CN108285490A - A kind of the gst fusion protein preparation and its application of high-affinity combination I-type collagen - Google Patents

A kind of the gst fusion protein preparation and its application of high-affinity combination I-type collagen Download PDF

Info

Publication number
CN108285490A
CN108285490A CN201810075693.9A CN201810075693A CN108285490A CN 108285490 A CN108285490 A CN 108285490A CN 201810075693 A CN201810075693 A CN 201810075693A CN 108285490 A CN108285490 A CN 108285490A
Authority
CN
China
Prior art keywords
gst
fusion protein
pbs
plgf
solution
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
Application number
CN201810075693.9A
Other languages
Chinese (zh)
Inventor
李遂焰
董育红
陈渝萍
孙源
刘佳
刘一佳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Southwest Jiaotong University
Original Assignee
Southwest Jiaotong University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Southwest Jiaotong University filed Critical Southwest Jiaotong University
Priority to CN201810075693.9A priority Critical patent/CN108285490A/en
Publication of CN108285490A publication Critical patent/CN108285490A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01018—Glutathione transferase (2.5.1.18)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/9116—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • G01N2333/91165—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1)
    • G01N2333/91171—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1) with definite EC number (2.5.1.-)
    • G01N2333/91177—Glutathione transferases (2.5.1.18)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Toxicology (AREA)
  • Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

It prepares and its applies the invention discloses a kind of gst fusion protein of high-affinity combination I-type collagen.The albumen carries the PlGF 2 that can be combined with I-type collagen high-affinity at the ends C of GST‑123‑144Small peptide.The high-affinity to Type I collagen albumen is shown in ELISA detections；The fusion protein can assign high-affinity of the small peptide to Type I collagen albumen of the ends C connection.The protein-specific is strong, molecular weight is small, easily prepared, can be used for being transformed a variety of anti-fibrosis medicines, with improve its in the enrichment and delay at fibrosis position, to extend its action time.

