CN102382850A - Novel human tumor necrosis factor receptor-Fc fusion gene and product protein thereof - Google Patents
Novel human tumor necrosis factor receptor-Fc fusion gene and product protein thereof Download PDFInfo
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Abstract
The invention discloses a novel human tumor necrosis factor receptor-Fc fusion gene and a product protein thereof. The human tumor necrosis factor receptor fusion gene complementary deoxyribonucleic acid (cDNA) is cloned and expressed at a carrier pCI-gs. The novel human tumor necrosis factor receptor-Fc fusion gene has the advantages that amplification marks of eukaryotic cells are introduced onto the pCI-gs carrier, and the copying number of target genes in host cells can be greatly increased, so the expression quantity of the target protein is improved. Simultaneously, CHO cells constructed in the invention realize pressurization amplification screening through sulfur ammonia methionine (MSX), so the expression quantity of the product protein of the fusion gene is obviously improved.
Description
Technical field
The invention belongs to the genetic engineering pharmaceutical field, be specifically related to a kind of novel TNFR-Fc fusion gene and expression product thereof.
Background technology
(Rheumatoid Arthritis RA) is one of human modal autoimmune disorder to rheumatoid arthritis, and morbidity is about 1%, and is obviously relevant with human leucocyte antigen DR4/DR1, has certain genetic predisposition.Mostly the Clinical symptoms of RA is the synovium of joint inflammation of hand, foot, wrist, knee, stern etc., finally causes joint injury.Synovitis causes a large amount of lymphocytic infiltrations, mainly comprises scavenger cell, T lymphocyte and plasmocyte, simultaneously blood vessel hyperplasia.Joint injury mainly betides synovial membrane and cartilage and bone and adjoins the place.
RA is a kind of to involve the systemic autoimmune disorder that the joint is the master.Recent study shows that tumor necrosis factor-alpha (TNF-α) is key factor (the Feldmann M 2001 in the RA pathogenesis; RavinderN Maini, 2001; RavinderN Maini, 199), it can stimulate body to produce a series of factors, like the generation of interleukin-(IL)-1, IL-6, IL-8 and granulocyte colony-stimulating factor etc., and inducing endothelial cell expression adhesion molecule, attract white corpuscle to the joint of getting involved.TNF-α also stimulates synovial membrane scavenger cell, fibroblast, osteoclast and chondrocyte to produce matrix metalloproteinase, and suppresses the synthetic of chondroproteoglycan, and can cause the damage of synovial membrane inflammation and joint cartilage.Blocking-up TNF-α can reach the purpose of treatment RA in the blocking-up inflammatory reaction of the pathogenetic upper reaches of RA.
(Lymphotoxin is LT) through combining to bring into play biological action with its coreceptor TNFR75 and TNFR55 for tumor necrosis factor alpha (Tumor Necrosis Factor Alpha, TNF α) and lymphotoxin.Human tumor necrosis factor receptor 75 (TNFR75) is made up of 461 amino acid, and terminal 235 amino-acid residues of its N-are cytolemma outskirt fragments that the TNFR75 protease hydrolysis gets off, and are also referred to as soluble TNF R75 (sTNFR75).
A large amount of experimental evidences show that soluble TNF R75 and TNFR55 (being the film outside part of acceptor) can be through combining to block TNF α and LT biological action (Hunns-Martin Lorenz, 2002 in vivo with TNF α and LT effective; Smith, C.A. etc. (1990), science, 248:1019-1023), and TNFR75 distributes and more extensively to send out, and affinity is stronger.But the TNFR transformation period is shorter.
The human IgG immunoglobulin like protein transformation period can be up to 21 days, and they are richs in protein in the human blood, are divided into IgA, IgG, IgM, IgE and IgD, and wherein IgG has IgG1, IgG2, IgG3 and four hypotypes of IgG4.The IgG1Fc section is made up of 232 amino-acid residues, comprises hinge area, CH2 district and CH3 district.Existing reporting combines the cysteine residues in the Fc hinge region of IgG and forms fusion rotein with other protein (like various cytokines and soluble receptors), make the protein-based IgG of being similar to molecule but do not have CH1 zone and light chain.Because structural homology, the Fc fusion rotein shows the interior medicine dynamics characteristic suitable with the human IgG of similar isotype.
