CN108424459A - The fusion protein and its preparation method and application of human serum albumins and people's saltant type hepatocyte growth factor - Google Patents
The fusion protein and its preparation method and application of human serum albumins and people's saltant type hepatocyte growth factor Download PDFInfo
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Abstract
The present invention discloses the fusion protein and its preparation method and application of human serum albumins and people's saltant type hepatocyte growth factor, the fusion protein includes secondth area in the firstth area and people's saltant type hepatocyte growth factor HGF compositions of human serum albumin HSA composition, and people's saltant type hepatocyte growth factor refers to that the amino acid R of hepatocyte growth factor the 494th is mutated into N, D, K, Q or G.The present invention, which is utilized, without followed by action of proteolytic enzymes just there is the HGF mutant of biological activity to prepare HSA HGF fusion proteins, the fusion protein maintains on the basis of human hepatocyte growth factor bioactivity, its expression quantity in CHO systems is improved, simplifies and isolates and purifies operation.The fusion protein of the present invention extends its half-life period in vivo under the premise of maintaining the bioactivity of human hepatocyte growth factor, improves the expression quantity of HGF, has good application prospect in pharmaceutical field.
Description
Technical field
The invention belongs to long-acting fusion protein technical field of pharmaceuticals, more particularly, it is prominent with people to be related to human serum albumins
The fusion protein and preparation method thereof of modification hepatocyte growth factor.
Background technology
Human serum albumins (human serum albumin, HSA) is protein most abundant in human plasma, accounts for blood plasma
60% or so of total protein.Ripe HSA is made of 585 amino acid, molecular weight 66.5kDa, contains 17 pairs of disulfide bond, and one
As be single-stranded aglycosylated protein.Under normal circumstances, it is not easy by glomerular filtration, plasma half-life was at 20 days or so.HSA
It is widely used in Plasma volume expansion agent, is alternatively arranged as excipient and stabilizer, is used for various pharmaceutical preparations and serum free medium
Component.HSA is a kind of inert protein of stabilization, can be used as pharmaceutical carrier, assign the better physicochemical property of drug.Meanwhile HSA
It is a kind of ideal biological activity protein carrier without zymetology and immunologic competence in human body.When desired polypeptides are merged with HSA
When expression, the expression quantity of polypeptide drugs can be not only improved, the stability of polypeptide drugs can also be improved.
Hepatocyte growth factor (hepatocyte growth factor, HGF) is necessary one in growth and development process
Kind active proteic substance, has and promotes hepatic cell growth and liver development effect, is mainly generated and is secreted by liver.The eighties,
T.Nakamura etc. has found a kind of protein that can promote hepatocyte growth from the serum of partially hepatectomized rat first, and
It is named as hepatocyte growth factor.Hepatocyte growth factor is mainly thin by the depot fat of liver as a kind of multi-functional growth factor
Born of the same parents generate, it can not only cell cultured supernatant DNA synthesis, participate in the proliferation of various kinds of cell, migration and form and occur, to a variety of
Ripe organ-/ tissue has repair.HGF is synthesized and is secreted with single chain precursor in vivo, under followed by action of proteolytic enzymes
It is hydrolyzed into active duplex molecule.The heterodimer that ripe HGF is made of a 69kDa α chain and 34kDa β chains.It is single-stranded
HGF can also be combined with receptor c-Met high-affinities, but be unable to activated receptor, not have biological activity.HGF genes are one
Kind of multi-functional gene, under normal circumstances, HGF/c-Met are of short duration activation, production and normal fetus formation, regeneration, wound
Wound reparation and angiogenesis etc..When body kidney, liver or heart injury, HGF/c-Met gene expressions are raised, and are implied
The protective effect of HGF, maintains internal balance in tissue damage.As a kind of multi-functional growth factor, HGF can not only
The synthesis of cell cultured supernatant DNA, proliferation, migration and the form for also participating in various kinds of cell occur, to the organ-/ tissue of a variety of maturations
There is repair.HGF acts on unique receptor c-MET, and after HGF is combined with c-Met, autophosphorylation, phosphorus occur for catalysis c-Met
After acidification its tyrosine kinase activity enhance, cause a variety of substrate proteins such as PLC-r, PI3-K, Gab1, Grb2, STAT3 and
The tyrosine residue phosphorylation of SHP2 etc..These substrate proteins play various biological activity through different signal paths, such as thin
Born of the same parents' increment, angiogenesis, cell migration, Apoptosis etc..In addition, the half-life period of HGF monomers in vivo is shorter, only 3-5 points
Clock, so short half-life period need large dosage or inject repeatedly, cause treatment cost to increase difficult with therapeutic process, merge egg
White HSA-HGF improves the competitiveness of practical application.