Description

A kind of the gst fusion protein preparation and its application of high-affinity combination I-type collagen
Technical field
The invention belongs to biomedical engineering technology field more particularly to a kind of GST of high-affinity combination I-type collagen Fusion protein prepares and its application.
Background technology
Fibrotic disease is almost related to clinical departments, including systemic sclerosis, idiopathic pulmonary fibrosis, hepatic sclerosis, progress Property nephrosis, myelofibrosis etc., have seriously threatened health of people.It is mainly characterized by extracellular matrix protein (ECM) especially I The over-deposit of collagen type.With liver fibrosis, pulmonary fibrosis and kidney fibrosis patient in current all kinds of fibrotic diseases It mostly and is not easy to treat, last progression of the disease crisis patients ' lives, especially pulmonary fibrosis are because its special structure medicament is in lesion hardly possible Into, it is difficult be detained more become disease particularly difficult to treat in all kinds of fibrotic diseases.Have all kinds of trials at present：TGF β are anti- Body, TGF beta antagonists, colchicin, taxol etc. all show certain therapeutic effect, and IFN-γ is because with adjusting macrophage Cell and Fibroblast Function and as inhibit fibrosis strong candidate drug.But because drug lacks targeting, in lesion The position residence time is short so that curative effect is unsatisfactory.After personal injury would generally self-regeneration damaged tissues, this process be by What a kind of protein being referred to as growth factor was controlled.Intracellular growth factor can avoid losing blood with accelerating wound healing Excessive and complication, it is also critically important to embryonic development.Growth factor is then by polymerizeing a kind of entitled " extracellular matrix (ECM) " Protein promote the growth of body tissue cell.Extracellular matrix cans be compared to " frame " of body tissue, it is generally the case that raw The ability of long factor polymerization extracellular matrix is stronger, and wound healing must be faster.2017, Lausanne federal Institute of Technology taught Jie Fu In breathe out Bell leader 25 kinds of growth factors of research group pair carried out zoopery, as a result, it has been found that, " PIGF-2 " growth because The polymerizing power of sub- extracellular matrix is most strong, promotes the efficiency highest of histoorgan reparation.The present invention then according to presently, there are The problem of and current research, finding can amino acid 1 23-144 knot in the Human plactnta growth factor-2 of high affine combination ECM protein Structure domain (PlGF-2123-144), by a large amount of Software for Design, successful design can in protokaryon bacterium high expression, bacteria lysis Largely it is present in the gene order of the gst fusion protein of supernatant afterwards, especially find out energy high efficient expression and purifies this solvable The experiment condition of albumen.Further, by literature survey, it is found that bicyclic beta platelet derived growth factor receptor identifies peptide (BiPPB) β platelet derived growth factor receptors can specifically be identified, the polypeptide is then connected to gst fusion protein The ends C-, and detection fusion albumen is used to be transformed the feasibility of other albumen, polypeptide etc..The experiment proved that working as PlGF-2123-144It is short After Peptide C-end connects other small peptides (BiPPB), still maintain to I-type collagen height parent's property.It is expected that the fusion protein is available In transformation anti-fibrosis medicine to improve its enrichment and delay at fibrosis position, to extend drug treating time, improve Curative effect of medication.
Invention content
The object of the present invention is to provide a kind of extracellular matrix ECM especially I-type collagens (Collagen I) to have The gst fusion protein of high affine combination includes the PlGF- of the gst fusion protein of the high affine combination I-type collagen of coding 2123-144DNA sequence dna；It is a further object to provide a kind of GST of high affine combination I-type collagen to merge egg White preparation method and applications.
Realize that the technical solution of the object of the invention is as follows：A kind of GST fusion eggs of high-affinity combination I-type collagen In vain, amino acid sequence such as SEQ ID NO：Shown in 1.The expressed sequence of the gst fusion protein, gene order is such as SEQ ID NO：Shown in 2.
The further gst fusion protein, the fusion protein carry Human plactnta growth factor-2 at the ends C- of GST Short peptide stretch PlGF-2123-144, combined with mediated fusion albumen and I-type collagen high-affinity.
The further gst fusion protein, described carries PlGF-2123-144The fusion protein of small peptide refers to GST- PlGF-2123-144Recombinant plasmid obtains after most suitable induced expression, purification condition.
Further above-mentioned gst fusion protein, described most suitable induced expression condition be for IPTG to final concentration 1mM, 30 DEG C, 6h.
Further above-mentioned gst fusion protein GST-PlGF-2123-144To the ELISA of Type I collagen albumen compatibility Detection method is as follows：
ELISA experiment flows are as follows：
1, I-type collagen Collagen I combines the preparation of liquid
(1) storing liquid 0.1% (w/v) acetic acid of 0.1M is diluted 10 times, 0.01% protein solution is made.
(2) it is carefully added into the chloroform for being equivalent to protein solution 10% in vial, not rock or vibrate, in 4 DEG C of ice Case is stood overnight, and collects the solution that albumen is contained on upper layer.
2, ELISA is tested
(1) elisa plate combines liquid to cover with 100 μ l/ hole Collagen I, and 4 DEG C overnight, suck supernatant, are dried overnight.
(2) 37 DEG C of closing 2h of PBS-T of 2% skim milk of 200 μ l are added per hole.PBS-T solution：PBS+0.05% Tween-20。
(3) solution is sucked, is rinsed 3 times with PBS-T per hole
(4) GST and GST-PlGF-2 are separately added into the solution of PBS-T+0.1% skim milks123-144Albumen makes it Final concentration is followed successively by 0.5 μm of ol, 3 μm of ol, 6 μm of ol, per 100 μ l of hole, 37 DEG C of incubation 2h.
(5) PBS-T is rinsed 3 times, 200 μ l every time.
(6) 2% milk powder (PBS-T) that 200 μ l are added per hole closes 45min in 37 DEG C.
(7) it is rinsed 1 time with 200 μ l of PBS-T per hole.
(8) primary antibody 1 that 100 μ l have diluted is added:5000 (2% milk powder (PBS-T)), 4 DEG C of overnight incubations.
(9) 200 μ l of PBS-T are rinsed 3 times, each 3min.
