:1; Described have promote that the polypeptide of angiogenic action is as shown in sequence table SEQ IDNO:2.
2. one has promotion osteogenesis and angiopoietic fusion polypeptide according to claim 1, it is characterized in that, described in have and promote that the polypeptide of osteogenesis effect has the polypeptide promoting osteogenesis effect through the replacement of 1 to 3 amino-acid residue, disappearance or interpolation for the amino acid residue sequence shown in sequence table SEQ ID NO:1.
3. one has promotion osteogenesis and angiopoietic fusion polypeptide according to claim 1, it is characterized in that, described in have and promote that the polypeptide of angiogenic action has through the replacement of 1 to 3 amino-acid residue, disappearance or interpolation a polypeptide promoting angiogenic action for the amino acid residue sequence shown in sequence table SEQ ID NO:2.
4. the fusion polypeptide described in claim 1-3 promotes the application in osteogenesis and angiopoietic medicine in preparation.
5. one kind comprises the pharmaceutical composition of fusion polypeptide described in claim 1-3.
6. pharmaceutical composition according to claim 5, is characterized in that, described pharmaceutical composition, except containing except fusion polypeptide, also comprises the carrier of pharmaceutical acceptable, vehicle or adjuvant.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111320682A (en) * | 2020-02-27 | 2020-06-23 | 广州领晟医疗科技有限公司 | Application of polypeptide in preparation of medicine for promoting cartilage repair and/or treating osteoarthritis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1752103A (en) * | 2005-10-27 | 2006-03-29 | 华中科技大学同济医学院附属协和医院 | Delicious peptide 2 bioactive peptides and preparation method and application |
| US20110129611A1 (en) * | 2009-12-01 | 2011-06-02 | Wisconsin Alumni Research Foundation | Decorating hydroxyapatite biomaterials with modular biologically active molecules |
| CN103665143A (en) * | 2013-12-16 | 2014-03-26 | 北京博恩康生物科技有限公司 | Induced osteogenesis polypeptide, and preparation method and purposes thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1752103A (en) * | 2005-10-27 | 2006-03-29 | 华中科技大学同济医学院附属协和医院 | Delicious peptide 2 bioactive peptides and preparation method and application |
| US20110129611A1 (en) * | 2009-12-01 | 2011-06-02 | Wisconsin Alumni Research Foundation | Decorating hydroxyapatite biomaterials with modular biologically active molecules |
| CN103665143A (en) * | 2013-12-16 | 2014-03-26 | 北京博恩康生物科技有限公司 | Induced osteogenesis polypeptide, and preparation method and purposes thereof |
Non-Patent Citations (2)
| Title |
|---|
| ATSUHIRO SAITO等: "Repair of 20-mm long rabbit radial bone defects using BMP-derived peptide combined with an α-tricalcium phosphate scaffold", 《JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A》 * |
| 乔琳: "促血管生成功能化自组装多肽的筛选及细胞学评价", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111320682A (en) * | 2020-02-27 | 2020-06-23 | 广州领晟医疗科技有限公司 | Application of polypeptide in preparation of medicine for promoting cartilage repair and/or treating osteoarthritis |
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Effective date of registration: 20210517 Address after: 100048 51 Fucheng Road, Haidian District, Beijing Patentee after: Fourth Medical Center, General Hospital of the Chinese PLA Patentee after: BEIJING AOSITESAI MEDICAL TECHNOLOGY Co.,Ltd. Address before: 100142 zone 1, 3 / F, No. 160, North West Fourth Ring Road, Haidian District, Beijing Patentee before: BEIJING AOSITESAI MEDICAL TECHNOLOGY Co.,Ltd. |