Chinese hamster ovary cell (Chinese Hamster Ovary, CHO) is nineteen fifty-seven Univ Colorado-Boulder USA
Dr.Theodore T.Puck are obtained from Adult female Hamster Qvary separation, are the adherent type cell of epithelium, are on bioengineering
Widely used cell line.Whole world approval is formally applied to human disease treatment or the gene engineering product of prevention about more than 30
Kind, wherein only a kind of (hepatitis B vaccine) is produced by saccharomycete, remaining all product is all by Chinese hamster ovary celI and Escherichia coli
Production.Escherichia coli are easily operated, and yield is high, at low cost, but the pharmaceutical protein of its production is aglycosylated, cannot be formed with work
The dimer of property, is easily degraded, and be also easy to produce endotoxin and inclusion body problem in human body.Chinese hamster ovary celI belongs to fibroblast,
It is nonsecreting type cell, since itself seldom secretes intrinsic protein, destination protein can be simplified and isolate and purify operation, it can be
Recombinant pharmaceutical proteins are efficiently produced in serum free medium, are the ideal hosts for expressing large biological molecule.It is widely used to resist
The research and development and production of the biological technology products such as body, genetic recombination drug, viral vaccine.
The ingredient of serum is sufficiently complex, in addition to containing somatotrophic active constituent, also contains certain cytotoxic substance
And inhibiting substances influence the expression of cell function, while its complicated ingredient is also under genetic engineering to the cell effect of dedifferenting
Trip work brings very big difficulty.Therefore definite ingredients, the serum free medium of price economy become the inevitable requirement of CHO cultures.
Chinese hamster ovary celI can in serum free medium suspension growth, simplify downstream separation purification process, reduce and be produced into
This.
HGF monomers expression quantity in CHO is relatively low, by albumin fusion technology, realizes the hypersecretion table in CHO
It reaches, obtains HSA-HGF with bioactivity and highly stable.
Invention content
First of the present invention is designed to provide merging for human serum albumins and people's saltant type hepatocyte growth factor
Albumen.
Second object of the present invention is to provide merging for human serum albumins and people's saltant type hepatocyte growth factor
The preparation method of albumen.
Third object of the present invention is to provide merging for human serum albumins and people's saltant type hepatocyte growth factor
The application of albumen.
First purpose, the present invention disclose following technical scheme to realize the present invention：Human serum albumins and people's saltant type
The fusion protein of hepatocyte growth factor, which is characterized in that the fusion protein includes the first of human serum albumin HSA composition
Secondth area in area and people's saltant type hepatocyte growth factor HGF compositions, C-terminal and the people saltant type liver cell of human serum albumins
The N-terminal of growth factor connects, and people's saltant type hepatocyte growth factor refers to the amino of hepatocyte growth factor the 494th
Sour R is mutated into N, D, K, Q or G.
As a preferred embodiment, people's saltant type hepatocyte growth factor refers to hepatocyte growth factor the 494th
Amino acid R be mutated into N.
Second purpose, the present invention disclose following technical scheme to realize the present invention：Human serum albumins and people's saltant type
The preparation method of the fusion protein of hepatocyte growth factor, which is characterized in that include the following steps：
(1) overlapping pcr is utilized, the 494th amino acid R of human hepatocyte growth factor HGF is mutated into N, D, K, Q
Or G, obtain the HGF mutant just without digestion with biological activity；
(2) overlapping pcr is utilized, the HGF mutants cDNAs of HSA cDNA and step (1) acquisition, centre addition are connected
Enterokinase cleavage site, obtained HSA-HGF antigen-4 fusion protein genes are inserted into expression vector, then convert recombinant vector to place
It is expressed in chief cell CHO, is isolated and purified from expression product and obtain the fusion protein.
As a preferred embodiment, the amino acid R of human hepatocyte growth factor HGF the 494th is mutated into step (1)
N。
As a preferred embodiment, the expression vector is pMH3.