(10) secondary antibody (1 that 100 μ l have diluted is added:2000WB, 2% (5%) milk powder (PBS-T)), room temperature 1h.
(11) 200 μ l PBS-T are rinsed 3 times, each 3min.
(12) 0.3% H2O2100 μ l of methanol solution close 15min.
(13) substrate developing solution A, developing solution B are added per hole, each 50 μ L (A, B liquid are first mixed and added in hole) are gently mixed It is even, it is incubated 30 minutes at 37 DEG C.
100 μ l 2M H are added per hole2SO4Color development stopping, the interior OD values that each hole solution is measured at 450nm wavelength of 15min.
A kind of application of the gst fusion protein of high-affinity combination I-type collagen is coupled other short with the fusion protein Peptide or the protein active factor can assign high parent property of the active factors to I-type collagen.
The fusion protein can be used for largely preparing PIGF-2122-224Functional oligopeptides and then each with functional oligopeptides transformation Class anti-fibrosis medicine, improve drug in the enrichment and delay at fibrosis position, to extend effect of the drug in diseased region Time.
The PIGF-2122-224Functional oligopeptides are carried out with the functional oligopeptides and other label proteins or label small peptide Fusion, can be used for the preparation of other expression systems and other function products.
Advantages of the present invention is with advantageous effect：
1, fusion protein of the present invention can assign the small peptide that the ends C- connect and still maintain affine to the height of Type I collagen albumen Property, the protein-specific is strong, molecular weight is small, easily prepared, can be used for being transformed a variety of anti-fibrosis medicines, to improve it in fiber Change position enrichment and delay, to extend its action time.
2, the DNA encoding sequence of fusion protein of the invention can be used as in the other transformation anti-fibrosis protein factors of structure Prokaryotic expression plasmid prepares all kinds of fibrosis medicines that curative effect is strengthened.
3, fusion protein GST-PlGF-2 of the invention123-144It can be used as to other albumen or even in other anti-fibrosis medicines Object is transformed, with raising they fibrosis position enrichment and delay, extend its action time, finally greatly improve drug Therapeutic effect.
4, with the fusion protein GST-PlGF-2 of acquisition123-144-BiPPB is follow-up further investigation and other genes or egg White interaction is laid a good foundation.
Description of the drawings
Fig. 1 is pMD19-PlGF-2 in invention123-144Plasmid enzyme restriction qualification result.
Fig. 2 is pGEX4T1-PlGF-2 in invention123-144Bacterium colony PCR qualification results.
Fig. 3 is pGEX4T1-PlGF-2 in invention123-144- BiPPB bacterium colony PCR qualification results.
Fig. 4 is GST, GST-PlGF-2 in invention123-144And GST-PlGF-2123-144- BiPPB albumen is in Escherichia coli The SDS-PAGE of induced expression is analyzed and the Western Blot analyses of GST labels.Wherein Fig. 4-1-1 and Fig. 4-1-2 are GST eggs White inductive condition is groped as a result, Fig. 4-2-1 and Fig. 4-2-2 are the protein induced conditions of GST-CBPP gropes as a result, Fig. 4-3 is GST- PlGF-2123-144The protein induced conditions of-BiPPB grope result.
Fig. 5 is GST, the GST-PlGF-2 purified in invention123-144And GST-PlGF-2123-144The SDS-PAGE of-BiPPB Analysis and the Western Blot analyses of GST labels.Wherein Fig. 5-1-1 and Fig. 5-1-2 are GST protein purifications as a result, Fig. 5-2-1 It is GST-PlGF-2 with Fig. 5-2-2123-144Protein purification is as a result, Fig. 5-3-1 and Fig. 5-3-2 are GST-PlGF-2123-144-BiPPB Protein purification result.
Fig. 6 is GST-PlGF-2 in invention123-144To Type I collagen albumen compatibility elisa assay.Wherein Fig. 6-1 is behaviour Make flow chart, Fig. 6-2 is ELISA data and result.
Fig. 7 is GST-PlGF-2 in invention123-144- BiPPB is to Type I collagen albumen compatibility ELISA data and analysis.
Specific implementation mode
The method that the present invention utilizes molecular biology, by 23 to 144 peptide of amino acid 1 of placenta growth factor -2 (PlGF-2) Section be connected to can on the prokaryotic vector of expressed fusion protein, in Escherichia coli expression and purity go out protein molecule GST, GST-PlGF-2123-144And GST-PlGF-2123-144-BiPPB.By experiment test, fusion protein has to Type I collagen albumen High affine activity, and work as PlGF-2123-144The ends small peptide C- connect other small peptides (BiPPB), GST-PlGF-2123-144- BiPPB still maintains high parent's property to I-type collagen.
Method for transformation used in the present invention is heat-shock transformed method, filters out positive bacterium colony by resistance marker, then leads to again The method for crossing bacterium solution PCR is identified to successful conversion positive bacterium colony.By sequencing and alignment, determines and carry amino acid encoding The positive bacteria of the correct fusion protein expression plasmid of sequence, is expressed.
In the fusion protein of the present invention, isopropylthiogalactoside (IPTG) is used as described in derivant progress The induced expression of albumen, while also expressing GST and GST-PlGF-2123-144- BiPPB albumen.In the induction table for carrying out albumen In reaching, we grope different condition, have found the highest condition of each albumen expression quantity in supernatant and carry out Great expression.
The fusion protein of the present invention carries GST label proteins, by the fine jade for being crosslinked with glutathione ligand when isolating and purifying Lipolysaccharide captures the purpose gst fusion protein with ligand binding；Reduced glutathione is then added to carry out purpose fusion protein Elution, successfully completes isolating and purifying for destination protein.
With reference to specific embodiment, the present invention will be further described.
Embodiment 1：pMD19-PlGF-2123-144Plasmid construction, PlGF-2123-144Segment is obtained using primer dimer mode , it is connected on carrier pMD19.
(1) PlGF-2 is designed123-144Primer.Primer numbers：1 and 2.
PlGF-2123-144Primer carries out PCR, PCR kit (TIANGEN), reaction system：50 μ l systems, ddH2O 32.2 μl；10xExTaqBuffer 5μl；10mMdNTP 1μl；Primer(F+R)8μlMgCl2/25mm 3μl；0.8 μ l of Taq enzyme. PCR response parameters：94℃3min；94℃30s；65 DEG C of 30s, after 35 recycle.72℃10min.
Connection, uses pMD19-T connection kits (TAKARA), junctor system：1 μ l of PCR product；T-Vector 1μl； ddH2O 3μl；Solution- I 5 μ l, 16 DEG C of reaction 30min.
Linked system：1 μ l of PCR product；T-Vector 1μl；ddH2O 3μl；I 5 μ l of Solution-, 16 DEG C of reactions 30min。
