CN109402130A - A kind of recombinant human horny cell growth factor-2-1 and its preparation method and application - Google Patents
A kind of recombinant human horny cell growth factor-2-1 and its preparation method and application Download PDFInfo
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- CN109402130A CN109402130A CN201811408920.1A CN201811408920A CN109402130A CN 109402130 A CN109402130 A CN 109402130A CN 201811408920 A CN201811408920 A CN 201811408920A CN 109402130 A CN109402130 A CN 109402130A
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- 238000001959 radiotherapy Methods 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Classifications
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- 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
- C07K14/50—Fibroblast growth factor [FGF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/10—Drugs for disorders of the urinary system of the bladder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of recombinant human horny cell growth factor-2-1 (KGF-1) production methods.The recombinant plasmid of the gene containing recombinant human horny cell growth factor-2-1 is also disclosed, the expression and purification step of the plasmid electrotransfection rear human horny cell growth factor-2 in Chinese hamster ovary line (CHO).The present inventor's keratinocyte growth factor-1 can remarkably promote 4MBr-5 cell Proliferation, suitable for treatment is because of skin and mucous membrane tissue damages relevant disease, pulmonary lesion related disease and treatment bladder or urothelial damages relevant disease.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to recombinant human horny cell growth factor-2-1 (rhKGF-1) and its
Preparation method and purposes.
Background technique
Human horny cell growth factor-2-1 (KGF-1 or KGF), also referred to as fibroblast growth factor-7 (FGF-7) are
People's Thorium Lung Burden proliferation, migration and a kind of important paracrine confactor of differentiation, pass through junctional epithelium cell
Receptor KGFR plays critical function.In the bronchial epithelial cell cultivated in vitro, KGF can accelerate epithelial cell to heal, enhancing
The reparation of human airway epithelial cells and the mobilization and migration for recycling epithelial cell.KGF promotes human airway epithelial cells proliferation and to activity
The removing toxic substances of oxygen is of great significance.In animal model, KGF can reduce alveolar damage and the death rate, can also reduce acute
Influence of the injury of lungs to gas exchanges region.In addition, cell division will be whole when primary urothelial lacks KGF culture
Only；It is knocked out in mouse in KGF, ureteric bud occurs, and kidney aggregation system is significantly less than the wild-type mice of control；KGF strikes
The bladder of mouse or urothelial thickness are also more considerably thinner than wild-type mice out.In the rat ulcer wing of cyclophosphamide induction
Guang inflammation model can almost improve exedens symptom with recombination KGF treatment completely.These prompt KGF in treatment pulmonary lesion phase
Related disorders, and treatment bladder or urothelial damage relevant disease.
Recombined human KGF (rhKGF) is a kind of KGF mutant or the like by N-terminal missing, the missing of N-terminal by
Proof has the function of enhancing protein stability, was ratified to list (Chinese by FDA as therapeutic recombinant proteins in 2004
Name: Pa Lifuming), for treating the serious canker sore because of high dose caused by radiotherapy and chemotherapy.The rhKGF of approved listing is by big
The production preparation of enterobacteria expression system, the system process is simple, low in cost, but extracts KGF from Escherichia coli and usually require
Active soluble protein can be just obtained by complex processes such as renaturation.Moreover, there is also potential for coli expression system
Safety problem.Chinese patent application (201210032424.7 and 201610458957.X) successively discloses and utilizes insect cell
With Pichia pastoris as host, KGF is recombinantly expressed.Relative to Escherichia coli, insect cell and Pichia anomala expression system it is excellent
Gesture is posttranslational modification and the product purification without protein renaturation.But either insect cell or Pichia pastoris, it is purified
KGF and native protein between still have activity difference.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of recombinant human horny cell growth factor-2s -1 (rhKGF-1).
The present invention provides a kind of nucleotide sequences as shown in SEQ ID NO:1.
A kind of recombinant vector is additionally provided, it includes nucleotide sequence shown in SEQ ID NO:1.Preferably, described
Recombinant vector is recombination pCMV plasmid.
A kind of recombinant cell is additionally provided, it includes recombinant vector above-mentioned.Preferably, the recombinant cell is recombination
Chinese hamster ovary Chinese hamster ovary celI.
