CN106279427A - There is antagonist protein CBD-EphA4LBD and the application of the neuranagenesis Inhibitory molecules of collagen specificity binding ability - Google Patents
There is antagonist protein CBD-EphA4LBD and the application of the neuranagenesis Inhibitory molecules of collagen specificity binding ability Download PDFInfo
- Publication number
- CN106279427A CN106279427A CN201510315850.5A CN201510315850A CN106279427A CN 106279427 A CN106279427 A CN 106279427A CN 201510315850 A CN201510315850 A CN 201510315850A CN 106279427 A CN106279427 A CN 106279427A
- Authority
- CN
- China
- Prior art keywords
- epha4lbd
- sequence
- fusion protein
- cbd
- section
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000008186 Collagen Human genes 0.000 title claims abstract description 38
- 108010035532 Collagen Proteins 0.000 title claims abstract description 38
- 229920001436 collagen Polymers 0.000 title claims abstract description 38
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 30
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 16
- 230000002401 inhibitory effect Effects 0.000 title abstract description 10
- 239000005557 antagonist Substances 0.000 title abstract description 6
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 60
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 60
- 150000001413 amino acids Chemical group 0.000 claims abstract description 10
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 8
- 239000002773 nucleotide Substances 0.000 claims description 19
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- 239000013612 plasmid Substances 0.000 claims description 18
- 208000020431 spinal cord injury Diseases 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 4
- 238000003780 insertion Methods 0.000 claims description 2
- 230000037431 insertion Effects 0.000 claims description 2
- 230000001737 promoting effect Effects 0.000 claims description 2
- 230000009261 transgenic effect Effects 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 210000002569 neuron Anatomy 0.000 abstract description 12
- 102000008763 Neurofilament Proteins Human genes 0.000 abstract description 8
- 108010088373 Neurofilament Proteins Proteins 0.000 abstract description 8
- 210000005044 neurofilament Anatomy 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 30
- 108020004414 DNA Proteins 0.000 description 16
- 241000700159 Rattus Species 0.000 description 13
- 238000000034 method Methods 0.000 description 13
- 239000000843 powder Substances 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 210000000278 spinal cord Anatomy 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 239000007864 aqueous solution Substances 0.000 description 10
- 230000006378 damage Effects 0.000 description 10
- 102000006386 Myelin Proteins Human genes 0.000 description 9
- 108010083674 Myelin Proteins Proteins 0.000 description 9
- 238000011534 incubation Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 230000029087 digestion Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000012460 protein solution Substances 0.000 description 8
- 230000001954 sterilising effect Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 6
- 238000007654 immersion Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 239000012634 fragment Substances 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 5
- 210000002161 motor neuron Anatomy 0.000 description 5
- 230000010355 oscillation Effects 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 210000003205 muscle Anatomy 0.000 description 4
- 210000004126 nerve fiber Anatomy 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 241000254173 Coleoptera Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000001537 neural effect Effects 0.000 description 3
- 239000001103 potassium chloride Substances 0.000 description 3
- 235000011164 potassium chloride Nutrition 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108091029865 Exogenous DNA Proteins 0.000 description 2
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 210000001638 cerebellum Anatomy 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 101800000263 Acidic protein Proteins 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000050554 Eph Family Receptors Human genes 0.000 description 1
- 108091008815 Eph receptors Proteins 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 102000053171 Glial Fibrillary Acidic Human genes 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 description 1
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000002567 autonomic effect Effects 0.000 description 1
- 238000005284 basis set Methods 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229940096422 collagen type i Drugs 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000019628 coolness Nutrition 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 210000002418 meninge Anatomy 0.000 description 1
- 210000005012 myelin Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229940005654 nitrite ion Drugs 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 235000019615 sensations Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses antagonist protein CBD-EphA4LBD and the application of a kind of neuranagenesis Inhibitory molecules with collagen specificity binding ability.The fusion protein that the present invention provides, named fusion protein CBD-EphA4LBD, including CBD section and EphA4LBD section；Described CBD section is made up of from amino terminal the 22nd to 28 amino acids residue sequence in sequence table 2；Described EphA4LBD section is made up of from amino terminal the 40th to 213 amino acids residue sequence in sequence table 2.Fusion protein CBD-EphA4LBD has good collagen specificity binding ability, can promote that neuronal cell neurofilament extends, and has fine repairing effect and good potential applicability in clinical practice.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the neuranagenesis Inhibitory molecules with collagen specificity binding ability
Antagonist protein CBD-EphA4LBD and application.
Background technology
Spinal cord is the important component part of nervus centralis, and major function is to transmit the nerve information between brain and periphery simultaneously
Also it is the low-level center of many simple reflex activities.Spinal cord injury (spinal cord injury, SCI) is more common in one
Among a little natural disasters, such as earthquake and mine disaster etc., send out it addition, the most often have in vehicle accident and some motional injuries
Raw.Generally below impaired joint position, body autonomic movement function and the disappearance of sensation is shown after spinal cord injury.At ridge
After marrow is impaired, damage and the normal spinal cord neuron of remaining be difficult to spontaneous regeneration and enter or cross over damage zone.
Summary of the invention
It is an object of the invention to provide the antagonist protein of a kind of neuranagenesis Inhibitory molecules with collagen specificity binding ability
CBD-EphA4LBD and application.