As a preferred embodiment, the host cell is CHO-KS cells.
As a preferred embodiment, the conversion refers to being converted to host cell using electroporation.
It is described to isolate and purify using ion-exchange chromatography and affinity chromatography as a preferred embodiment, the ion
Displacement chromatography filler is SP Sepharose Fast Flow, and for the affinity chromatography using affinity column, filler is liver
Plain Sepharose Fast Flow.
Third purpose to realize the present invention, the present invention disclose following technical scheme：Human serum albumins and people's saltant type
The fusion protein of hepatocyte growth factor is preparing the postoperative reparation for the treatment of hepatic fibrosis-renal tubular ectasia syndrome, liver, liver diffusivity damage medicine
In application.
HGF is synthesized and is secreted with inactive single chain precursor in vivo, is hydrolyzed under followed by action of proteolytic enzymes active
Duplex molecule, by overlapping pcr, using people HGF as template, obtain without followed by action of proteolytic enzymes just have biology
Active HGF mutant；Overlapping pcr is recycled, HGF mutants cDNAs and HAS cDNA, obtained HSA-HGF are connected
CDNA, antigen-4 fusion protein gene are inserted by EcoRI and NotI in pMH3 expression vectors, and electricity is transferred in CHO-KS cells, HSA-
HGF fusions, which are integrated into host chromosome, is expressed.
The preferred animal cell expression system of the present invention, the system can instruct the correct folding of protein, provide complexity
A variety of posttranslational modification functions such as glycosylation, expression product is closest to natural higher organism protein macromolecule.More preferably
CHO expression systems have the function of product excretion, seldom secrete own endogenous albumen, are purified convenient for downstream separation；Can efficiently it expand
Increase and express recombination, suspension culture can be carried out, expression is higher.More preferably CHO-KS cells, can be adherent
Growth, and the culture that can suspend.Preferred CHO secreted expression carriers pMH3 (Fig. 1), novel super-high expression regulation element are broken away from
Integrate position effect；GC-rich high efficient expressions core technology (WO2008/091276)；Natural high Expression element chick beta
Actin gene intron-1 (GC content average 75.3%, the highest 90.8%), superelevation G/C content
So that external source target gene is constantly in transcriptionally active state, do not influenced by position is integrated, continues high expression.
Conversion containing fusion cDNA expression vector to host cell in, have PEG methods, protoplasm body, electroporation or
Chemical transformation etc..The preferred electroporation of the present invention improves transfection efficiency.Electricity is pressurizeed by G418 after turning and screens positive transformants
Son.Clonal supernatants are collected, the methods of Dot blotting, Western blot, SDS-PAGE analysis fusioning protein table are passed through
It reaches.The DNA host cells that can be built by cultivating the present invention, to produce the fusion protein of the present invention.Host cell preferably exists
It is carried out on bioreactor, in two stages, the first stage is mainly used for host cell growth, and second stage is mainly used for mesh for culture
Albumen synthesis.It can be detached from cell fermentation liquid, purified fusion albumen.
The advantage of the invention is that：The present invention utilizes the HGF just without followed by action of proteolytic enzymes with biological activity
Mutant prepares HSA-HGF fusion proteins, on the basis of which maintains human hepatocyte growth factor bioactivity, improves
Its expression quantity in CHO systems, simplifies and isolates and purifies operation.The fusion protein of the present invention is maintaining human liver cell life
Its half-life period in vivo is extended under the premise of the bioactivity of the long factor, improves the expression quantity of HGF, is had very in pharmaceutical field
Good application prospect.
Description of the drawings
Fig. 1 is pMH3 Vector maps.
The detection Dot Blotting analyses that Fig. 2 is 96 orifice plate endonexin HSA-HGF, indicate the high expression picked out
Clone.
Fig. 3 is the HSA antibody test Western Blot analyses of the fusion protein of 24 orifice plates.
Fig. 4 is the Fed-batch Growth curve in batch cultivation of pMH3-HSA-HGF.
Fed-batch batch culture 8 days SDS-PAGE analyses that Fig. 5 is pMH3-HSA-IGF-1, M：Marker；1-8：
Stream plus 8 days samples.