External existing people has made up people TNFRII receptor part and the segmental fusion rotein of the Fc of IgG by genetic engineering means, and this albumen has neutralizing effect (USP 5,605,590) to TNF under isolated condition.Domestic also have make up this type of fusion rotein, Ma Jing etc. are at the recombination of CN1417334A soluble part in tumor necrosis factor acceptor, and have made up the fusion rotein of a kind of linker of containing in antigen-4 fusion protein gene and the product; The Liu Chang gate of a village etc. has made up a kind of fusion rotein of brachymemma in the novel TNFR-Fc fusion rotein of CN1502632A; Xu Bin etc. utilize bicistronic mRNA to make up a kind of human tumor necrosis factor soluble receptor II-antibody FC section fusion rotein in CN101003575.
This type medicine humanization degree is high, and the immunogenic response that causes is low, and the treatment works well, than depending merely on more effectively disease controlling of anodyne and antiphlogiston.But this type medicine ubiquity preparation method is complicated at present, and expression amount is low, and assistant officer's problem to be solved such as cost an arm and a leg.
Summary of the invention
The problem that the present invention solves has provided a kind of novel recombinant human TNFR75-Fc fusion gene, and the expressed protein product.
Second problem that the present invention solves is to have made up a kind of segmental recombinant vectors pCI-gs-TNFR75-Fc of recombinant human TNFR75-Fc fusion gene that contains; Behind the transfection mammalian cell; Its eukaryotic amplification label; Can significantly improve the copy number of goal gene in host cell, thereby improve the expression amount of target protein.
The 3rd problem that the present invention solves be, a kind of mammalian cell that contains recombinant vectors pCI-gs-TNFR75-Fc is provided, can stably express TNFR75-Fc.Said mammalian cell can be mammalian cell engineering cell strains commonly used such as Chinese hamster ovary celI, NSO cell.
The 4th problem that the present invention solves is through sulphur ammonia methionine(Met) (MSX) pressurization amplification screening; Make expression target protein cell strain expression amount improve at least one one magnitude; When efficiently expressing the cell strain screening; MSX concentration is brought up to 400～500 μ m the most at last, and screening mono-clonal expression amount reaches 20～30pg/cell24hr.
For addressing the above problem, the present invention implements through following technical scheme:
Total RNA of extracting HL-60 cell strain, RT-PCR amplification people sTNFR75 gene fragment; Pcr amplification human IgG1 Fc gene fragment.Adopt Overlap extention round pcr two gene fragments to be spliced the full length cDNA sequence of the rhTNFR:Fc fusion rotein that obtains encoding.This cDNA sequence that obtains is inserted clone's intermediate carrier Teasy, after sequencing confirms correctly, carry out Construction of eukaryotic.
RhTNFR:Fc fusion rotein cDNA fragment cloning is in expression vector; Employing liposome transfection technology; Aseptic extractive recombinant plasmid rhTNFR:Fc/pCI-gs is imported in the Chinese hamster ovary celI,, obtained the gene engineering monoclonal cell strain of the rhTNFR:Fc fusion rotein of high expression level amount through MSX pressurization screening; When MSX concentration was brought up to 400～500 μ m, screening mono-clonal expression amount reached 20～30pg/cell24hr.
The present invention has synthesized a kind of human tumor necrosis factor receptor fusion gene TNFR75-Fc through the method for OVERLAP PCR; Employing contains the eukaryotic cell efficient expression vector of GS system; In mammalian cell, make this Expression of Fusion Protein amount improve 20 percentage points through MSX pressurization screening, the fusion rotein activity has improved 8 percentage points.
Embodiment
Below in conjunction with embodiment the present invention is done further explain, but the present invention is not limited to following embodiment.
The preparation of embodiment 1 human soluble TNFR75 cDNA
1, pcr amplification TNFR75 cDNA
Design primer A:5 ' TC
AAG CTTATG GCT CCC GTC GCC GTC TGG 3 ' primer B:5 ' GTC ACA AGATTT GGG CTC GTC GCC AGT GCT CCC TTC AGC 3 ',
Design other one group of primer; In primer B, will introduce the linker sequence; This sequence (Gly4Ser) 3 is 15 amino acid whose hydrophobicity polypeptide of a coding, and their mobility is better; Do not influence the natural three-dimensional structure of TNFR and Fc, the dna sequence dna of its linker is 5 '-GGT GGC GGT GGA AGC GGC GGT GGC GGA AGC GGC GGT GGCGGC AGC-3 '.