Present invention firstly provides a kind of fusion protein, named fusion protein CBD-EphA4LBD, including CBD section
With EphA4LBD section；Described CBD section is residual from amino terminal the 22nd to 28 amino acids by sequence in sequence table 2
Basis set one-tenth；Described EphA4LBD section by sequence in sequence table 2 from amino terminal the 40th to 213 amino acids residue
Composition.
In described fusion protein, described CBD section is positioned at the upstream of described EphA4LBD section.
Described fusion protein is concretely following (a) or (b): in (a) sequence table, sequence 2 is from amino terminal the 22nd
Protein to 213 amino acids residue compositions；Protein shown in sequence 2 in (b) sequence table.
The gene of encoding said fusion protein falls within protection scope of the present invention.
In described gene, the DNA molecular encoding described CBD section can be if the sequence 1 of sequence table is from 5 ' ends the 64th
To shown in 84 nucleotide, the DNA molecular encoding described EphA4LBD section can be if the sequence 1 of sequence table is from 5 '
Shown in the 118th to 639 nucleotide of end.
Described gene is concretely following (c) or (d): the sequence 1 of (c) sequence table is from 5 ' ends the 64th to 639
Position DNA molecular shown in nucleotide；The DNA molecular shown in sequence 1 of (d) sequence table.
Expression cassette, recombinant vector, transgenic cell line or recombinant bacterium containing described gene belongs to the protection of the present invention
Scope.
Described recombinant vector can be pET28a (+) sequence 1 of vector multiple cloning site insertion sequence table is from 5 ' ends
The recombiant plasmid that DNA molecular shown in the nucleotide of 64-642 position obtains.Described recombinant vector concretely by pET28a (+)
Small fragment between carrier Nde I and XhoI restriction endonuclease recognition sequence replaces with the sequence 1 of sequence table from 5 ' ends
The recombiant plasmid pET28a-6his-CBD-EphA4LBD that DNA molecular shown in the nucleotide of 64-642 position obtains.
Described recombinant bacterium can be for importing, by recombiant plasmid described in any of the above, the recombinant bacterium that e. coli bl21 (DE3) obtains.
Gene described in fusion protein described in any of the above or any of the above is in preparation promotes the medicine of repair of spinal cord injury
Application falls within protection scope of the present invention.Described promotion repair of spinal cord injury can be to promote that neuronal cell neurofilament is stretched
At length and/or promotion spinal cord injury, nerve fiber increases and/or promotes that at spinal cord injury, 5-HT type motor neuron increases.
The present invention also protects a kind of medicine promoting repair of spinal cord injury, and its active component is following (e) or (f): (e)
Fusion protein described in any of the above；F () is combined with the collagen scaffold of the fusion protein described in any of the above.Described rush
Entering repair of spinal cord injury can be to promote the elongation of neuronal cell neurofilament and/or promote that at spinal cord injury, nerve fiber increases
And/or promote that at spinal cord injury, 5-HT type motor neuron increases.
The collagen scaffold being combined with described fusion protein specifically can be prepared as follows: by collagen scaffold with containing
State the solution incubated at room of fusion protein.The concretely half an hour time of described incubated at room." containing described fusion egg
White solution " concretely dissolve, with PBS, the solution that described fusion protein obtains.Every 20 microlitres are " containing
State the solution of fusion protein " in can be containing fusion protein described in 5 micrograms.
The preparation method of described collagen scaffold can comprise the steps: to take the Corii Bovis seu Bubali skin after peeling off corium,
Immersion treatment 24-72h in the SDS aqueous solution of 1-4g/100ml, then with the Triton X-100 of 1-8% (volume fraction)
Aqueous solution soaking processes 24-72h, then with the Tris-HCl buffer containing 0.5-1.5mol/L NaCl
(25-100mmol/L, pH7.6-8.5) immersion treatment 24-72h, lyophilizing after cleaning with water, it is collagen scaffold.
The preparation method of described collagen scaffold specifically can comprise the steps: to take the Corii Bovis seu Bubali skin after peeling off corium, at 3g/100ml
SDS aqueous solution in immersion treatment 48h, then with at the Triton X-100 aqueous solution soaking of 5% (volume fraction)
Reason 48h, then by Tris-HCl buffer (50mmol/L, pH8.0) immersion treatment 48h containing 1mol/L NaCl,
Lyophilizing after cleaning with deionized water, is collagen scaffold.The preparation method of described collagen scaffold also includes will be prepared into
To collagen scaffold carry out the step of sterilizing.Described sterilizing method particularly includes: Co60Radiation sterilization.
Fusion protein CBD-EphA4LBD has good collagen specificity binding ability, can promote in the presence of having inhibitor
Neuronal cell neurofilament extends.Transplant the rat three months after surgery of the collagen scaffold being combined with CBD-EphA4LBD
Relatively blank group and simple collagen support group can show more preferable nerve fiber and the regeneration of 5-HT type neuron.
Result shows, fusion protein CBD-EphA4LBD has potentiality and the potential applicability in clinical practice well repairing spinal cord injury.
The present invention advantageously accounts for the antagonist protein of spinal cord regeneration Inhibitory molecules and spreads the low activity and excessive risk caused in vivo,
Reparation for spinal cord injury provides new method.