(4) connection product is converted, taking the connection product of 7 μ l, (it is new that section is held up in Beijing with 50 μ l DH5 α competent bacterias Industry Bioisystech Co., Ltd) mixing, 30min is placed on ice, and 42 DEG C of water-bath 70s place 1min on ice.100 μ l SOC are added Culture medium.37 DEG C of 300rpm cultures 30min on shaking table.The bacterium solution after 75 μ l cultures is taken to drop in Amp+Solid LB media On (X-gal (20%) 40ul, IPTG (2%) 4ul), coating is uniform, and 37 DEG C of constant temperature incubations are stayed overnight.
(5) identification of conversion results：The positive bacterium colony of white grown after conversion is risen with sterile lancet choicest be put into added with A certain amount of Amp+In culture medium, overnight, with reference to plasmid extraction specification, (Beijing village ally border biological gene science and technology has for 37 DEG C of shakings Limit company), part bacterium solution plasmid is extracted, plasmid molecule amount size is identified in digestion.Positive bacterium solution remainder send company to be surveyed Sequence.Sequencing result correct plasmid, structure are completed.
(6) A of attached drawing 1 is pMD19-PlGF-2123-144Plasmid enzyme restriction result.What wherein M electrophoresis band represented is TAKARA 1000bp marker, 1 swimming band represent positive plasmid single endonuclease digestion as a result, 1' represents the non-digestion result of positive plasmid.
Embodiment 2：pGEX4T1-PlGF-2123-144Plasmid construction.
(1) primer of PCR amplification is designed.Primer numbers：Segment prepares primer 3 and 4；Bacterium colony PCR primer 5 and 6.
(2) plasmid containing target fragment carries out PCR, with pMD19-PlGF-2123-144Target fragment is carried out for masterplate PCR amplification, PCR kit (NEB), segment reaction system：50 μ l systems, ddH2O 33μl；5×Q5buffer 10μl； 10mMdNTP 1μl；5 μ l, Q5 enzymes of Primer (F+R), 0.5 μ l；0.5 μ l of template.PCR response parameters：98℃30s；98℃8s；65 DEG C 15s, 35 cycles.72℃2min.After the completion of PCR clone PCRs, 2 μ l PCR products are taken, carry out 2% Ago-Gel electricity Swimming detection, detection amplification situation.
(3) after electrophoresis determines that stripe size is correct, precipitation recycling is carried out.Operation is as follows：50 μ l products；+ 125 anhydrous second of μ l Alcohol；+ 10 μ l (3M pH5.2) sodium acetate.After mixing, it stays overnight for -80 DEG C.12,000g×15min.Supernatant is abandoned, 1ml is added 75% ethyl alcohol shakes mixing, and supernatant is abandoned in 12,000g × 10min centrifugations, and room temperature is dried, and 60 μ l ddH are added2O is re-dissolved DNA, after with UV detector measure DNA concentration.
(4) pGEX-4T-1 is prepared with reference to plasmid extraction specification (border biological gene Science and Technology Ltd. of Beijing village ally), Digestion, which is carried out, using EcoRI and XhoI prepares linearized vector.The PCR fragment recycled above is carried out using EcoRI and XhoI Digestion prepares PlGF-2123-144DNA is embedded in segment.
(5) after the completion of digestion, glue recycling is carried out.Plasmid enzyme restriction product returns in the UV lamp into row agarose gel electrophoresis Digestion carrier is received, glue recycling is carried out with kit.PlGF-2123-144PAGE electrophoresis, electrophoresis knot are carried out after-BiPPB segment digestions EB is carried out after beam and dyes 10min, is pressed clip size in the UV lamp and is recycled target fragment.It is transferred in tubule, 2 times of gels is added Solvent soln (3.85g ammonium acetates, 0.215g magnesium acetates, the 0.2ml 0.5M EDTA (pH=8.0), 1.0ml10% of volume SDS is settled to 100ml), 37 DEG C of incubation 3h.12,000g × 1min is centrifuged, and is shifted in supernatant to new EP pipes.It is added in precipitation The solution vortex oscillation of 0.5 times of volume, 12,000g × 1min centrifugations.Supernatant merges, 2 times of volumes of addition into new EP pipes - 80 DEG C of 6h of ethyl alcohol.12,000g × 10min outwells supernatant.It is re-dissolved with 200 μ l TE solution, 25 μ l 3M sodium acetates is added Solution is uniformly mixed.- 80 DEG C of the ethyl alcohol of two volumes is added overnight.12,000g × 10min is centrifuged, and outwells supernatant.70% second Alcohol is washed once again, 12,000g × 4min centrifugations, 20 μ l ddH of precipitation2O is re-dissolved.
(6) digestion products connect, linked system：10×T4buffer 1μl；2 μ l of linear carrier；4 μ l of Insert Fragment； 0.5 μ l of T4DNA ligases；ddH22.5 μ l of O, 4 DEG C overnight.Connection product is converted afterwards, takes the connection product and 50 of 7 μ l μ l DH5 α competent bacterias mix, and place 30min on ice, 42 DEG C of water-bath 70s place 1min on ice.100 μ l SOC trainings are added Support base.37 DEG C of 300rpm cultures 30min on shaking table.The bacterium solution after 50 μ l cultures is taken to drop in Amp+Solid LB media on, Coating is uniform, and 37 DEG C of constant temperature incubations are stayed overnight.
(7) identification of conversion results：The bacterium colony grown after conversion is risen with sterile lancet choicest and is put into added with a certain amount of Amp+In culture medium, 37 DEG C of shakings overnight, take part to carry out bacterium colony PCR, PCR system：10 μ l systems, ddH2O 4.9μl；5× Q5buffer 2μl；10mMdNTP 1μl；1 μ l, Q5 enzymes of Primer (F+R), 0.1 μ l；1 μ l of template.PCR response parameters：98℃ 30s；98℃8s；65 DEG C of 15s, 35 cycles.72℃2min.After PCR, 2% Ago-Gel is examined.Positive bacterium solution takes portion Dispensing company is sequenced, and plasmid is small to put forward remaining bacterium solution.Sequencing result plasmid is correct, and structure is completed.
Attached drawing 2 is pGEX4T1-PlGF-2123-144Plasmid bacterium colony PCR qualification results.What wherein M electrophoresis band represented is Themo100bp marker, negative control are the controls of pGEX4T1 bacterium solutions, and 1 swimming band represents positive findings.
Embodiment 3：pGEX4T1-PlGF-2123-144- BiPPB plasmid constructions.
(1) PlGF-2 needed for123-144- BiPPB segments are synthesized by Sheng Gong companies, are inserted into pUC57 carriers.
(2) primer of PCR amplification is designed.Primer numbers：Segment prepares primer 3 and 7；Bacterium colony PCR primer 5 and 6.
(3) plasmid containing target fragment carries out PCR, and operation is as described in Example 2, response parameter：98℃30s；98℃ 8s；55 DEG C of 18s, 35 cycles.72℃2min.
(4) after electrophoresis determines that stripe size is correct, precipitation recycling is carried out, is operated with embodiment 2.