Fig. 6 is the purity analysis of albumen.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.Experimental method used in following embodiments for example without
Specified otherwise is conventional method.The materials, reagents and the like used in the following examples unless otherwise specified can be from business way
Diameter obtains.It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.In the following example
Test method without specific conditions, usually according to normal condition, such as J. Pehanorm Brookers (Joseph Sambrook) etc.
It writes《Molecular Cloning:A Laboratory guide》Described in condition, or tested according to the condition that manufacturer is recommended.
The preparation of embodiment 1.HSA-HGF fusion proteins
(1) acquisition of HGF mutant
It is expanded with PCR method from the plasmid of the cDNA of HGF containing someone, primer is as follows：
P1：5’TCTTGTGCCAAAACGAAACAATTGGAAGTTGTAAATGGGATTCCAACACGAAC 3’(SEQ ID
NO.1)
A：5’actggtcaaaggaaaagaagaaatacaattcatgaattcaa 3’(SEQ ID NO.2)
B：5’AgcggccgcCTATGACTGTGGTACCTTATATGTTAAAATAA 3’(SEQ ID NO.3)
P2：5’GTTCGTGTTGGAATCCCATTTACAACTTCCAATTGTTTCGTTTTGGCACAAGA 3’(SEQ ID
NO.4)
PCR method is as follows：It is added in 50 μ L systems：10 μM of each 1 μ L of primer, 0.1 μ L template plasmids, 25 μ L
Primestar (2 ×), 23 μ L water.PCR conditions are：95 DEG C of pre-degenerations 2 minutes, 95 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 70
DEG C extend 30 seconds, 30 cycle；
The PCR product of P1 and A recycles, and 1:1 is merged plus P2 with the PCR recovery products of B, and fusion reaction is：95℃
Pre-degeneration 2 minutes, 95 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 45 seconds, and 70 DEG C extend 30 seconds, 18 cycles；
Using fusion product as template, primer A and B expands HGF mutant sequences, and PCR method is as follows：Add in 50 μ L systems
Enter：10 μM of each 1 μ L of primer A and B, 3 μ L fusion products, 25 μ L primestar (2 ×), 20 μ L water.PCR conditions are：95℃
Pre-degeneration 2 minutes, 95 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 70 DEG C extend 30 seconds, 30 cycles；
(2) connection of HSAcDNA and HGF mutants cDNAs
HSA cDNA and HGF mutants cDNAs are merged using over-lap PCR, after purification and HGF by the PCR product of HSA cDNA
Mutants cDNA PCR product presses 1 after purification:It is used as template after 1 molar ratio mixing, is added in 50ul reaction systems：PCR
Reaction condition is：PCR method is as follows：It is added in 50 μ L systems：10 μM of each 1 μ L of primer A and B, 3 μ L fusion products, 25 μ L
Primestar (2 ×), 20 μ L water.PCR conditions are：95 DEG C of pre-degenerations 2 minutes, 95 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 70
DEG C extend 30 seconds, 30 cycle；
EcoRI and NotI restriction enzyme sites are added respectively in HSA-HGF cDNA segments N-terminals and C-terminal simultaneously.It purifies
HSA-HGF cDNA segments and pMH3 empty carriers respectively by EcoRI and NotI double digestions, with the direct reclaim reagent of PCR product
Box purifies digestion products.Carrier is according to 1 after target fragment and digestion:7 molar ratio is connected with T4 ligases, connection product conversion
Bacillus coli DH 5 alpha competent cell, conversion product are coated in the LB culture mediums containing 100ug/ml ammonia benzyl mycins, 37 DEG C of cultures
Overnight, picking single bacterium colony is inoculated in the LB culture mediums of the 5ml benzyl mycins of ammonia containing 100ug/ml, and 37 DEG C of shaking table cultures are stayed overnight, extraction
Plasmid, by PCR, EcoRI and NotI double digestions, agarose gel electrophoresis identifies positive colony, and to the recombination of positive colony
Plasmid carries out DNA sequencing.Correct positive colony is sequenced to freeze in -30 DEG C, recombinant plasmid is named as pMH3-HSA-HGF.
(3) cell transfecting and colony screening
Cell transfecting
1) plasmid prepares：PMH3-HSA-HGF plasmids 12000rpm centrifuges 10min, sucts in layer plasmid to sterile EP tube,
For transfecting.
2) Chinese hamster ovary celI is collected, it is each to react with 3 × 106A cell is preferable, and the cell PBS being collected into washed once, from
Cell is resuspended with 200 μ L PBS after the heart.