Accompanying drawing explanation
Fig. 1 is the result of embodiment 2.
Fig. 2 is the result of embodiment 3.
Fig. 3 is the schematic diagram performed the operation in embodiment 4 and be administered.
Fig. 4 is nearly rostral in embodiment 4, nearly caudal and the immunofluorescence of damage zone neural thread protein NTP specific markers NF
Coloration result；Scale is 50 μm, and " Hoechst/GFAP/NF " is that " nucleus fluorescent dye Hoechst/ colloid is fine
Dimension acidic protein/neural thread protein NTP.
Fig. 5 is the immunofluorescence dyeing result of damage zone motor neuron specific markers 5-HT in embodiment 4；Scale table
Show 50 μm, " Hoechst/GFAP/5-HT " be " nucleus fluorescent dye Hoechst/ glial fibrillary acidic protein/
Tyrosine hydroxylase ".
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions,
It is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats in fact
Test, results averaged.
PET28a (+) carrier: Novagen company.E. coli bl21 (DE3): Beijing full formula gold biotechnology is limited
Company (article No. CD601-01).Anti-his:sigma (article No. H1029).Anti-mouse IgG (has alkalescence
Phosphatase enzyme mark): sigma (article No. A1418).EphrinB3 (Inhibitory molecules in myelin source): ProSpec company
(article No. PRO-497).PBS in embodiment is the PBS of pH7.4-7.6,0.01M.Derive from
The anti-NF antibody of mice: Zhong Shan Golden Bridge (article No. ZM-0198).Derive from the anti-5-HT antibody of mice:
Immunostar (article No. 20080).
Embodiment 1, the preparation of fusion protein CBD-EphA4LBD
One, the structure of recombiant plasmid pET28a-6his-CBD-EphA4LBD
1, the double chain DNA molecule shown in sequence 1 of composition sequence table.
2, the double chain DNA molecule obtained with step 1 is as template, use the primer of S1 and X1 composition to carrying out PCR amplification,
Obtain pcr amplification product.
S1:5 '-GCGAAGCTTGCGAAGTTACCTTATTGG-3 '；
X1:5 '-CTCCTCGAGTCACTTTTTATAGAACACAC-3′；
3, with restricted enzyme HindIII and the pcr amplification product of XhoI double digestion step 2, digestion products is reclaimed.
4, by pET28a (+) DNA fragmentation between carrier Nde I and BamH I restriction endonuclease recognition sequence replaces with sequence table
Sequence 1, from the DNA fragmentation shown in 5 ' end 64-84 position nucleotide, obtains recombiant plasmid pET28a-6his-CBD.
5, with restricted enzyme HindIII and XhoI double digestion recombiant plasmid pET28a-6his-CBD, about 5kb is reclaimed
Carrier framework.
6, the carrier framework of the digestion products step 5 of step 3 is connected, obtain recombiant plasmid
pET28a-6his-CBD-EphA4LBD.According to sequencing result, to recombiant plasmid pET28a-6his-CBD-EphA4LBD
Carry out structure to be described as follows: by pET28a (+) small fragment between carrier Nde I and XhoI restriction endonuclease recognition sequence replaces
In order to the sequence 1 of sequence table is from the DNA molecular shown in 5 ' end 64-642 position nucleotide.
In recombiant plasmid pET28a-6his-CBD-EphA4LBD, exogenous dna fragment merges with the DNA set out in plasmid,
The sequence 1 of formation sequence table, from the fusion gene CBD-EphA4LBD shown in 5 ' end 1-642 position nucleotide, expresses sequence
The fusion protein CBD-EphA4LBD shown in sequence 2 of list.
In the sequence 2 of sequence table, form HIS from N-terminal the 5th to 10 amino acids residue6Label, the 22nd to 28 bit amino
Acid residue composition collagen binding domain (CBD section), the 40th to 213 amino acids residue composition people EPH receptor
The receptor binding domain (EphA4LBD section) of A4.
In the sequence 1 of sequence table, it is start codon from the 1st to 3 nucleotide of 5 ' end, the 13rd to 30 nucleotide
Coding HIS6Label, the 64th to 84 nucleotide coding CBD section, the 118th to 639 nucleotide coding EphA4LBD district
Section, the 640th to 642 nucleotide is termination codon.
Two, the structure of recombiant plasmid pET28a-6His-EphA4LBD
1, the double chain DNA molecule shown in sequence 3 of composition sequence table.
2, the double chain DNA molecule obtained with step 1 is as template, use the primer of S2 and X1 composition to carrying out PCR amplification,
Obtain pcr amplification product.
S2:5 '-CGCGGATCCGAAGTTACCTTATTGGATT-3 '.
X1:5 '-CTCCTCGAGTCACTTTTTATAGAACACAC-3′；
3, with restricted enzyme BamHI and the pcr amplification product of XhoI double digestion step 2, digestion products is reclaimed.
4, with restricted enzyme BamHI and XhoI double digestion pET28a (+) carrier, reclaim the carrier framework of about 5kb.