(5)PlGF-2123-144- BiPPB PCR post-fragments use EcoRI and XhoI digestions.
(6) after the completion of digestion, glue recycling is carried out, PAGE glue recycling step is the same as embodiment 2.
(7) digestion products connect, and linked system prepares carrier with embodiment 2, digestion carrier using embodiment 2.Conversion behaviour Make embodiment 2.
(8) identification of conversion results, specific steps and primer are the same as embodiment 2.Sequencing proves successfully to build plasmid.
Attached drawing 3 is pGEX4T1-PlGF-2123-144- BiPPB plasmid bacterium colony PCR qualification results.Wherein M electrophoresis band represents Be Themo100bp marker, 1 swimming band represents positive findings.
Embodiment 4：GST、GST-PlGF-2123-144And GST-PlGF-2123-144- BiPPB expression plasmids are in Escherichia coli The SDS-PAGE of induced expression is analyzed and the Western Blot analyses of GST labels.
The prokaryotic expression of fusion protein grope and optimize to promote fusion protein to be present in bacteria lysis In supernatant component afterwards, the purifying convenient for the later stage and the active reservation of fusion protein.Below mainly with GST-PlGF-2123-144Table It is illustrated for up to the induced expression of plasmid.
PGEX4T1, the pGEX4T1-PlGF-2 that will first build123-144And pGEX4T1-PlGF-2123-144- BiPPB matter Grain converts BL21 competence respectively, and picking single bacterium colony shakes bacterium after (Beijing Qing Kexin industry Bioisystech Co., Ltd), will contain There are correct plasmid BL21 fungi preservations, is carried out at the same time induced expression.
IPTG induced concentrations are groped：
(1) pGEX4T1-PlGF-2 is chosen123-144Transformed clone, in 37 DEG C of the LB of 4ml, 250rpm shaking overnights will contain Correct plasmid BL21 carries out fungi preservation.
(2) 5 50ml centrifuge tubes are separately added into 5ml and contain Amp+2XYT culture mediums, by 1:1000 are added shaking overnight PGEX4T1-PlGF-2123-144Bacterium solution shakes bacterium to OD550=0.8.
(3) IPTG is separately added into bacterium solution respectively to final concentration 0.1mM, 0.5mM, 1.0mM, 2.0mM and 4.0mM, 30 DEG C, 250rpm shakes 6h.For wherein IPTG final concentrations 1.0mM bacterium solutions per 0h, 2h, 4h, 6h takes 100 μ l bacterium solutions, 12,000g × 2min from The heart is resuspended bacterial precipitation with 25 μ l loading, directly carries out Western Blot detections.0.1mM, 0.5mM, 2.0mM and 4.0mM 6h take 100 μ l bacterium solutions, 12,000g × 2min centrifugations that bacterial precipitation is resuspended with 25 μ l loading, directly carries out Western Blot detections.
(4) each concentration of 0.1mM, 0.5mM, 1.0mM, 2.0mM and 4.0mM takes 4ml bacterium solutions, 12,000g × 2min to centrifuge, With 1ml GST lysis buffers (150mM NaCl2, 20mM HEPES pH7.5,5mM MgCl2, 1%Triton-X 100, 10ml working solutions using when 10 μ l protease inhibitors and DTT be added make its final concentration of 1mM) bacterial precipitation is resuspended, surpass on ice Sound cracks 20s, is spaced 10s, altogether ultrasound 10min.Room temperature 12000rmp centrifuges 2min, supernatant is transferred in new EP pipes, takes 25 Loading is added in μ l, and 40 μ l (- 80 DEG C of preservations of remaining solution) are supplied with GST lysis buffers.
(5) 4 DEG C of 12000rmp centrifuge 2min, supernatant are transferred in new EP pipes, take 25 μ l that albumen loading buffer is added Liquid supplies 40 μ l (- 80 DEG C of preservations of remaining solution) with GST lysis buffers.
(6) part lysate is taken to carry out SDS-PAGE protein electrophoresis and GST label proteins together with the sample of above-mentioned collection Western Blot detection.
GST and GST-PlGF-2123-144Most suitable induced expression condition be IPTG to final concentration 1mM, 30 DEG C, 6h.GST- PlGF-2123-144The most suitable induced expression conditions of-BiPPB be IPTG to final concentration 0.5mM, 30 DEG C, 6h.Such as attached drawing 4.
Embodiment 5：The purifying of fusion protein.
Below mainly with GST-PlGF-2123-144Purifying for illustrate.Purification condition is all that room temperature carries out.
(1) the mild bottle for shaking Glutathione Sepharose 4B makes its mixing, takes 200 μ l solution to clean In EP pipes.
(2) 500g × 5min is centrifuged, and carefully gently outwells supernatant.
(3) precipitation is washed with being equivalent to 1ml PBS, overturn mixing, 500g × 5min centrifugations.This step is repeated 2 times.
(4) the 20ml bacterium solutions of condition induced expression as above are pressed, 12,000g × 10min centrifugations are resuspended in 1ml PBS, add Enter the lysozyme lysis bacterium of final concentration of 1mg/ml, 12,000g × 10min centrifugations, supernatant is transferred in new EP pipes.Bacterium is split Solution liquid, which is added in ready Glutathione Sepharose 4B, is incubated at room temperature 30min.
(5) 500g × 5min is centrifuged, and carefully gently outwells supernatant, preserve carry out after supernatant PAGE-SDS glue and WesternBlot is detected.
(6) 1ml PBS washing precipitations are added, 500g × 5min centrifugations are carefully gently outwelled supernatant, carried out after preserving supernatant PAGE-SDS glue detects and Western Blot detections.This step is repeated 3 times.
(7) 100 μ l elution solution are added and elute albumen, are incubated at room temperature 10min.500g × 5min is centrifuged, and supernatant is shifted Into new EP pipes, the detection of PAGE-SDS glue and Western Blot detections are carried out after preserving supernatant.This step is repeated 3 times.
GST、GST-PlGF-2123-144And GST-PlGF-2123-144The SDS-PAGE of-Peptide is analyzed and GST labels are anti- Property Western Blot analysis it is as shown in Fig. 4.
Embodiment 6：GST-PlGF-2123-144ELISA detections to Type I collagen albumen compatibility.
The ELISA method is transformed by double antibody sandwich method, and experiment flow schematic diagram is as in Figure 6-1, is as follows：
1, Collagen I combines the preparation of liquid
(1) storing liquid 0.1% (w/v) acetic acid of 0.1M is diluted 10 times, 0.01% protein solution is made.
(2) it is carefully added into the chloroform for being equivalent to protein solution 10% in vial, not rock or vibrate, in 4 DEG C of ice Case is stood overnight, and collects the solution that albumen is contained on upper layer.
2, ELISA is tested
(1) elisa plate combines liquid to cover with 100 μ l/ hole Collagen I, and 4 DEG C overnight, suck supernatant, are dried overnight.
(2) 37 DEG C of closing 2h of PBS-T of 2% skim milk of 200 μ l are added per hole.PBS-T solution：PBS+0.05% Tween-20。
(3) solution is sucked, is rinsed 3 times with PBS-T per hole