Recombinant human horny cell growth factor-2-1 (rhKGF-1) provided by the invention, its core as shown in SEQ ID NO:1
Nucleotide sequence coding.Its amino acid sequence is as shown in SEQ ID NO:2.
The present invention also provides a kind of method for preparing aforementioned recombinant human horny cell growth factor-2-1 (rhKGF-1), packets
Containing following steps:
(1) recombinant cell above-mentioned is taken, business is inoculated in and is cultivated with suspending in serum-free addition culture medium A cti Pro, the phase
Between flow feeding Cell Boost 7a powder supplement twice, 37 DEG C of cultivation temperature, feed time be the 3rd day and
5th day, culture solution being collected under the conditions of 4 DEG C within the 7th day, being centrifuged, filtering obtains supernatant；
(2) take supernatant, isolated and purified with ion-exchange chromatography: first with anion-exchange chromatography, cation is exchanged again
Chromatography；
Mobile phase A is detected as equilibration buffer for rinsing tomographic system flow velocity 1.5mL/min when anion chromatography
Wavelength 254nm and 280nm；Gradient elution is carried out to its destination protein with Mobile phase B, collects elution samples, the gradient elution
Mode are as follows: 100-50%A, 0-50%B, elute 20min；
Mobile phase A when cation chromatography are as follows: 20mM citric acid, pH6.0；Mobile phase B are as follows: 20mM citric acid, chlorination containing 1M
Sodium, pH6.0；Flow velocity 1.5mL/min, Detection wavelength 254nm and 280nm；Gradient is carried out to its destination protein with Mobile phase B to wash
It is de-, collect elution samples to get；The mode of the gradient elution are as follows: 100-70%A, 0-30%B elute 20min；
The mobile phase A is the Tris-HCl solution of 20mM；The Mobile phase B is the Tris-HCl of the 20mM of 1M sodium chloride
Solution.
The present invention also provides aforementioned rhKGF-1 on preparation skin injury, mucous membrane tissue damage, pulmonary lesion, bladder
Purposes in skin lesion wound or the drug of urothelial damage.
The present invention also provides a kind of drugs, it is using rhKGF-1 as active constituent, in addition pharmaceutically acceptable auxiliary material
The preparation being prepared.
Sterling rhKGF-1 has been prepared in the present invention by way of genetic engineering, and bioactivity is good, and preparation method
Simply, it can be achieved that big industrial production, has good market application prospect.
Under the premise of not departing from the above-mentioned basic fundamental thought of the present invention, modification, the replacement of other diversified forms can also be made
Or change.
Detailed description of the invention
Fig. 1 plasmid pCMV schematic diagram.
Fig. 2 recombinant plasmid pCMV-KGF-1 schematic diagram.
Fig. 3 DNA gel electrophoretic analysis.1-9 swimming lane is the pCMV-KGF-1 monoclonal recombinant bacterial strain PCR product selected,
In, 2 and 9 swimming lanes are the recombinant clone bacterial strain of the sequence containing KGF-1.
Fig. 4 SDS-PAGE analysis.1 swimming lane is fermentation liquid, and M swimming lane is Marker, and 2 swimming lanes are to penetrate liquid, and 3-5 swimming lane is
Eluent.
Specific embodiment
The specific embodiment of form by the following examples makees further specifically above content of the invention
It is bright.But this should not be interpreted as to the scope of the above subject matter of the present invention is limited to the following embodiments.It is all above-mentioned interior based on the present invention
Hold realized technology to all belong to the scope of the present invention.
The preparation of the recombinant protein of the present invention of embodiment 1
1, the building of recombinant plasmid
(1) synthesis of human horny cell growth factor-2-1 (KGF-1) gene
Gene sequencing
Carry out KGF-1 coding region gene sequence (GenBank :) optimization, obtain KGF-1 gene order (SEQ ID NO:
1) and KGF-1 gene expression after amino acid sequence (SEQ ID NO:2).By Shanghai, Sheng Gong bioengineering Co., Ltd is synthesized,
The gene order of synthesis is recombinated into plasmid vector pUC57, and obtaining the plasmid comprising target gene is pUC57-KGF-1.