The primer B of linker sequence is introduced in design.Primer Linker-B:5 ' GCT GCC GCC ACC GCC GCT TCCGCC ACC GCC GCT TCC ACC GCC ACC GTC.
With the human promyelocytic leukemia cell total rna is template, is template, is template with A and Linker-B with primer A and B respectively, carries out the RT-PCR reaction; 94 ℃ of sex change 5min begin circulation, 94 ℃ of 30sec, 58 ℃ of 30sec; 72 ℃ of 1min, totally 32 circulations, last 72 ℃ are extended 8min.STNFR75 and the gene fragment that contrasts sTNFR75-linker obtain encoding.
2, the evaluation of PCR product and gel reclaim
Above-mentioned condition is carried out PCR, on agarose gel electrophoresis, identify, find the band of the about 800bp of target protein molecular weight in the product, this fragment is reclaimed.
The segmental clone of embodiment 2 human IgG1's CH Fc fragment genes
Design primer C:5 ' GAG CCC AAA TCT TGT GAC GAG CCC AAA TCT TGT GAC AAA 3 ', primer D:5 ' AGT
GAA TTCTCA TTT ACC CGG AGA CAG GGA 3 ';
Design primer linker-C:GGT GGC GGT GGA AGC GGC GGT GGC GGA AGC GGC GGTGGC GGC AGC GAG.
With IgG1 Fc/Teasy vector plasmid is template, is primer, is primer with Linker-C and D with C and D respectively, carries out the RT-PCR reaction; 94 ℃ of sex change 5min begin circulation, 94 ℃ of 20sec, 62 ℃ of 30sec; 72 ℃ of 2min, totally 35 circulations, last 72 ℃ are extended 10min.Amplification obtains the gene fragment of coding human IgG1 Fc and contrast linker-IgG1Fc.
The acquisition of embodiment 3 TNFR75-Fc fusion genes
1, the full-length gene fragment of pcr amplification coding rhTNFR-Fc fusion rotein/rhTNFR-Iinker-Fc
With sTNFR75PCR amplified production and human IgG1 Fc section pcr amplification product is template, with A:5 ' TC
AAG CTTATG GCT CCC GTC GCC GTC TGG 3 ' and D:5 ' AGT
GAA TTCTCA TTT ACC CGG AGA CAG GGA 3 ' is a primer, carries out the RT-PCR reaction.
Be template with sTNFR75-linker pcr amplification product and people linker-IgG1 Fc section pcr amplification product simultaneously, with A:5 ' TC
AAG CTTATG GCT CCC GTC GCC GTC TGG 3 ' and D:5 ' AGT
GAA TTCTCA TTT ACC CGGAGA CAG GGA3 ' is a primer, carries out the RT-PCR reaction.
RT-PCR amplified reaction step: 94 ℃ of sex change 5min begin circulation, 94 ℃ of 40sec, and 62 ℃ of 30sec, 70 ℃ of 1min, totally 30 circulations, last 72 ℃ are extended 10min.
Amplification obtains the full-length cDNA fragment of rhTNFR-Fc and rhTNFR-linker-Fc fusion rotein, and its gene fragment length is respectively 1470bp and 1485bp.
2, rhTNFR-Fc/rhTNFR-linker-Fc fusion rotein cDNA clones in intermediate carrier
RhTNFR-Fc/rhTNFR-linker-Fc fusion rotein cDNA pcr amplification product and linear T easy vector (Promega) carry out ligation.Connect product Transformed E .coli intestinal bacteria competence host cell, coat and carry out blue hickie screening on the LB dull and stereotyped (containing 100 μ g/ml penbritins and 15 μ g/ml tsiklomitsins) that adds X-gal and IPTG in advance.From transforming the dull and stereotyped single bacterium colony of random choose white of going up, inoculation propagation is respectively carried out the plasmid Rapid identification, obtains TNFR75-Fc and contrast rhTNFR-linker-Fc fusion gene.
