CN108623690A - A kind of fusion protein of thrombopoietin and its preparation method and application - Google Patents
A kind of fusion protein of thrombopoietin and its preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
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Abstract
The invention belongs to genetic engineering pharmaceutical fields, it is related to a kind of fusion protein of thrombopoietin and its preparation method and application, more particularly to thrombopoietin simulating peptide (the Thrombopoietin Mimetic Peptide of primary yeast preferred codons coding, TMP) the fusion protein and its preparation method and application of dyad and human serum albumins molecule (Human serum Albumin, HSA).The fusion egg includes the TMP dyads that a HSA molecule and a yeast preferred codons encode, wherein HSA molecules are located at the ends N of fusion protein, and the TMP dyads of yeast preferred codons coding are located at the ends C of fusion protein.The fusion protein high-caliber stabilization can express in yeast, can be applied to industrial production；Meanwhile the fusion protein has longer half-life period with thrombopoietic characteristic is remarkably promoted in human body, can be used for preparing the drug for treating the diseases such as a variety of primary or secondary thrombocytopenia.
Description
Technical field
The invention belongs to genetic engineering pharmaceutical field, it is related to fusion protein and its preparation side of a kind of thrombopoietin
Method and application, and in particular to the thrombopoietin simulating peptide (Thrombopoietin of primary yeast preferred codons coding
Mimetic Peptide, TMP) dyad and human serum albumins molecule (Human serum Albumin, HSA) fusion egg
In vain.
Background technology
The primary caused by a variety of causes and secondary thrombocytopenia clinically can be often encountered, such as primary blood
Platelet reduction property purpura, alpastic anemia and decrease of platelet caused by anti-tumor/radiotherapy etc..For this kind of disease,
Compare and be suitble to be given treatment to using long-acting blood platelet-increasing drug, on the one hand, times for spraying can be reduced, reduce the needle pain of patient
It is bitter；On the other hand, it is possible to reduce drug dose reduces medical expense.
Blood platelet is generated by megacaryocyte, and it is to improve the primary hand of platelet levels to promote the proliferation of megacaryocyte, differentiation
Section.Thrombopoietin (Thrombopoietin, TMP), also known as megakaryocyte growth development facor (MGDF), are that marrow is huge
Monocyte proliferation, differentiation and thrombopoietic most important regulatory factor.TMP genes in 1994 lack annotation by successful for the first time gram
After grand soon, people are to develop a kind of recombination function type TMP gene engineering products-PEG- that polyethylene glycol (PEG) is changed
rHuMGDF.Results of animal shows that PEG-rHuMGDF not only has rush thrombopoietic activity outstanding, but also in vivo
With longer half-life period, single application is that can reach the effect of notable increased platelets counts.However, people during clinical test
Find, some health volunteers generated antibody after receiving the drug therapy exists with endogenous TMP report to the leadship after accomplishing a task in and instead
It answers, subject is caused decrease of platelet occur, so that the development of PEG-rHuMGDF or even recombined human TMP were at 1998
Halted by U.S. FDA.It is affected by it, in U.S., Europe, Japan and other countries, recombined human TMP is not approved for entering clinic so far.