3) prepare ice water.Configuration electricity turns reaction system：+ 20 μ g plasmids of 200 μ L cell suspensions+10 μ g (5 μ L) salmon sperm dna
(improve transfection efficiency) is added in 2mm pole cups after mixing.
4) pole cup ice bath 1min, 500V, 500us are shocked by electricity four times, often shock by electricity an ice bath 1min.
5) culture medium (DMEM/F12=1 of 10mL is respectively added in two dish:1, contain 10%FBS), it will be in pole cup
Suspension divide equally in two dish.
6) dish is placed in 37 DEG C of incubators and is cultivated.
Colony screening
1) electricity changes liquid (D/F+10%FBS), 2.8mg/L G418 pressurization screenings for second day after turning；
2) drug effect 2-4 days, if there are a large amount of dead cells, and survivaling cell is more, then changes liquid and add new G418 again
(secondary pressurized) directly changes liquid and is not pressurized if survivaling cell is seldom, waits for that clone grows；
3) after about 5 days, cell monoclonal is grown to suitable size, and clone is chosen in preparation；
4) 96 orifice plates are taken, add 100 μ L of culture solution (D/F+10%FBS) per hole；
5) culture solution in culture dish is siphoned away, washed once with PBS and (washed away dead cell), add 5mL culture solutions, is moved using 10 μ L
Liquid rifle is adjusted to 10 μ L, clone is chosen into 96 orifice plates；
6) it sets in incubator and cultivates one week or so, wait for that clone is long and pay attention to often shaking orifice plate to 80% bottom hole is paved with, make thin
Born of the same parents disperse growth.
7) culture solution containing FBS is removed to when being paved with 80% bottom hole when clone's length, adds (the expression training of 100 holes μ L/ sky D/F culture solutions
Nutrient solution), after 48h, pass through Dot blot and detect expression (Fig. 2,7 plants of cells of number are to select cell strain)；
8) it by positive clone digestion, is transferred in 24 orifice plates, is cultivated with D/F+10%FBS, pay attention to making every hole cell as possible
Measure close, culture 2 days or so；
9) when cell is paved with bottom hole 80% in 24 orifice plates, change 500 holes μ L/ expression culture solution, for 24 hours after again with inspections such as WB
Survey expression (in Fig. 3,4,6, No. 7 are to select cell strain).
10) two time clonings of the postdigestive unicellular paving of high-expression clone, secondary high table is selected by a colony screening method
Dyclonine.
(4) the shaking flask stream of high-expression clone adds expression
High expressed positive colony is selected, cell is transferred in T75 culture bottles and is cultivated, waits for cell confluency degree to 90% or so
When, T75 cell dissociations are transferred in 250mL shaking flasks, 20mL suspending nutrient solutions are added, 37 DEG C, 100rpm is cultivated, and is waited for
Cell is double and in good condition daily, and expression suspends successfully.
Cell density is adjusted to 1.0 × 106A/mL is inoculated in 250ml triangular flasks, and 30ml suspending nutrient solutions are added,
Sample batch experiment is carried out, is sampled within every two days, cell state is observed and counts, express 7-8 days, until under Cell viability is apparent
Drop, is detected by SDS-page, WB, Elisa etc..The clone of selection most advantage of expression carries out 3L Fed-batchs table in batches
It reaches.
The highest clone of selection batch expression quantity carries out 3L Fed-batchs and expresses in batches.Prepare two bottles of 40mL seed cells,
Density is up to 4.0 × 106When a/mL, merge in two bottles of cells to 3L glass shaking flasks, adding suspending nutrient solution to cell density is
2.0×106A/mL.Observation cell state daily detects cell density, cell density is made to be always held at 2.0 × 106A/mL,
Until total volume expands to 1L.Wait for that cell density is grown to 4.0-6.0 × 106When a/mL, starts to supplement supplemented medium, make grape
Sugared concentration is maintained at 3.0g/L or so, cell go to 34 DEG C of cultures (referring to Fig. 4, the cell growth in fed-batch process
Curve).It is sampled daily in fed-batch process, fermented liquid supernatant is collected by centrifugation, feed supplement is expressed 8 days, and expression culture is collected
Base simultaneously detects expression of results (referring to Fig. 5, the protein expression situation in fed-batch process).