KGF-1 gene order (SEQ ID NO:1):
AGCTATGACTACATGGAAGGAGGCGATATCAGAGTGAGAAGACTGTTCTGTCGGACACAGTGGTACCTG
AGGATCGACAAGAGAGGCAAGGTGAAGGGCACCCAGGAGATGAAGAATAACTACAACATCATGGAAATCAGGACAGT
GGCCGTCGGAATCGTGGCCATCAAAGGAGTGGAAAGTGAATTCTATCTCGCCATGAACAAGGAAGGAAAGCTCTATG
CTAAGAAGGAGTGCAATGAAGATTGTAACTTCAAGGAACTCATTCTGGAAAACCATTACAACACATATGCCTCTGCT
AAGTGGACACACAACGGAGGCGAAATGTTCGTCGCCTTGAATCAGAAGGGCATTCCTGTCAGAGGAAAGAAAACCAA
GAAAGAACAGAAGACAGCCCACTTTCTGCCTATGGCTATCACTTAA
Amino acid sequence (SEQ ID NO:2) after KGF-1 gene expression
SYDYMEGGDIRVRRLFCRTQWYLRIDKRGKVKGTQEMKNNYNIMEIRTVAVGIVAIKGVESEFYLAMNK
EGKLYAKKECNEDCNFKELILENHYNTYASAKWTHNGGEMFVALNQKGIPVRGKKTKKEQKTAHFLPMAIT
The plasmid portion sequence fragment (SEQ ID NO:3) of raw work biosynthesis gene order containing KGF-1:
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAG
TCGACTCTAGCTATGACTACATGGAAGGAGGCGATATCAGAGTGAGAAGACTGTTCTGTCGGACACAGTGGTACCTG
AGGATCGACAAGAGAGGCAAGGTGAAGGGCACCCAGGAGATGAAGAATAACTACAACATCATGGAAATCAGGACAGT
GGCCGTCGGAATCGTGGCCATCAAAGGAGTGGAAAGTGAATTCTATCTCGCCATGAACAAGGAAGGAAAGCTCTATG
CTAAGAAGGAGTGCAATGAAGATTGTAACTTCAAGGAACTCATTCTGGAAAACCATTACAACACATATGCCTCTGCT
AAGTGGACACACAACGGAGGCGAAATGTTCGTCGCCTTGAATCAGAAGGGCATTCCTGTCAGAGGAAAGAAAACCAA
GAAAGAACAGAAGACAGCCCACTTTCTGCCTATGGCTATCACTTGATTAATTAAGCG
(2) building of recombinant expression carrier
Double digestion is carried out to pCMV plasmid (as shown in Figure 1) and pUC57-KGF-1 plasmid, digestion system is centrifuged in 1.5ml
Following ingredient is established in pipe: above-mentioned 40 μ l of plasmid, 10 Cutsmart μ l, Avr II and each 5 μ l of Pac I (A/P), 50 μ of aqua sterilisa
L, 37 DEG C of reactions overnight, are recycled using QIAGEN Product Purification Kit after mixing, and recycling obtains vector linearization plasmid pCMV
(A/P) and target gene fragment KGF-1 (A/P).
Under the action of T4 ligase, plasmid pCMV (A/P) and KGF-1 (A/P) base that above-mentioned digestion recycling is obtained
Because segment connects, formed recombinant plasmid pCMV-KGF-1 (as shown in Figure 2).Such as lower body is established in connection reaction in 1.5mlEP pipe
System: 1 μ l of pCMV (A/P), 7 μ l of KGF-1 (A/P) genetic fragment, 10 × T4Buffer, 1 μ l, 1 T4DNAligase μ l, after mixing
4h or more is reacted under room temperature (20 DEG C or so), connection product is converted into Top10 competent escherichia coli cell, is applied to 2YT
(AMP) on plating medium, 37 DEG C of static overnight incubations of constant incubator, plate number is respectively pCMV-KGF-1.