(4) GST and GST-PlGF-2 are separately added into the solution of PBS-T+0.1% skim milks123-144Albumen makes GST albumen and GST-PlGF-2123-144Final concentration of protein is respectively 0.5 μm of ol, 3 μm of ol and 6 μm of ol, is incubated per 37 DEG C of 100 μ l of hole Educate 2h.
(5) PBS-T is rinsed 3 times, 200 μ l every time.
(6) 2% milk powder (PBS-T) that 200 μ l are added per hole closes 45min in 37 DEG C.
(7) it is rinsed 1 time with 200 μ l of PBS-T per hole.
(8) primary antibody 1 that 100 μ l have diluted is added:5000 (2% milk powder (PBS-T)), 4 DEG C of overnight incubations.
(9) 200 μ l of PBS-T are rinsed 3 times, each 3min.
(10) secondary antibody (1 that 100 μ l have diluted is added:2000WB, 2% (5%) milk powder (PBS-T)), room temperature 1h.
(11) 200 μ l PBS-T are rinsed 3 times, each 3min.
(12) 0.3% H2O2100 μ l of methanol solution close 15min.
(13) substrate developing solution A, developing solution B are added per hole, each 50 μ L (A, B liquid are first mixed and added in hole) are gently mixed It is even, it is incubated 30 minutes at 37 DEG C.
100 μ l 2M H are added per hole2SO4Color development stopping, the interior OD values that each hole solution is measured at 450nm wavelength of 15min.
ELISA testing results are as shown in Fig. 6, as a result show GST-PlGF-2123-144Height is shown to Type I collagen albumen The compatibility of degree.
Embodiment 7：GST-PlGF-2123-144- BiPPB detects the ELISA of Type I collagen albumen compatibility.
Concrete operation method is same as above, and is only separately added into GST and GST- in the solution of PBS-T+0.1% skim milks PlGF-2123-144- BiPPB albumen, it is 0.5 μm of ol, GST-PlGF-2 to make GST final concentration of protein123-144- Peptide albumen is dense eventually Degree is 0.5 μm of ol, 1 μm of ol, 2 μm of ol and 4umol, per 100 μ l of hole, 37 DEG C of incubation 2h.
ELISA testing results are as shown in Fig. 7, as a result show GST-PlGF-2123-144- BiPPB is to Type I collagen albumen table Reveal the compatibility of height.
Here is the experimental data of Fig. 6 and Fig. 7
。
SEQUENCE LISTING
<110>Southwest Jiaotong University
<120>A kind of gst fusion protein of high-affinity combination I-type collagen
<130> 2018
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 713
<212> PRT
<213>Artificial sequence
<400> 1
Ser Glu Gln Ile Asp Asn Met Glu Thr Ser Glu Arg Pro Arg Ile Leu
1 5 10 15
Glu Leu Glu Gly Leu Tyr Thr Tyr Arg Thr Arg Pro Leu Tyr Ser Ile
20 25 30
Leu Glu Leu Tyr Ser Gly Leu Tyr Leu Glu Val Ala Leu Gly Leu Asn
35 40 45
Pro Arg Thr His Arg Ala Arg Gly Leu Glu Leu Glu Leu Glu Gly Leu
50 55 60
Thr Tyr Arg Leu Glu Gly Leu Gly Leu Leu Tyr Ser Thr Tyr Arg Gly
65 70 75 80
Leu Gly Leu His Ile Ser Leu Glu Thr Tyr Arg Gly Leu Ala Arg Gly
85 90 95
Ala Ser Pro Gly Leu Gly Leu Tyr Ala Ser Pro Leu Tyr Ser Thr Arg
100 105 110
Pro Ala Arg Gly Ala Ser Asn Leu Tyr Ser Leu Tyr Ser Pro His Glu
115 120 125
Gly Leu Leu Glu Gly Leu Tyr Leu Glu Gly Leu Pro His Glu Pro Arg
130 135 140
Ala Ser Asn Leu Glu Pro Arg Thr Tyr Arg Thr Tyr Arg Ile Leu Glu
145 150 155 160
Ala Ser Pro Gly Leu Tyr Ala Ser Pro Val Ala Leu Leu Tyr Ser Leu
165 170 175
Glu Thr His Arg Gly Leu Asn Ser Glu Arg Met Glu Thr Ala Leu Ala
180 185 190
Ile Leu Glu Ile Leu Glu Ala Arg Gly Thr Tyr Arg Ile Leu Glu Ala
195 200 205
Leu Ala Ala Ser Pro Leu Tyr Ser His Ile Ser Ala Ser Asn Met Glu
210 215 220
Thr Leu Glu Gly Leu Tyr Gly Leu Tyr Cys Tyr Ser Pro Arg Leu Tyr
225 230 235 240
Ser Gly Leu Ala Arg Gly Ala Leu Ala Gly Leu Ile Leu Glu Ser Glu
245 250 255
Arg Met Glu Thr Leu Glu Gly Leu Gly Leu Tyr Ala Leu Ala Val Ala
260 265 270
Leu Leu Glu Ala Ser Pro Ile Leu Glu Ala Arg Gly Thr Tyr Arg Gly
275 280 285
Leu Tyr Val Ala Leu Ser Glu Arg Ala Arg Gly Ile Leu Glu Ala Leu
290 295 300
Ala Thr Tyr Arg Ser Glu Arg Leu Tyr Ser Ala Ser Pro Pro His Glu
305 310 315 320
Gly Leu Thr His Arg Leu Glu Leu Tyr Ser Val Ala Leu Ala Ser Pro
325 330 335
Pro His Glu Leu Glu Ser Glu Arg Leu Tyr Ser Leu Glu Pro Arg Gly
340 345 350
Leu Met Glu Thr Leu Glu Leu Tyr Ser Met Glu Thr Pro His Glu Gly
355 360 365
Leu Ala Ser Pro Ala Arg Gly Leu Glu Cys Tyr Ser His Ile Ser Leu
370 375 380
Tyr Ser Thr His Arg Thr Tyr Arg Leu Glu Ala Ser Asn Gly Leu Tyr
385 390 395 400
Ala Ser Pro His Ile Ser Val Ala Leu Thr His Arg His Ile Ser Pro
405 410 415
Arg Ala Ser Pro Pro His Glu Met Glu Thr Leu Glu Thr Tyr Arg Ala
420 425 430
Ser Pro Ala Leu Ala Leu Glu Ala Ser Pro Val Ala Leu Val Ala Leu
435 440 445
Leu Glu Thr Tyr Arg Met Glu Thr Ala Ser Pro Pro Arg Met Glu Thr
450 455 460
Cys Tyr Ser Leu Glu Ala Ser Pro Ala Leu Ala Pro His Glu Pro Arg
465 470 475 480
Leu Tyr Ser Leu Glu Val Ala Leu Cys Tyr Ser Pro His Glu Leu Tyr
485 490 495
Ser Leu Tyr Ser Ala Arg Gly Ile Leu Glu Gly Leu Ala Leu Ala Ile
500 505 510
Leu Glu Pro Arg Gly Leu Asn Ile Leu Glu Ala Ser Pro Leu Tyr Ser
515 520 525
Thr Tyr Arg Leu Glu Leu Tyr Ser Ser Glu Arg Ser Glu Arg Leu Tyr
530 535 540
Ser Thr Tyr Arg Ile Leu Glu Ala Leu Ala Thr Arg Pro Pro Arg Leu
545 550 555 560
Glu Gly Leu Asn Gly Leu Tyr Thr Arg Pro Gly Leu Asn Ala Leu Ala
565 570 575
Thr His Arg Pro His Glu Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Ala
580 585 590
Ser Pro His Ile Ser Pro Arg Pro Arg Leu Tyr Ser Ser Glu Arg Ala
595 600 605
Ser Pro Leu Glu Val Ala Leu Pro Arg Ala Arg Gly Gly Leu Tyr Ser
610 615 620
Glu Arg Pro Arg Gly Leu Pro His Glu Ala Arg Gly Ala Arg Gly Ala
625 630 635 640
Arg Gly Pro Arg Leu Tyr Ser Gly Leu Tyr Ala Arg Gly Gly Leu Tyr
645 650 655
Leu Tyr Ser Ala Arg Gly Ala Arg Gly Ala Arg Gly Gly Leu Leu Tyr
660 665 670
Ser Gly Leu Asn Ala Arg Gly Pro Arg Thr His Arg Ala Ser Pro Cys
675 680 685
Tyr Ser His Ile Ser Leu Glu Leu Glu Gly Leu Ala Arg Gly Pro Arg
690 695 700
His Ile Ser Ala Arg Gly Ala Ser Pro
705 710
<210> 2
<211> 786
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (8)..(8)
<223> n is a, c, g, or t
<400> 2
sdngstdnaa tgtcccctat actaggttat tggaaaatta agggccttgt gcaacccact 60
cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa 120
ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa tcttccttat 180
tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta tatagctgac 240
aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga 300
gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga ctttgaaact 360
ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga agatcgttta 420
tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat gttgtatgac 480
gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc aaaattagtt 540
tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa atccagcaag 600
tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga ccatcctcca 660
aaatcggatc tggttccgcg tggatccccg gaattccgtc gtcgtccgaa aggtcgtggt 720
aaacgtcgtc gtgaaaaaca gcgtccgacc gactgccacc tgctcgagcg gccgcatcgt 780
gactga 786