5, the digestion products of step 3 and the carrier framework of step 4 are connected, obtain recombiant plasmid
pET28a-6His-EphA4LBD.According to sequencing result, recombiant plasmid pET28a-6His-EphA4LBD is carried out structure
Be described as follows: by pET28a (+) small fragment between carrier B amHI and XhoI restriction endonuclease recognition sequence replaces for sequence
The sequence 3 of table is from the DNA molecular shown in the 103rd to 627 nucleotide of 5 ' end.
In recombiant plasmid pET28a-6His-EphA4LBD, exogenous dna fragment merges with the DNA set out in plasmid, is formed
The sequence 3 of sequence table is from the fusion gene EphA4LBD shown in 5 ' end 1-627 position nucleotide, the sequence of expressed sequence table
Fusion protein EphA4LBD shown in row 4.
Three, the acquisition of recombinant bacterium
Recombiant plasmid pET28a-6his-CBD-EphA4LBD is imported e. coli bl21 (DE3), obtains fungus beetle of recombinating.
Recombiant plasmid pET28a-6His-EphA4LBD will be obtained and import e. coli bl21 (DE3), obtain recombinant bacterium second.
Four, fusion protein CBD-EphA4LBD and the preparation of fusion protein EphA4LBD
1, recombinant bacterium (restructuring fungus beetle or recombinant bacterium second) monoclonal is inoculated in 10ml LB fluid medium, 37 DEG C,
200rpm shaken cultivation 12 hours.
2, after completing step 1, take cultivating system, be seeded to LB fluid medium according to the volume ratio of 1:50,37 DEG C,
200rpm shaken cultivation, to OD600nm=0.8-1, is subsequently adding IPTG (concentration making IPTG in cultivating system is 1mM)
And inducing culture 5 hours (condition of inducing culture: 37 DEG C, 200rpm vibration).
3, after completing step 2, take cultivating system, 4 DEG C, 8000g be centrifuged 10min, collect thalline.
4, ultrasonication (100W supersound process 15min) thalline, then 4 DEG C, 12000g be centrifuged 30min, in collection
Clear liquid.
5, take the supernatant that step 4 obtains, carry out affinity chromatograph, collect target liquid.
The concrete steps of affinity chromatograph: 500 μ L His fillers (GE, article No. 17-5268-01) are loaded in duckpin, use
5ml binding buffer (25mM Tris-HCl, pH8.0,250mM potassium chloride, 5mM imidazoles, 2mM β-sulfydryl
Ethanol) rinse；Then supernatant loading step 4 obtained, abandons effluent；Then with 5ml washing buffer
(25mM Tris-HCl, pH 8.0,250mM potassium chloride, 20mM imidazoles, 2mM beta-mercaptoethanol) rinses pillar,
Abandon effluent；Then with 2.5ml elution buffer (25mM Tris-HCl, pH8.0,250mM potassium chloride, 600mM
Imidazoles, 2mM beta-mercaptoethanol) rinse, collect effluent (being target liquid).
6, the target liquid that step 5 obtains is taken, with desalting column (GE company；Article No. 17-0851-01) by specification takes off
Salt treatment, it is thus achieved that protein solution.
Using restructuring fungus beetle to carry out above-mentioned steps, the protein solution obtained is fusion protein CBD-EphA4LBD solution, cold
Freeze and obtain fusion protein CBD-EphA4LBD dry powder after drying.
Using recombinant bacterium second to carry out above-mentioned steps, the protein solution obtained is fusion protein EphA4LBD solution, freezing dry
Fusion protein EphA4LBD dry powder is obtained after dry.
Embodiment 2, fusion protein and the analysis of collagen binding characteristic
One, collagen dry powder is prepared
1, take the Collagen type-I of SD rat, be placed in neutral salt solution (containing 0.1M Tris, 0.2M NaCl and 0.1M EDTA
Aqueous solution, pH7.6) in, room temperature, 50rpm oscillation incubation 3 hours, then 4 DEG C, 12000g be centrifuged 30 minutes,
Take precipitation.
2, taking the precipitation that step 1 obtains, be dissolved in 0.5M aqueous acetic acid, room temperature stands 12 hours, then 4 DEG C, 13000g
Centrifugal 1 hour, collect supernatant.
3, taking the supernatant that step 2 obtains, mix with the NaCl aqueous solution equal-volume of 4M, room temperature stands 20 minutes, so
Latter 4 DEG C, 13000g be centrifuged 30 minutes, collect precipitation.
4, take the precipitation that step 3 obtains, dissolve with 0.2M acetic acid aqueous solution, dialyse to remove NaCl in water, obtain
Collagen solution.
5, collagen solution lyophilization step 4 obtained, obtains collagen dry powder.
Two, fusion protein and the analysis of collagen binding characteristic
1, take the collagen dry powder that step one obtains, dissolve with PBS, obtain the collagen solution of 100 μ g/ml.
2, taking 96 hole ELISA Plate, add the collagen solution (100 μ l/ hole) that step 1 obtains, 4 DEG C stand 12 hours, abandon
Supernatant, washs 3 times with PBS.
3, after completing step 2, take described ELISA Plate, add the PBS (100 containing 3% (mass percent) BSA
μ l/ hole), 37 DEG C, 60rpm oscillation incubation 1.5 hours, abandon supernatant, wash 4 times with PBS.