The expression of embodiment 4 TNFR75-Fc/rhTNFR-linker-Fc fusion genes
1, rhTNFR-Fc/rhTNFR-linker-Fc fusion gene fragment cloning is in expression vector
RhTNFR-Fc/rhTNFR-linker-Fc expressing fusion protein type construction of recombinant plasmid is to utilize the EcoR I single endonuclease digestion site of the pCI-gs that transforms through commercial expression vector pCI-neo; Insertion is through the correct rhTNFR-Fc/rhTNFR-linker-Fc gene fragment of determined dna sequence result; Identify the segmental both forward and reverse directions of insertion with the Spe1 single endonuclease digestion; Connect product Transformed E .coli competent cell, after Screening and Identification, obtain rhTNFR-Fc/pCI-gs/E.coli Top10F ' and contrast rhTNFR-linker-Fc/pCI-gs/E.coli Top10F ' reorganization bacterium.
2, a large amount of preparations of expression type recombinant plasmid
With the intestinal bacteria bacterial classification inoculation that carries recombinant plasmid rhTNFR-Fc/pCI-gs/rhTNFR-linker-Fc/pCI-gs in the 200ml liquid nutrient medium; After the cultivation of spending the night, adopt Qiagen Plasmid Maxi Kits, carry out a large amount of preparations of recombinant plasmid to specifications.The gained DNA, through 70% washing with alcohol, aseptic air-dry after, be dissolved in the 500 μ l sterilized waters, get 2 μ l plasmids dilutions and detect A260 value and A280 value respectively for 100 times, the ratio of A260/A280 then is used for cell transfecting with this plasmid between 1.75-1.85 the time.
3, cell strain transfection
After the Chinese hamster ovary celI of cultivating in the culturing bottle two days digests with 0.25% trypsin solution, get and carry out the trypan blue dyeing counting in right amount, with 3 * 10
5Individual cell count is inoculated the single hole of 6 orifice plates; Cell inoculation 6 orifice plates reach 90% full layer after 24 hours, can carry out transfection; Respectively get 250 μ l OPTI-MEM I (Gibco); Add in two aseptic centrifuge tubes; In two pipes, add 10 μ lLipofectamine (Invitrogen) and 4 μ g dilution plasmid respectively; In liquid mixing to the same pipe in two pipes, room temperature leaves standstill and made it form DNA in 20 minutes: liposome complex; Meanwhile, inhale the cell culture medium of abandoning in 6 orifice plates, in each hole, add the 2ml cell growth medium in twice back of washed cell gently with cell growth medium (DMEM/F12/10%FCS); With 500 μ lDNA: liposome complex solution adds in the respective aperture in 6 orifice plates, places 37 ℃, 5%CO
2Overnight cultures in the incubator.
The screening of embodiment 5 TNFR75-Fc/TNFR75-linker-Fc fusion rotein high expressing cell strains
After the resultant recombinant cell strain of embodiment 4 screening digested with 0.25% trypsin solution, import overnight cultures in three 100mm petridish into 1: 3 ratio; After 24 hours; Substratum in the 100mm petridish is replaced by the selection substratum; (the thiamines methionine(Met) is Sigma) as selective agent promptly in DMEM (no Stimulina) nutrient solution that contains 10% dialysis foetal calf serum (Gibco BRL), to add final concentration and be the MSX of 25 μ m; Select substratum according to every replacing of necrocytosis situation at a distance from 2-4 days; About 7-10 days cell begins mass mortality; Cell clone begins to occur after about 14 days; Transfer 24 orifice plates with clone's ring picking mono-clonal from flat board when treating that the cell clone diameter reaches 1-2mm, about 50 single cell clones of picking are dyed in every approving and forwarding.
When single cell clone grew to the full layer of 50-70% in 24 orifice plates, the culture supernatant of getting each clone was carried out ELISA and is detected, and according to cell growing state and expression amount, chose expression amount and carried out medicine pressurization amplification screening greater than the cell clone of 1pg/cell24hr; The concentration that progressively improves selective agent MSX increases the recombinant plasmid that is integrated in the karyomit(e), and when treating that drug level is increased to 500 μ m, screening mono-clonal expression amount reaches 20～30pg/cell24hr.Get each clone's culture supernatant, use the ELISA method and detect its fusion protein expression, 50 of each picking mono-clonals carry out expression amount mensuration altogether, and the result sees table 1.
Table 1, expression amount contrast and experiment
Calculate and to know by table 1, produce novel reorganization TNFR75-Fc expression amount comparison through the present invention and according to the facts test and improved 20 percentage points, more help this proteic expansion production.