Human serum albumins molecule (Albumin Human, HSA) is the primary protein component in blood plasma, and it is total to account for about blood plasma
The 50-60% of albumen.HSA molecules are a kind of non-glycosylated single chain proteins, are made of altogether 585 amino acid residues, molecular weight
For 66.5kDa (A.Dugaiczyk et al., PNAS, 1982,79：71-75), plasma half-life is up to two weeks or more.HSA points
Son be in host cell synthesized in the form of a kind of former peptide, wherein containing 24 amino acid residues composition signal peptide and propetide,
It is removed with signal peptide in secretion process and propetide transporting.HSA molecules are a stable inert proteins, can be used as drug
Carrier.HSA molecules are the same below successfully to express (EP330451 and EP361991) in more hosts, especially in yeast cells
HSA molecule higher levels can be achieved stablizes expression.Polypeptide can be not only improved when desired polypeptides and HSA molecule amalgamation and expressions
The stability of drug can also increase the half-life period of polypeptide drugs in vivo.The dimer of patent No. CN201310084212.8
Change in the preparation and application of fusion protein and discloses Dimerized thrombopoietin (TPO) simulating peptide TMP dyads-people's blood
The preparation and application of pure protein molecular fusion protein, but to there is expressing fusion protein in it effect is undesirable, in order to
The bioactivity for improving expression yield, enhancing fusion protein encodes present invention employs yeast preferred codons and promotees blood platelet life
At the strategy of plain simulating peptide TMP dyads.Based on the studies above present situation, the present invention now provides a kind of melting for thrombopoietin
Hop protein, the thrombopoietin simulating peptide TMP dyads be by yeast preferred codons encode made of, it is described to melt
Hop protein high-caliber stabilization can express in yeast, can be applied to industrial production；Meanwhile the fusion protein has significantly rush
Into thrombopoietic characteristic, there is longer half-life period in human body.Present invention simultaneously provides the preparation sides of the fusion protein
Method and application.
The heterogenous expression of recombinant protein is by gene own sequence feature such as codon preference, G/C content, mRNA two levels
The influence of the factors such as structure, mRNA stability.Research shows that the replacement of synonym can influence protein translation extension
Rate [1-2] illustrates that the optimization of codon may play the influence run counter to desire to the expression of recombinant protein.On the other hand,
The expression of recombinant protein is also influenced by factors such as host, condition of culture, secretory pathway, promoters, existing gene optimization
Theoretical and design method is also immature, and there is various limitations, table of the gene optimization strategy to raising recombinant protein
It is necessary condition up to amount, rather than adequate condition.In conclusion being transformed by yeast preferred codons to improve genetic engineering
There is great uncertainties in production application for the strategy of bacterial strain expression quantity, are changed for different target gene
It makes, it is final the result is that unforeseen.
Bibliography：
[1]Komar AA,Lesnik T,Reiss C.Synonymous codon substitutions affect
ribosome traffic and protein folding during in vitro translation.FEBS Lett,
1999,462(3):387-391.
[2]Trinh R,Gurbaxani B,Morrison SL,et al.Optimization of codon pair
use within the(GGGGS)3linker sequence results in enhanced protein
expression.Mol Immunol,2004,40(10):717-722.
Invention content
The purpose of the present invention is to provide a kind of fusion protein of thrombopoietin, the fusion protein long half time,
Expression can be stablized by high level in host.
It is a further object to provide the preparation methods of above-mentioned fusion protein.
It is a further object to provide the applications of above-mentioned fusion protein.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of fusion protein of thrombopoietin disclosed by the invention, including human serum albumin HSA molecule, rush blood
Platelet generates plain simulating peptide TMP dyads, the amino acid sequence such as SEQ ID NO of the human serum albumin HSA molecule：4
The sequence with said function, encodes the amino acid sequence of the HSA molecules after the shown or amino acid sequence is mutated
DNA sequence dna such as SEQ ID NO：Shown in 3, the amino acid sequence such as SEQ of the thrombopoietin simulating peptide TMP dyads
ID NO：Sequence shown in 2 or with said function after the amino acid sequence is mutated, thrombopoietin simulating peptide TMP
Dyad is made of being encoded by a yeast preferred codons, to encode the thrombopoietin simulating peptide TMP dyads
Amino acid sequence DNA sequence dna such as SEQ ID NO：Shown in 1, which further includes that can form Dimerized structural domain
Peptide fragment.
A kind of fusion protein of thrombopoietin disclosed by the invention, can form in the peptide fragment of Dimerized structural domain
Contain at least two cysteines.
A kind of fusion protein of thrombopoietin disclosed by the invention, can form in the peptide fragment of Dimerized structural domain
Containing there are two cysteines.