4, after completing step 3, take described ELISA Plate, add testing protein solution (100 μ l/ hole), 37 DEG C, 60rpm
Oscillation incubation 2.5 hours, abandons supernatant, washs 4 times with PBS.Testing protein solution is fusion protein
CBD-EphA4LBD solution or fusion protein EphA4LBD solution (use PBS dissolve and dilute CBD-EphA4LBD
Protein dry powder or EphA4LBD protein dry powder), protein concentration is 0.5,1.0,2.0,4.0,6.0,8.0 or 10.0
μM.Using PBS as the negative control of testing protein solution.
5, after completing step 4, taking described ELISA Plate, addition one is anti-, and (anti-his is diluted to 2000 with PBS
Times volume；100 μ l/ holes), 37 DEG C, 60rpm oscillation incubation 1.5 hours, abandon supernatant, wash 4 times with PBS.
6, after completing step 5, taking described ELISA Plate, addition two is anti-, and (anti-mouse IgG, dilutes with PBS
To 10000 times of volumes；100 μ l/ holes), 37 DEG C, 60rpm oscillation incubation 1 hour, abandon supernatant, wash with PBS
Wash 4 times.
7, after completing step 6, take described ELISA Plate, add AP nitrite ion (i.e. 2mg/ml P-NPP solution；80μl/
Hole), lucifuge stands 8.5 minutes, is subsequently adding equal-volume 0.2M NaOH aqueous solution to terminate reaction.
8, after completing step 7, every hole takes 100 μ l supernatant, measures the OD value under 405nm by microplate reader.
Carry out five times repeating test, results averaged.
Result is shown in Fig. 1.Fusion protein CBD-EphA4LBD is significantly higher than fusion protein EphA4LBD with the binding ability of collagen.
Embodiment 3, the Bioactivity detection of fusion protein
One, neuronal cell is prepared
1, take the SD rat of newborn 7 days, soak 2-5 minute in 75% (volume ratio) ethanol water.
2, after completing step 1, rat is placed on super-clean bench, broken end, takes cerebellum, put in the PBS of pre-cooling,
Reject blood vessel and meninges.
3, after completing step 2, take cerebellum, shred and be placed in DMEM height sugar culture fluid, blow and beat gently with dropper until
Tissue agglomerate disappears, and then filters with 400 mesh nylon membranes of sterilizing, then 4 DEG C, 1000rpm be centrifuged 5min, collect thin
Born of the same parents are precipitated, and are cerebellar granule neuron cell (abbreviation neuronal cell).
Two, myelin protein is prepared
1, taking the brain of 4 adult SD rats, in 0.32M aqueous sucrose solution, homogenate is broken, obtains 2-3ml tissue fluid.
2, take 12ml ultracentrifugation pipe, be initially charged 6ml 0.85M aqueous sucrose solution, be subsequently adding the tissue that step 1 obtains
Liquid (2-3ml), is subsequently adding 0.32M aqueous sucrose solution full to centrifuge tube, then 4 DEG C, 75000g be centrifuged 45min, from
It is divided into three layers in heart pipe, takes intermediate layer, be crude extract.
3, take the crude extract that step 2 obtains, wash twice with the water of 4 DEG C of pre-coolings that (each washing methods is as follows: after adding water
Stand half an hour, then 4 DEG C, 12000g be centrifuged 30 minutes, abandon supernatant).
4, taking 12ml ultracentrifugation pipe, be initially charged 6ml 0.85M aqueous sucrose solution, be subsequently adding that step 3 obtains slightly carries
Thing, is subsequently adding 0.32M aqueous sucrose solution full to centrifuge tube, then 4 DEG C, 75000g be centrifuged 45min, in centrifuge tube point
Become three layers, take intermediate layer system.
5, intermediate layer system step 4 obtained mixes with 3ml water, with 0.2 micron membrane filter filtration sterilization, collects filtrate
And carry out lyophilization, be myelin protein dry powder, be placed in-80 DEG C standby.
Three, the Bioactivity detection of fusion protein
1, taking myelin protein dry powder, be dissolved in PBS, (every 10 microlitre myelin proteins are molten to obtain myelin protein solution
Containing 200ng myelin protein dry powder in liquid).
2, take ephrinB3, be dissolved in PBS, obtain ephrinB3 solution (in every 10 microlitre ephrinB3 solution
Containing 20ng ephrinB3).
3, taking 48 orifice plates, the 1st to 15 every hole, hole adds the myelin protein solution that 10 microlitre steps 1 obtain, 16-30 hole
Every hole adds the ephrinB3 solution that 10 microlitre steps 2 obtain, and it is (right that every hole, 31-45 hole adds 10 microlitre PBS
According to first), dry up.
4, after completing step 3, described 48 orifice plates, the neuronal cell (20000-30000 of inoculation step one preparation are taken
Individual cells/well), it is subsequently adding the DMEM high glucose medium containing 10% (volume ratio) hyclone.