By culture, the plasmid for carrying out recombinant bacterium is extracted, is then carried out the several recombination single colonies of picking from above-mentioned bacterium colony
Digestion, digestion system are that following ingredient is added in 1.5ml EP pipe: 7 μ l of plasmid, 1 Cutsmart μ l, Avr II and Pac I
(A/P) each 0.2 μ l, benefit aqua sterilisa react 4h at 37 DEG C after mixing to 10 μ l.Agarose gel electrophoresis analyzes result (as schemed
3).After digestion identification is correct, expression plasmid is largely extracted using QIAGEN Plasmid Midi Kit, and save backup.
2, it recombinantly expresses
Recombinant plasmid pCMV-KGF-1 (such as Fig. 2) is transfected through electric robin into CHO DG44 cell, cell after electricity is turned
Mixed liquor is transferred in the 6 orifice plates of the culture medium containing SFM4, is placed in 37 DEG C of incubators and is cultivated, and MTX (10nM) is added after 48h and is added
Pressure screening, restores to increase to 100nM to 90% or more, MTX to Cell viability；MTX to 500nM is added in cell, restores to motility rate
To after 90% or more, cell is transferred to shaking flask and suspended in business serum-free addition culture medium A cti Pro and is cultivated.Yu Pei
Support the 3rd and the 5th day respectively flow feeding Cell Boost 7a powder supplement continue to cultivate, the 7th day harvest is cultivated
Liquid is centrifuged 15min under the conditions of 4 DEG C, with 5000rpm, collects supernatant and is purified after 0.45 μm of membrane filtration.
3, it isolates and purifies:
It is purified using supernatant of the ion-exchange chromatography to above-mentioned expression cell, concrete operations are as follows:
Anion-exchange chromatography: 1mL HiTrapTM Q HP prepacked column is connected with AKTA purifier tomographic system.
Mobile phase A are as follows: 20mM Tris-HCl, pH 8.0；Mobile phase B are as follows: 20mM Tris-HCl, sodium chloride containing 1M, pH 8.0；Flow velocity
1.5mL/min, Detection wavelength 254nm and 280nm.Chromatographic column 5-10CV first is balanced with mobile phase A, to the cell for having collected processing
Supernatant carries out loading, and destination protein is incorporated on medium, and partial impurities penetrate.After end of the sample, using mobile phase A cleaning layer
Column 3-5CV is analysed, finally its destination protein is carried out gradient elution (100-50%A, 0-50%B, 20min) with Mobile phase B, is collected
Elution samples and by buffer exchange into 20mM citric acid (pH6.0).Chromatographic column is regenerated with the 1M sodium chloride of 3-5CV, is used in combination
0.1M sodium hydroxide is cleaned.
Cation-exchange chromatography: 1ml HitrapTM SP HP prepacked column is connected with AKTA purifier tomographic system.
Mobile phase A are as follows: 20mM citric acid, pH6.0；Mobile phase B are as follows: 20mM citric acid, sodium chloride containing 1M, pH6.0；Flow velocity 1.5mL/
Min, Detection wavelength 254nm and 280nm.Chromatographic column 5-10CV first is balanced with mobile phase A, anion chromatography is dialysed on sample
Sample, destination protein are incorporated on medium, and partial impurities penetrate.After end of the sample, chromatographic column 3- is rinsed using mobile phase A
5CV, linear gradient elution 100-70%A, 0-30%B, 20min.
The recombined human KGF-1 (rhKGF-1) that purifying is obtained using ion-exchange chromatography is examined according to SDS-PAGE electrophoresis
It surveys, as a result rhKGF-1 molecular weight of the invention meets expected (Fig. 4) about in 19-20KD.
The bioactivity of the recombined human KGF-1 (rhKGF-1) of the present invention of embodiment 2 detects
Recombined human KGF-1 and commercially available KGF-1 prepared by Example 1, compares their bioactivity:
Using F-12 culture medium culture 4MBr-5 rhesus macaque pulmonary branches tracheal epithelial cell, be then transferred to 96 well culture plates after
Continuous culture, is added the rhKGF-1 of beforehand dilution for 24 hours, makes its final concentration in 1-100ng/mL range, and total volume is 100 μ L,
Continue culture 48 hours in 37 DEG C of constant incubators.CCK8 solution is added after culture, 37 DEG C of incubation 1.5h set multifunctional examining
It surveys instrument and measures absorbance in 450nm, draw curve graph and calculate EC50 (medium effective concentration to stimulate cellular proliferation).