Claims (9)

1. a kind of gst fusion protein of high-affinity combination I-type collagen, amino acid sequence such as SEQ ID NO：Shown in 1.
2. the expressed sequence of gst fusion protein according to claim 1, gene order such as SEQ ID NO：2 institutes Show.
3. gst fusion protein according to claim 1, which is characterized in that the fusion protein is in the ends C- of GST band someone 2 short peptide stretch PlGF-2 of placenta growth factor123-144, combined with mediated fusion albumen and I-type collagen high-affinity.
4. gst fusion protein according to claim 3, which is characterized in that described carries PlGF-2123-144Small peptide melts Hop protein refers to GST-PlGF-2123-144Recombinant plasmid obtains after most suitable induced expression, purification condition.
5. gst fusion protein according to claim 4, which is characterized in that described most suitable induced expression condition be for IPTG to final concentration 1mM, 30 DEG C, 6h.
6. gst fusion protein GST-PlGF-2 according to claim 4123-144To the ELISA of Type I collagen albumen compatibility Detection method is as follows：
ELISA experiment flows are as follows：
1, I-type collagen Collagen I combines the preparation of liquid
(1) storing liquid 0.1% (w/v) acetic acid of 0.1M is diluted 10 times, 0.01% protein solution is made.
(2) it is carefully added into the chloroform for being equivalent to protein solution 10% in vial, not rock or vibrate, is put in 4 DEG C of refrigerators It sets overnight, collects the solution that albumen is contained on upper layer.
2, ELISA is tested
(1) elisa plate combines liquid to cover with 100 μ l/ hole Collagen I, and 4 DEG C overnight, suck supernatant, are dried overnight.
(2) 37 DEG C of closing 2h of PBS-T of 2% skim milk of 200 μ l are added per hole.PBS-T solution：PBS+0.05% Tween-20。
(3) solution is sucked, is rinsed 3 times with PBS-T per hole
(4) GST and GST-PlGF-2 are separately added into the solution of PBS-T+0.1% skim milks123-144Albumen keeps it dense eventually Degree is followed successively by 0.5 μm of ol, 3 μm of ol, 6 μm of ol, per 100 μ l of hole, 37 DEG C of incubation 2h.
(5) PBS-T is rinsed 3 times, 200 μ l every time.
(6) 2% milk powder (PBS-T) that 200 μ l are added per hole closes 45min in 37 DEG C.
(7) it is rinsed 1 time with 200 μ l of PBS-T per hole.
(8) primary antibody 1 that 100 μ l have diluted is added:5000 (2% milk powder (PBS-T)), 4 DEG C of overnight incubations.
(9) 200 μ l of PBS-T are rinsed 3 times, each 3min.
(10) secondary antibody (1 that 100 μ l have diluted is added:2000WB, 2% (5%) milk powder (PBS-T)), room temperature 1h.
(11) 200 μ l PBS-T are rinsed 3 times, each 3min.
(12) 0.3% H2O2100 μ l of methanol solution close 15min.
(13) it is added substrate developing solution A per hole, developing solution B, each 50 μ L (A, B liquid are first mixed and added in hole), gently mixing, It is incubated 30 minutes at 37 DEG C.
100 μ l 2M H are added per hole2SO4Color development stopping, the interior OD values that each hole solution is measured at 450nm wavelength of 15min.
7. a kind of application of the gst fusion protein of high-affinity combination I-type collagen, which is characterized in that with the fusion protein It is coupled other small peptides or the protein active factor, high parent property of the active factors to I-type collagen can be assigned.
8. a kind of application of the gst fusion protein of high-affinity combination I-type collagen, which is characterized in that the fusion protein It can be used for preparing PIGF-2122-224Functional oligopeptides and then all kinds of anti-fibrosis medicines are transformed with the functional oligopeptides, improve drug and exist The enrichment and delay at fibrosis position, to extend drug diseased region action time.
9. PIGF-2 according to claim 8122-224Functional oligopeptides, which is characterized in that with the functional oligopeptides and other labels Albumen or label small peptide are merged, and the preparation of other expression systems and other function products is can be used for.
CN201810075693.9A 2018-01-26 2018-01-26 A kind of the gst fusion protein preparation and its application of high-affinity combination I-type collagen Pending CN108285490A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810075693.9A CN108285490A (en) 2018-01-26 2018-01-26 A kind of the gst fusion protein preparation and its application of high-affinity combination I-type collagen