The active simultaneous test of embodiment 6 TNFR75-Fc/TNFR75-linker-Fc fusion roteins
To carry out Sepharose-A affinity chromatography and hydrophobic chromatography with the fusion rotein TNFR75-Fc/TNFR75-linker-Fc that the cell strain that filters out is expressed.Concrete steps are (1) through albumin A affinitive layer purification albumen, in the albumin A affinity chromatography, add the urea of 1mol/L on the albumen during appearance, in proteic elution process, also add the urea of 1mol/L urea in the elutriant.Target protein under about 3.5 o'clock wash-outs of low pH; (2) (1) gained sample is directly passed through appearance on the hydrophobic chromatography, used salts solution is an ammonium sulfate, and used filler is the filler that contains butyl or phenyl.Obtain purity greater than 90% fusion rotein.
Carry out active determination test then.Through the kill and wound BA that detect rhTNFR-Fc of rhTNFR-Fc antagonism TNF-α to target cell L929 cell strain.
Active (AU/ml)=[the standard substance ED of sample
50(ng/ml)/product to be tested ED
50(ng/ml)] * standard substance (AU/ml) * testing sample extension rate of tiring
The result sees table 2.
The L929 cell of 1, taking the logarithm vegetative period, with the abundant cell dispersion in the conventional digestion of 0.25% trypsinase back, each survey live (1 96 orifice plate) gets about 2 * 10
6Cell, the centrifugal supernatant of abandoning, adding nutrient solution B is adjusted to 1.5 * 10 with the cell density of cell culture fluid
5Behind/the ml, add in 96 well culture plates, the 0.1ml/ hole places 37 ℃, 5% carbonic acid gas incubator overnight cultures (18～24h).
2, preparation standard substance: get the TNFR:Fc working standard of one 10 μ g/ml, it is 500ng/ml that the dactinomycin solution that adds the 10 μ g/ml that dilute with TNF-α (40U/ml) is diluted to concentration with it.
3, according to the protein quantification of sample to be determined; At first be diluted to 1mg/ml with sterile water for injection; The dactinomycin solution of the 10 μ g/ml that in the aseptic centrifuge tube of 1.5ml, dilute with TNF-α (40U/ml) progressively is diluted to 500ng/ml, notices that each extension rate is no more than 10 times.
4, the dilution of standard substance and trial-product
Doubling dilution working standard or dilution of sample liquid (promptly dilute the TNF-α and the actinomycetes D that contain same concentrations in all pipes of back, but the concentration of TNFR:Fc are different continuously.When next step adding contains in the hole of equal-volume nutrient solution, actual extent of dilution will be 2 times of this moment).
5, every hole adds the various dilution samples of 100 μ l, 3 holes of each extent of dilution.Negative control adds the dactinomycin solution with the 40U/ml of TNF-α dilution.Add dactinomycin solution in the positive control with 10 μ g/ml of serum-free RPMI-1640/DMEM (1: 1) nutrient solution dilution.
6,37 degree were cultivated 18-24 hour in the 5%CO2 incubator.
7, endpoint determination: every hole adds 40.0 μ l violet staining liquid, dyes ten minutes, and is with tap water rinsing 3～4 times, colourless until tap water.Bat is dried as far as possible with 96 orifice plates, and making does not have residual moisture in the plate, add destainer to 96 orifice plates, every hole 100 μ l, and mixing, ELIASA 570nm reads absorption value in the place.The result sees table 2.
Table 2, active contrast and experiment
| LOGEC50 | |
| TNFR-Fc | 3.812 |
| TNFR-linker-Fc | 3.518 |
Calculated and can be known by table 2, under same protein concentration, TNFR75-Fc has improved 8 percentage points than the protein-active of TNFR75-linker-Fc.
Claims (9)
1. novel recombinant human TNFR75-Fc fusion gene is characterized in that it contains the described base sequence of SEQ ID NO:3.
2. recombinant human fusion gene as claimed in claim 1 is characterized in that, it is made with containing the people Ig antibody Fc gene fusion of the described sequence of SEQ ID NO:2 by the people TNFR75 gene that contains the described sequence of SEQ ID NO:1.
3. according to claim 1 or claim 2 recombinant human fusion gene is characterized in that, in the fusion gene building process, has introduced 18 base complementrities that 18 bases and human IgG1 Fc gene fragment 5 ' are held in 3 ' the end primer of TNFR75.