Testing result see the table below shown, and the present invention has in 1-100ng/mL concentration range obvious promotees 4MBr-5 cell Proliferation
Effect, effect present concentration dependent.RhKGF-1 of the invention, which is calculated, according to the result of 3 repetition experiments promotes 4MBr-
The medium effective concentration EC50 of 5 cell Proliferations is about 2.87 ± 0.32ng/mL, lower than reference substance KGF-1 (R&D company, activity
Unit is 1.3 × 106IU/mg EC50 (3.29 ± 0.28ng/mL)) shows the rhKGF biology using expressing cho cell
It is active high, there is good market application prospect.
RhKGF-1 of the invention is compared with the bioactivity of reference substance rhKGF-1
The experiment results show that the recombined human KGF-1 that the present invention is prepared, promotes the activity of epithelial cell growth excellent
It is good, hence it is evident that be better than commercially available KGF-1.
Sterling rhKGF-1 has been prepared in the present invention by way of genetic engineering, and bioactivity is good, and preparation method
Simply, it can be achieved that big industrial production, has good market application prospect.
SEQUENCE LISTING
<110>Hospital Affiliated To Chengdu Traditional Chinese Medicine Univ
<120>a kind of recombinant human horny cell growth factor-2-1 and its preparation method and application
<130> GY025-18P1711
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 423
<212> DNA
<213> Artificial Sequence
<220>
<223>KGF-1 gene
<400> 1
agctatgact acatggaagg aggcgatatc agagtgagaa gactgttctg tcggacacag 60
tggtacctga ggatcgacaa gagaggcaag gtgaagggca cccaggagat gaagaataac 120
tacaacatca tggaaatcag gacagtggcc gtcggaatcg tggccatcaa aggagtggaa 180
agtgaattct atctcgccat gaacaaggaa ggaaagctct atgctaagaa ggagtgcaat 240
gaagattgta acttcaagga actcattctg gaaaaccatt acaacacata tgcctctgct 300
aagtggacac acaacggagg cgaaatgttc gtcgccttga atcagaaggg cattcctgtc 360
agaggaaaga aaaccaagaa agaacagaag acagcccact ttctgcctat ggctatcact 420
taa 423
<210> 2
<211> 140
<212> PRT
<213> Artificial Sequence
<220>
<223>amino acid sequence after KGF-1 gene expression
<400> 2
Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile Arg Val Arg Arg Leu Phe
1 5 10 15
Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp Lys Arg Gly Lys Val Lys
20 25 30
Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn Ile Met Glu Ile Arg Thr
35 40 45
Val Ala Val Gly Ile Val Ala Ile Lys Gly Val Glu Ser Glu Phe Tyr
50 55 60
Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr Ala Lys Lys Glu Cys Asn
65 70 75 80
Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu Glu Asn His Tyr Asn Thr
85 90 95
Tyr Ala Ser Ala Lys Trp Thr His Asn Gly Gly Glu Met Phe Val Ala
100 105 110
Leu Asn Gln Lys Gly Ile Pro Val Arg Gly Lys Lys Thr Lys Lys Glu
115 120 125
Gln Lys Thr Ala His Phe Leu Pro Met Ala Ile Thr
130 135 140
<210> 3
<211> 511
<212> DNA
<213> Artificial Sequence
<220>
<223>the plasmid portion sequence fragment of raw work biosynthesis gene order containing KGF-1
<400> 3
gcagatctcc tagggccacc atggcctgga tgatgcttct cctcggactc cttgcttatg 60
gatcaggagt cgactctagc tatgactaca tggaaggagg cgatatcaga gtgagaagac 120
tgttctgtcg gacacagtgg tacctgagga tcgacaagag aggcaaggtg aagggcaccc 180
aggagatgaa gaataactac aacatcatgg aaatcaggac agtggccgtc ggaatcgtgg 240
ccatcaaagg agtggaaagt gaattctatc tcgccatgaa caaggaagga aagctctatg 300
ctaagaagga gtgcaatgaa gattgtaact tcaaggaact cattctggaa aaccattaca 360
acacatatgc ctctgctaag tggacacaca acggaggcga aatgttcgtc gccttgaatc 420
agaagggcat tcctgtcaga ggaaagaaaa ccaagaaaga acagaagaca gcccactttc 480
tgcctatggc tatcacttga ttaattaagc g 511
Claims (10)
1. a kind of genetic fragment of recombined human KGF-1, it is characterised in that: its nucleotide sequence is as shown in SEQ ID NO:1.