A kind of fusion protein of thrombopoietin disclosed by the invention, the peptide fragment that can form Dimerized structural domain are
H peptide fragments, amino acid sequence such as Seq ID No:Shown in 8.
A kind of fusion protein of thrombopoietin disclosed by the invention, the peptide fragment that can form Dimerized structural domain are
H peptide fragments and peptide fragment selected from fip structural domains, the amino acid sequence such as Seq ID No of H peptide fragments:Shown in 8, selected from fip structural domains
The amino acid sequence of peptide fragment such as Seq ID No:Shown in 10.
A kind of fusion protein of thrombopoietin disclosed by the invention, wherein HSA molecules are connected to fusion protein
The ends C-, structural formula are expressed as HSA-L3-H-L4-TMP-L1-TMP, and L1, L3, L4 indicate that peptide linker, the amino acid sequence of L1 are
The amino acid sequence of GGPSG, L3 are GGGGSGL, and the amino acid sequence of L4 is EFGGGGS, and H expressions can form Dimerized structure
The H peptide fragments in domain.
A kind of fusion protein of thrombopoietin disclosed by the invention, wherein HSA molecules are located at the N- of fusion protein
End, structural formula are expressed as TMP-L1-TMP-L2-H-fip-HSA, L1 and L2 and indicate that peptide linker, the amino acid sequence of L1 are
The amino acid sequence of GGPSG, L2 are GGGGSRS, and H indicates that the H peptide fragments of Dimerized structural domain can be formed, and fip indicates to be selected from fip
The peptide fragment of structural domain；The amino acid sequence of the fusion protein such as SEQ ID NO：Shown in 6, the nucleosides of encoding said fusion protein
The DNA sequence dna of acid sequence such as SEQ ID NO：Shown in 5.
The invention discloses the Dimerized fusion proteins that a kind of fusion protein of thrombopoietin is formed.
The present invention discloses a kind of fusion protein of thrombopoietin, including human serum albumin HSA molecule, to promote blood small
Plate generates plain simulating peptide TMP dyads, the amino acid sequence such as SEQ ID NO of the human serum albumin HSA molecule：4 institutes
Show or the amino acid sequence it is mutated after with said function sequence, encode the DNA of the amino acid sequence of the HSA molecules
Sequence such as SEQ ID NO：Shown in 3, the amino acid sequence such as SEQ ID of the thrombopoietin simulating peptide TMP dyads
NO：Sequence shown in 2 or with said function after the amino acid sequence is mutated, the thrombopoietin simulating peptide
TMP dyads are made of being encoded by a yeast preferred codons, to encode the thrombopoietin simulating peptide TMP bis-
The DNA sequence dna of conjuncted amino acid sequence such as SEQ ID NO：Shown in 1, which further includes Dimerized structural domain.
A kind of fusion protein of thrombopoietin disclosed by the invention, the Dimerized structural domain is by two
The covalent effect of sulfide linkage keeps the fusion protein of thrombopoietin Dimerized.
The fusion protein of a kind of thrombopoietin disclosed by the invention, containing extremely in the Dimerized structural domain
Few two disulfide bond formed by cysteine.
A kind of fusion protein of thrombopoietin disclosed by the invention contains two in the Dimerized structural domain
A disulfide bond formed by cysteine.
A kind of fusion protein of thrombopoietin disclosed by the invention, the Dimerized structural domain include H peptides
Section, the amino acid sequence such as Seq ID No of H peptide fragments:Shown in 8.
A kind of fusion protein of thrombopoietin disclosed by the invention, Dimerized structural domain include H peptide fragments and choosing
From the peptide fragment of fip structural domains, the amino acid sequence such as Seq ID No of H peptide fragments:Shown in 8, the ammonia of the peptide fragment selected from fip structural domains
Base acid sequence such as Seq ID No:Shown in 10.