4. a recombinant vectors pCI-gs-TNFR75-Fc is characterized in that, it contains the described TNFR-Fc fusion gene of claim 1.
5. a mammalian cell is characterized in that, it contains the described recombinant vectors of claim 4.
6. the described mammalian cell of claim 5 is characterized in that, it is selected from mammalian host cells commonly used such as Chinese hamster ovary celI, NSO cell.
7. by the described fusion gene expressed protein of claim 1 product, it is characterized in that it comprises TNFR75 and human IgG1's antibody Fc fragment.
8. protein as claimed in claim 7 is characterized in that it comprises the said aminoacid sequence of SEQ ID NO:4.
9. like claim 7 or 8 described proteins, it is characterized in that in efficiently expressing the cell strain screening process, MSX concentration is brought up to 400～500 μ m the most at last.
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| CN201510691152.5A CN105200073B (en) | 2010-09-01 | 2010-09-01 | Human tumor necrosis factor receptor-Fc fusion and its product albumen |
| CN201510691126.2A CN105177032B (en) | 2010-09-01 | 2010-09-01 | New tumor necrosin receptor-Fc fusion gene and its product albumen |
| CN2010102684093A CN102382850A (en) | 2010-09-01 | 2010-09-01 | Novel human tumor necrosis factor receptor-Fc fusion gene and product protein thereof |
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| CN201510691152.5A Division CN105200073B (en) | 2010-09-01 | 2010-09-01 | Human tumor necrosis factor receptor-Fc fusion and its product albumen |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019029129A1 (en) * | 2017-08-07 | 2019-02-14 | 上海科新生物技术股份有限公司 | DUAL-SPECIFICITY HYBRID PROTEIN FOR IL-17 AND TNF-α |
| CN112494658A (en) * | 2020-12-04 | 2021-03-16 | 苏桥生物(苏州)有限公司 | Stable Fc fusion protein preparation |
| WO2023046085A1 (en) * | 2021-09-24 | 2023-03-30 | Sichuan Clover Biopharmaceuticals, Inc. | Tpo mimetic fusion proteins and methods of use submission of sequence listing as ascii text file |
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| CN108355141B (en) * | 2018-01-11 | 2019-06-18 | 广西壮族自治区人民医院 | A kind of drug and preparation method thereof of the treatment allergic disease based on TIM-4-Fc fusion protein |
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| CN100395339C (en) * | 2006-02-28 | 2008-06-18 | 中国人民解放军第二军医大学 | Method for expressing hepatitis C virus envelope protein E2 by mammal cell with high efficient secretion |
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- 2010-09-01 CN CN2010102684093A patent/CN102382850A/en active Pending
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| US5605690A (en) * | 1989-09-05 | 1997-02-25 | Immunex Corporation | Methods of lowering active TNF-α levels in mammals using tumor necrosis factor receptor |
| USRE36755E (en) * | 1989-09-05 | 2000-06-27 | Immunex Corporation | DNA encoding tumor necrosis factor-α and -β receptors |
| US7485456B1 (en) * | 1996-04-02 | 2009-02-03 | Yeda Research And Development Co. Ltd. | Modulators of TNF receptor associated factor (TRAF), their preparation and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019029129A1 (en) * | 2017-08-07 | 2019-02-14 | 上海科新生物技术股份有限公司 | DUAL-SPECIFICITY HYBRID PROTEIN FOR IL-17 AND TNF-α |
| CN112494658A (en) * | 2020-12-04 | 2021-03-16 | 苏桥生物(苏州)有限公司 | Stable Fc fusion protein preparation |
| WO2023046085A1 (en) * | 2021-09-24 | 2023-03-30 | Sichuan Clover Biopharmaceuticals, Inc. | Tpo mimetic fusion proteins and methods of use submission of sequence listing as ascii text file |
| WO2023044774A1 (en) * | 2021-09-24 | 2023-03-30 | Sichuan Clover Biopharmaceuticals, Inc. | Tpo mimetic fusion proteins and methods of use submission of sequence listing as ascii text file |
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| CN105177032A (en) | 2015-12-23 |
| CN105177032B (en) | 2019-05-10 |
| CN105200073A (en) | 2015-12-30 |
| CN105200073B (en) | 2019-03-01 |
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