5, after completing step 4, taking described 48 orifice plates, 1-5 hole, 16-20 hole, every hole, 31-35 hole add 5 microlitres and melt
Hop protein CBD-EphA4LBD solution (fusion protein CBD-EphA4LBD concentration in system is 20nM), 6-10
Hole, 21-25 hole, every hole, 36-40 hole add 5 microlitre fusion protein EphA4LBD solution, and (fusion protein EphA4LBD is at body
Concentration in system is 20nM), 11-15 hole, 26-30 hole, every hole, 41-45 hole add the (comparison of 5 microlitre PBS
Second), 37 DEG C stand 24 hours.Fusion protein CBD-EphA4LBD solution is by molten for fusion protein CBD-EphA4LBD dry powder
Obtain in PBS.Fusion protein EphA4LBD solution is that fusion protein EphA4LBD dry powder is dissolved in PBS buffering
Liquid obtains.
6, after completing step 5, the elongation situation of detection neurofilament.
Neurofilament immunofluorescence dyeing step is as follows: the culture medium in (1) exhaustion hole, with PBS washed cell 1-2
Secondary；(2) adding 4% paraformaldehyde solution, room temperature stands 20min, then washs 3 times with PBS；(3) add
Hyclone, room temperature stands 1 hour, then washs 1 time with PBS；(4) adding one, anti-(one resists for anti-β III
Tubulin, purchased from Millipore company, article No. 05-559,1:500 dilution during use), 4 DEG C of stationary incubation 12 hours,
Then wash 3 times with PBS；(5) adding fluorescence two, anti-(fluorescence two is anti-purchased from invitrogen company；Alexa488Donkey Anti-Mouse IgG (H+L), CA21202s), 37 DEG C of stationary incubation 40min, then
Wash 3 times with PBS；(6) add concentration 50 μ g/ml Hoechst 33342 (purchased from sigma company) to carry out
Core lining dye 10min, then washs 3 times with PBS；(7) basis of microscopic observation, photograph.
Result is shown in Fig. 2.Myelin protein and two kinds of inhibitive factor of ephrinB3 all can be stretched with the neurofilament of inhibitory neuron cell
Long.In the case of there is above-mentioned inhibitive factor, fusion protein CBD-EphA4LBD and fusion protein EphA4LBD
To promote the elongation of neuronal cell neurofilament, and the activity of fusion protein CBD-EphA4LBD is higher than fusion protein
EphA4LBD。
Embodiment 4, transplanting are combined with the collagen scaffold treatment complete cross-section damage of rat spinal cord T8 of fusion protein CBD-EphA4LBD
Wound
One, collagen scaffold is prepared
1, taking the Corii Bovis seu Bubali skin after peeling off corium, immersion treatment 48h in the SDS aqueous solution of 3g/100ml, then with 5%
The Triton X-100 aqueous solution soaking of (volume fraction) processes 48h, then with the Tris-HCl containing 1mol/L NaCl
Buffer (50mmol/L, pH8.0) immersion treatment 48h, lyophilizing after cleaning with deionized water, it is collagen scaffold.
2, collagen scaffold is cut into the suitable size of spinal cord section (a length of 2-3mm, a diameter of 1.5-2.5mm) Co afterwards60According to
Penetrate sterilizing.
Two, acute rat spinal cord T8 damage model is made
1, preparing body weight is the male adult SD rats of 250 ± 25g, lumbar injection 3% pentobarbital sodium (30-40mg/kg)
Anaesthetize.
2, after completing step 1, taking ventricumbent position, lower back loses hair or feathers, and the skin outside T9-T11 spinal column and skin are cut in sterilization
Under, separate the other muscle of bilateral spinous process, pull open the other muscle of spinous process with drag hook, expose T10 spinous process and vertebral plate.
3, after completing step 2, sting except spinous process with small size needle holder, then excise vertebral plate, gently mention with microforceps sudden and violent
The myeloid tissue of dew, then cuts off the myeloid tissue of T10 sections with microscissors, and the spinal cord after detachment is spaced about 2-3mm,
In art, available microscissors makees the most horizontal rotary-cut, to confirm complete detachment.
Three, packet transaction
After completing step 2, SD rat is divided into three groups (often groups 15), processes as follows respectively:
First group: muscle and skin are sewed up respectively, i.e. complete operation；
Second group: transplanting collagen scaffold after PBS incubated at room half an hour at Spinal Cord Defect, then by flesh
Meat and skin are sewed up respectively, i.e. complete operation；
3rd group: at Spinal Cord Defect, transplanting fusion protein CBD-EphA4LBD solution (dissolves and dilute with PBS
Release fusion protein CBD-EphA4LBD dry powder, containing 5 microgram fusion protein CBD-EphA4LBD in every 20 Al of Solution) room
Temperature hatches the collagen scaffold after half an hour, then muscle and skin is sewed up respectively, i.e. completes operation；
Fig. 3 is shown in by operation and the schematic diagram being administered.
Being put to death by rat after performing the operation 3 months, taken out by spinal cord rapidly, first in 4% paraformaldehyde solution, 4 DEG C of standings are solid
Fixed 48 hours, then in 30% aqueous sucrose solution, 4 DEG C of standings were dehydrated 12 hours, then use the embedding of OCT embedding medium
Carrying out frozen section after tissue, slice thickness is 10 μm.