2. a kind of recombinant vector, it is characterised in that: include nucleotide sequence shown in SEQ ID NO:1.
3. recombinant vector according to claim 2, it is characterised in that: the recombinant vector is pCMV plasmid.
4. a kind of recombinant plasmid, it is characterised in that: it includes nucleotide sequence and plasmid vector described in claim 2 and 3,
Be characterized in: the recombinant plasmid is expression plasmid pCMV-KGF-1.
5. a kind of recombinant cell for expressing recombined human KGF-1 gene, it is characterised in that include recombination described in claim 2 and 3
Plasmid.
6. requiring the expression cell according to right 4, it is characterised in that: the recombinant cell is the Chinese storehouse containing recombinant plasmid
Mouse ovary Chinese hamster ovary celI.
7. a kind of recombined human KGF-1, it is characterised in that: its amino acid sequence is as shown in SEQ ID NO:2.
8. a kind of method for preparing the recombined human KGF-1 of claim 1 or 7, it is characterised in that: comprise the following steps:
(1) recombinant cell described in claim 5 or 6 is taken, business is inoculated in and is hanged in serum-free addition culture medium A cti Pro
Floating culture, during which flow feeding Cell Boost 7a powder supplement twice, 37 DEG C of cultivation temperature, feed time
For the 3rd day and the 5th day, culture solution being collected under the conditions of 4 DEG C within the 7th day, being centrifuged, filtering obtains supernatant；
(2) supernatant is taken, is isolated and purified with ion-exchange chromatography: first with anion-exchange chromatography cation exchange layer again
Analysis；
Mobile phase A is as equilibration buffer when anion chromatography, for rinsing tomographic system flow velocity 1.5mL/min, Detection wavelength
254nm and 280nm；Gradient elution is carried out to its destination protein with Mobile phase B, collects elution samples, the side of the gradient elution
Formula are as follows: 100-50%A, 0-50%B elute 20min；
Mobile phase A when cation chromatography are as follows: 20mM citric acid, pH6.0；Mobile phase B are as follows: 20mM citric acid, sodium chloride containing 1M,
pH6.0；Flow velocity 1.5mL/min, Detection wavelength 254nm and 280nm；Gradient elution is carried out to its destination protein with Mobile phase B, is received
Collect elution samples to get；The mode of the gradient elution are as follows: 100-70%A, 0-30%B elute 20min；
The mobile phase A is the Tris-HCl solution of 20mM；The Mobile phase B is that the Tris-HCl of the 20mM of 1M sodium chloride is molten
Liquid.
9. the recombined human KGF-1 of claim 7 or 8 treats skin injury, mucous membrane tissue damage, pulmonary lesion, wing in preparation
Purposes in Guang epithelial damage or the drug of urothelial damage.
10. a kind of drug, it is characterised in that: it is using the recombined human KGF-1 of claim 7 or 8 as active constituent, in addition medicine
The preparation that acceptable auxiliary material is prepared on.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811408920.1A CN109402130A (en) | 2018-11-23 | 2018-11-23 | A kind of recombinant human horny cell growth factor-2-1 and its preparation method and application |
Applications Claiming Priority (1)
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| CN110339345A (en) * | 2019-07-30 | 2019-10-18 | 重庆派金生物科技有限公司 | A kind of recombined human truncated-type keratinocyte growth factor-1 eye drops and its preparation method and application |
| KR20220028520A (en) * | 2020-08-28 | 2022-03-08 | 한국해양과학기술원 | Thermally stable fgf7 polypeptide and use of the same |
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| CN110339345B (en) * | 2019-07-30 | 2022-11-29 | 重庆派金生物科技有限公司 | Recombinant human truncated keratinocyte growth factor-1 eye drops and preparation method and application thereof |
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