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810075693.9A CN108285490A (en) 2018-01-26 2018-01-26 A kind of the gst fusion protein preparation and its application of high-affinity combination I-type collagen

Publications (1)

Publication Number Publication Date
CN108285490A true CN108285490A (en) 2018-07-17

Family

ID=62835715

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810075693.9A Pending CN108285490A (en) 2018-01-26 2018-01-26 A kind of the gst fusion protein preparation and its application of high-affinity combination I-type collagen

Country Status (1)

Country Link
CN (1) CN108285490A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102917726A (en) * 2009-12-31 2013-02-06 毕欧丽安技术有限公司 Interferon analogs
CN104603148A (en) * 2012-07-03 2015-05-06 洛桑聚合联合学院 Conjugates containing sequences from placenta growth factor and their use as components of biomaterials and in medicine
CN108265070A (en) * 2016-12-30 2018-07-10 中国农业科学院上海兽医研究所 The method of one species specific detection Infection of Toxoplasma Gondii

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102917726A (en) * 2009-12-31 2013-02-06 毕欧丽安技术有限公司 Interferon analogs
CN104603148A (en) * 2012-07-03 2015-05-06 洛桑聚合联合学院 Conjugates containing sequences from placenta growth factor and their use as components of biomaterials and in medicine
CN108265070A (en) * 2016-12-30 2018-07-10 中国农业科学院上海兽医研究所 The method of one species specific detection Infection of Toxoplasma Gondii

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MIKAËL M. MARTINO等: "Growth Factors Engineered for Super-Affinity to the Extracellular Matrix Enhance Tissue Healing", 《SCIENCE》 *
程明亮: "《肝纤维化的基础研究及临床》", 31 August 1996 *

Similar Documents

Publication Publication Date Title
An et al. Collagen interactions: Drug design and delivery
CN110204608B (en) Yeast fermented small-molecule recombinant fibronectin peptide and preparation method and application thereof
JP4697982B2 (en) Modular transfection system
CN107074910A (en) Improve the senior macromolecule transduction domains of cell permeability（aMTD）The method that sequence, the method for encoding its specific characteristic of polynucleotides, identification comprising its aMTD, exploitation include its aMTD sequences
KR102494646B1 (en) Stabilized fibronectin based scaffold molecules
CN113186109A (en) Saccharomyces cerevisiae expression long-acting recombinant fibronectin and application thereof in cosmetics
ES2661662T3 (en)  Thermostable ligature of blunt ends and methods of use
CN110420334A (en) A kind of method of drug conjugate and raising drug molecule half-life period
CN108285490A (en) A kind of the gst fusion protein preparation and its application of high-affinity combination I-type collagen
CN107446022B (en) Polypeptide PIP-14 capable of antagonizing RNA binding activity of PARP1 protein and application thereof
CN106928363A (en) A kind of FAP nano antibodies Nb12
CN116925240A (en) Recombinant collagen and expression method and application thereof
CN108948208A (en) A kind of underwater albumen of injectable selfreparing and application thereof
CN107236046B (en) Recombinant human endostatin fusion protein and preparation method and application thereof
CN104031134B (en) Vector protein for gene therapy as well as preparation method and application of vector protein
CN116003635A (en) Long-acting fibronectin and preparation method and application thereof
CN108531492A (en) The method for building active sheep beta-alexin sbd-1 vivoexpressions using class elastin laminin elp
CN106913864A (en) The new application of fusion protein TAT DCF1
CN107216395A (en) A kind of TM4SF1 specific chimerics antigen receptor and its application
CN106928368A (en) A kind of FAP nano antibodies Nb57
CN105012951A (en) Method of linked adhesion molecule JAM-A in promotion of skin wound restoration, and application of linked adhesion molecule JAM-A
CN102146417B (en) Method and special expression vector thereof for promoting solubility expression of foreign protein by TAT (Trans-activator transduction) core peptide
CN108219003A (en) Fusion protein and its preparation method and application
Bennett et al. Genetically Encoded XTEN‐based Hydrogels with Tunable Viscoelasticity and Biodegradability for Injectable Cell Therapies
Patel et al. Linkers: a synergistic way for chimeric proteins

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20180717

WD01 Invention patent application deemed withdrawn after publication