Section statining: (1) takes section, washs one time with PBS, then closes 30min with lowlenthal serum, inhales
Abandon lowlenthal serum, wash twice with PBS；(2) add that 200 μ L mono-are anti-(derives from the anti-NF of mice
Antibody or derive from the anti-5-HT antibody of mice, dilution ratio is 1:500), make liquid that spinal cord group is completely covered
Knit, 4 DEG C of stationary incubation 12 hours, wash three times with PBS；(3) anti-(invitrogen of fluorescence two is added
Company, Alexa488Donkey Anti-Mouse IgG (H+L), CA21202s；According to two anti-explanations
Book dilutes), room temperature stationary incubation 1 hour, washs twice with PBS；(4) by Hoechst (1:800) dye
Core 10min, washs twice with PBS, and lucifuge is dried naturally；(5) dropping mountant, seals up coverslip, aobvious
Micro-Microscopic observation, photograph.
Quantitative result takes the meansigma methods of this group rat.
The immunofluorescence dyeing result of nearly rostral, nearly caudal and damage zone neural thread protein NTP specific markers NF is shown in Fig. 4.
The immunofluorescence dyeing result of damage zone motor neuron specific markers 5-HT is shown in Fig. 5.
Result shows, the rat having transplanted the collagen scaffold being combined with fusion protein CBD-EphA4LBD is grown in damage zone
Nerve fiber and the quantity of 5-HT type motor neuron be all remarkably higher than the rat transplanting simple collagen support.
Claims (10)
1. a fusion protein, including CBD section and EphA4LBD section；Described CBD section is by sequence in sequence table
Row 2 form from amino terminal the 22nd to 28 amino acids residue；Described EphA4LBD section is by sequence 2 in sequence table
Form from amino terminal the 40th to 213 amino acids residue.
2. fusion protein as claimed in claim 1, it is characterised in that: in described fusion protein, described CBD section
It is positioned at the upstream of described EphA4LBD section.
3. fusion protein as claimed in claim 2, it is characterised in that: described fusion protein is following (a) or (b):
A protein that in () sequence table, sequence 2 forms from amino terminal the 22nd to 213 amino acids residue；
Protein shown in sequence 2 in (b) sequence table.
4. the gene of fusion protein described in coding claim 1 or 2 or 3.
5. gene as claimed in claim 4, it is characterised in that: in described gene, encode described CBD section
DNA molecular, if the sequence 1 of sequence table is from shown in the 64th to 84 nucleotide of 5 ' end, encodes described EphA4LBD
The DNA molecular of section is if the sequence 1 of sequence table is from shown in the 118th to 639 nucleotide of 5 ' end.
6. gene as claimed in claim 5, it is characterised in that: described gene is following (c) or (d):
C the sequence 1 of () sequence table is from the DNA molecular shown in the 64th to 639 nucleotide of 5 ' end；
The DNA molecular shown in sequence 1 of (d) sequence table.
7. contain the expression cassette of gene, recombinant vector, transgenic cell line or restructuring described in claim 4 or 5 or 6
Bacterium.
8. recombinant vector as claimed in claim 7, it is characterised in that: described recombinant vector be pET28a (+) load
The sequence 1 of body multiple clone site insertion sequence table obtains from the DNA molecular shown in 5 ' end 64-642 position nucleotide
Recombiant plasmid.
9. the fusion protein described in claim 1 or 2 or 3, or, gene described in claim 4 or 5 or 6,
Preparation promotes the application in the medicine of repair of spinal cord injury.
10. promoting a medicine for repair of spinal cord injury, its active component is following (e) or (f):
Fusion protein described in (e) claim 1 or 2 or 3；
F () is combined with the collagen scaffold of the fusion protein described in claim 1 or 2 or 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510315850.5A CN106279427A (en) | 2015-06-10 | 2015-06-10 | There is antagonist protein CBD-EphA4LBD and the application of the neuranagenesis Inhibitory molecules of collagen specificity binding ability |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510315850.5A CN106279427A (en) | 2015-06-10 | 2015-06-10 | There is antagonist protein CBD-EphA4LBD and the application of the neuranagenesis Inhibitory molecules of collagen specificity binding ability |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106279427A true CN106279427A (en) | 2017-01-04 |
Family
ID=57659681
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510315850.5A Pending CN106279427A (en) | 2015-06-10 | 2015-06-10 | There is antagonist protein CBD-EphA4LBD and the application of the neuranagenesis Inhibitory molecules of collagen specificity binding ability |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106279427A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109081872A (en) * | 2017-06-13 | 2018-12-25 | 中国科学院遗传与发育生物学研究所 | A kind of regenerated function collagen biomaterial of induction eardrum and preparation method thereof |
| CN110787321A (en) * | 2018-08-01 | 2020-02-14 | 中国科学院遗传与发育生物学研究所 | Application of functional collagen scaffold LOCS + CBD-NT3 in repairing spinal cord injury |
| CN111978405A (en) * | 2019-05-21 | 2020-11-24 | 中国科学院苏州纳米技术与纳米仿生研究所 | Functional polypeptide, erythrocyte drug-carrying system capable of specifically binding collagen and application thereof |