(5) fusion protein isolates and purifies
Supernatant, membrane microfiltration of the supernatant through 0.22um, through SP Sepharose Fast Flow is collected by centrifugation in zymotic fluid
Chromatography, 20mMTris+0.4M Nacl (pH 8.5) elutions obtain HSA-HGF fusion proteins, using heparin
Sepharose Fast Flow chromatographies obtain purer fusion protein (referring to Fig. 6, albumen isolates and purifies).
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
<110>Medical College, Shanghai Communication Univ.
Southern Yangtze University
<120>The fusion protein and its preparation method and application of human serum albumins and people's saltant type hepatocyte growth factor
<130> /
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 53
<212> DNA
<213>It is artificial synthesized
<400> 1
tcttgtgcca aaacgaaaca attggaagtt gtaaatggga ttccaacacg aac 53
<210> 2
<211> 41
<212> DNA
<213>It is artificial synthesized
<400> 2
actggtcaaa ggaaaagaag aaatacaatt catgaattca a 41
<210> 3
<211> 41
<212> DNA
<213>It is artificial synthesized
<400> 3
agcggccgcc tatgactgtg gtaccttata tgttaaaata a 41
<210> 4
<211> 53
<212> DNA
<213>It is artificial synthesized
<400> 4
gttcgtgttg gaatcccatt tacaacttcc aattgtttcg ttttggcaca aga 53
Claims (10)
1. the fusion protein of human serum albumins and people's saltant type hepatocyte growth factor, which is characterized in that the fusion protein
Secondth area in the firstth area and people's saltant type hepatocyte growth factor HGF compositions including human serum albumin HSA composition, human serum
The C-terminal of albumin is connect with the N-terminal of people's saltant type hepatocyte growth factor, and people's saltant type hepatocyte growth factor is
The amino acid R for referring to hepatocyte growth factor the 494th is mutated into N, D, K, Q or G.
2. the fusion protein of human serum albumins according to claim 1 and people's saltant type hepatocyte growth factor, special
Sign is that people's saltant type hepatocyte growth factor refers to that the amino acid R of hepatocyte growth factor the 494th is mutated into N.
3. the preparation method of human serum albumins described in claim 1 and the fusion protein of people's saltant type hepatocyte growth factor,
It is characterised in that it includes following steps：
(1) overlapping pcr is utilized, the 494th amino acid R of human hepatocyte growth factor HGF is mutated into N, D, K, Q or G,
Obtain the HGF mutant just without digestion with biological activity；
(2) overlapping pcr is utilized, the HGF mutants cDNAs of HSA cDNA and step (1) acquisition are connected, centre addition intestines swash
Enzyme restriction enzyme site, obtained HSA-HGF antigen-4 fusion protein genes are inserted into expression vector, then convert recombinant vector thin to host
It is expressed in born of the same parents CHO, is isolated and purified from expression product and obtain the fusion protein.
4. preparation method according to claim 2, which is characterized in that by human hepatocyte growth factor HGF in step (1)
494 amino acid R are mutated into N.
5. preparation method according to claim 2, which is characterized in that the expression vector is pMH3.
6. preparation method according to claim 2, which is characterized in that the host cell is CHO-KS cells.
7. preparation method according to claim 2, which is characterized in that the conversion refers to being converted to place using electroporation
Chief cell.
8. preparation method according to claim 2, which is characterized in that described to isolate and purify using ion-exchange chromatography
And affinity chromatography.
9. preparation method according to claim 8, which is characterized in that the ion-exchange chromatography filler is SP
Sepharose Fast Flow, for the affinity chromatography using affinity column, filler is heparin Sepharose Fast
Flow。
10. human serum albumins described in claim 1 and the fusion protein of people's saltant type hepatocyte growth factor are preparing treatment
Application in the postoperative reparation of hepatic fibrosis-renal tubular ectasia syndrome, liver, liver diffusivity damage medicine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710077667.5A CN108424459A (en) | 2017-02-14 | 2017-02-14 | The fusion protein and its preparation method and application of human serum albumins and people's saltant type hepatocyte growth factor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| JP7246494B2 (en) | 2019-01-07 | 2023-03-27 | ベイジン・ノースランド・バイオテック・カンパニー・リミテッド | Human hepatocyte growth factor mutants and uses thereof |
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