| CN113121700A (en) * | 2019-12-30 | 2021-07-16 | 中国科学院苏州纳米技术与纳米仿生研究所 | Recombinant protein specifically combined with collagen, encoding gene and application thereof |
-
2015
- 2015-06-10 CN CN201510315850.5A patent/CN106279427A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| JUAN FAN ET AL: "Linear Ordered Collagen Scaffolds Loaded with Collagen-Binding Neurotrophin-3 Promote Axonal Regeneration and Partial Functional Recovery after Complete Spinal Cord Transection", 《JOURNAL OF NEUROTRAUMA》 * |
| QIN,H.ET AL: "2LW8_A", 《PDB》 * |
| XING LI: "Functionalized Collagen Scaffold Neutralizing the Myelin-Inhibitory Molecules Promoted Neurites Outgrowth in Vitro and Facilitated Spinal Cord Regeneration in Vivo", 《ACS APPL. MATER. INTERFACES》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109081872A (en) * | 2017-06-13 | 2018-12-25 | 中国科学院遗传与发育生物学研究所 | A kind of regenerated function collagen biomaterial of induction eardrum and preparation method thereof |
| CN110787321A (en) * | 2018-08-01 | 2020-02-14 | 中国科学院遗传与发育生物学研究所 | Application of functional collagen scaffold LOCS + CBD-NT3 in repairing spinal cord injury |
| CN111978405A (en) * | 2019-05-21 | 2020-11-24 | 中国科学院苏州纳米技术与纳米仿生研究所 | Functional polypeptide, erythrocyte drug-carrying system capable of specifically binding collagen and application thereof |
| CN111978405B (en) * | 2019-05-21 | 2023-01-03 | 中国科学院苏州纳米技术与纳米仿生研究所 | Functional polypeptide, erythrocyte drug-carrying system capable of specifically binding collagen and application thereof |
| CN113121700A (en) * | 2019-12-30 | 2021-07-16 | 中国科学院苏州纳米技术与纳米仿生研究所 | Recombinant protein specifically combined with collagen, encoding gene and application thereof |
| CN113121700B (en) * | 2019-12-30 | 2023-06-13 | 独步吾奇生物医疗科技(江苏)有限公司 | Recombinant protein specifically combined with collagen, encoding gene and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11879002B2 (en) | Bi-specific therapeutic proteins, in vivo methods of use thereof and encoding nucleic acids thereof | |
| RU2624036C2 (en) | Structures of fusion protein in spider silk for binding to organic target | |
| CN106279427A (en) | There is antagonist protein CBD-EphA4LBD and the application of the neuranagenesis Inhibitory molecules of collagen specificity binding ability | |
| JPH03501261A (en) | cellulose binding fusion protein | |
| CN111620952A (en) | Novel coronavirus vaccine based on chimeric virus-like particles | |
| JP2024023297A (en) | Solubilized apyrase, methods and use | |
| CN101516907A (en) | Muteins of tear lipocalin and methods for obtaining the same | |
| CN108753730A (en) | The umbilical cord mesenchymal stem cells and its construction method of a kind of NTF4 genetic modifications and application | |
| CN106279428A (en) | There is antagonist protein CBD-PlexinB1LBD and the application of the neuranagenesis Inhibitory molecules of collagen specificity binding ability | |
| WO2013170408A1 (en) | Tat ptd-endostatin recombinant protein, preparation method and application thereof | |
| CN110716035A (en) | Echinococcosis-resistant high-throughput drug screening method based on echinococcosis tubulin as target spot | |
| CN101870974A (en) | Preparation method of proteinase activated receptors agonist and use thereof | |
| WO2018024153A1 (en) | Preparation method and use of recombinant swine fever e2 protein and subunit vaccine thereof | |
| CN108379240A (en) | Anti- EGFR scFv::The application of FTH1/FTH1 protein nanos particle in medicine preparation | |
| CN104497118B (en) | A kind of insect protein Hig and its encoding gene and application | |
| CN105218683A (en) | A kind of Tat PTD-Endostatin-RGD recombinant protein and preparation method thereof and application | |
| CN106913864A (en) | The new application of fusion protein TAT DCF1 | |
| CN110183536B (en) | Composite stent material and application thereof | |
| KR102602561B1 (en) | Biosensor based on lateral flow analysis for 2019 novel coronavirus neutralizing antibody detection | |
| CN101591640B (en) | Method for inducing somatic cell to reprogram by penetrating stem cell transcription factor | |
| CN113354735B (en) | Specific Dectin-1 nano antibody and preparation method and application thereof | |
| CN103911388B (en) | Recombinate the production technology of Exenatide | |
| WO2022179556A1 (en) | Preparation method for modified toxin polypeptide | |
| Kheradmand-Hajibashi | Effect of Cargo Location on Translocation of Cell-Penetrating Peptide Fusion Proteins into Candida albicans Cells | |
| CN105418767A (en) | Fusion protein for preparing Abeta (Amyloidbeta-peptide) epitope vaccines, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180608 Address after: 101407 01-617, 6 floor, 13 building, No. 53 Yan Qi street, Yan Qi Economic Development Zone, Huairou, Beijing. Applicant after: Beijing Zhongke kunkang Biotechnology Co., Ltd. Address before: 100190 South Zhongguancun, Haidian District, Haidian District, Beijing Applicant before: Inst. of Genetics & Development Biology, Chinese Academy of Sciences |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170104 |
|
| RJ01 | Rejection of invention patent application after publication |