The fusion protein of thrombopoietin disclosed by the invention, the HSA molecules are connected to the ends C- of fusion protein
End, structural formula are expressed as HSA-L3-H-L4-TMP-L1-TMP, and L1, L3, L4 indicate that peptide linker, the amino acid sequence of L1 are
The amino acid sequence of GGPSG, L3 are GGGGSGL, and the amino acid sequence of L4 is EFGGGGS, and H expressions can form Dimerized structure
The H peptide fragments in domain.
A kind of fusion protein of thrombopoietin disclosed by the invention, the HSA molecules are located at the N- of fusion protein
End, structural formula are expressed as TMP-L1-TMP-L2-H-fip-HSA, L1 and L2 and indicate that peptide linker, the amino acid sequence of L1 are
The amino acid sequence of GGPSG, L2 are GGGGSRS, and H indicates that the H peptide fragments of Dimerized structural domain can be formed, and fip indicates to be selected from fip
The peptide fragment of structural domain；The amino acid sequence of the fusion protein such as SEQ ID NO：Shown in 6, the nucleosides of encoding said fusion protein
The DNA sequence dna of acid sequence such as SEQ ID NO：Shown in 5.
The Dimerized fusion protein that a kind of fusion protein of thrombopoietin disclosed by the invention is formed.
A kind of fusion protein of thrombopoietin disclosed by the invention, the fusion protein use yeast cell to express
It prepares.
A kind of fusion protein of thrombopoietin disclosed by the invention, the yeast are thermophilic pichia methanolica.
A kind of fusion protein of thrombopoietin disclosed by the invention is small in preparation treatment primary or secondary blood
Plate reduces the application in the drug of disease.
A kind of preparation method of the fusion protein of thrombopoietin disclosed by the invention, described steps are as follows：
1) the thrombopoietin simulating peptide TMP dyad sequences of full genome synthetic yeast preferred codons coding；
2) HSA molecular sequences are obtained by PCR amplification；
3) by digestion with restriction enzyme, connection and Escherichia coli are converted, obtained containing encoding the yeast preference password
The recombination of the thrombopoietin simulating peptide dyad and the DNA sequence dna of the fusion protein of human serum albumins molecule of son coding
Expression vector；
4) recombinant expression carrier described in step 3) is transformed into competent E.coli TOP10, then is transformed into host table
It is expressed up to system to get the fusion protein.
A kind of recombinant expression carrier of the fusion protein encoding gene containing above-mentioned thrombopoietin.It can be used for carrying
The expression vector of the gene of coding fusion protein of the present invention includes but is not limited to protokaryon, the common plasmid of eukaryotic expression system.
A kind of host expression system containing above-mentioned recombinant expression carrier.Host can be bacterium, yeast and mammal
Cell etc., wherein it is preferred that yeast, more preferably thermophilic pichia methanolica；For the fusion protein of the present invention of recombinant expression
It can be extracted, purified from corresponding cell culture by a variety of methods, these methods include centrifugation, ultrafiltration and liquid phase
The technologies such as chromatography, wherein liquid chromatography includes the chromatographic techniques such as ion exchange, hydrophobic and molecular sieve again.
Fusion protein of the present invention can form pharmaceutical preparation use together with pharmaceutical carrier, these pharmaceutical carriers include
Water, brine, carbohydrate, alcohols and amino acid etc..The pharmaceutical preparation made of fusion protein of the present invention is preferably water content and is less than
3% or water-free lyophilized preparation, administering mode include venoclysis, injection (including subcutaneous and intramuscular injection), intranasal, breathing
Road etc., wherein it is preferred that subcutaneously or intramuscularly injecting.
The fusion protein can in yeast stability and high efficiency expression, can be applied to industrial production；Meanwhile the fusion egg
It is white that there is longer half-life period in human body with thrombopoietic characteristic is remarkably promoted, it can be used for preparing and treat a variety of originals
The drug of the diseases such as hair property or secondary thrombocytopenia.
Specific embodiment presented below with realize a kind of thrombopoietin of the present invention fusion protein and its
Preparation method and application.But it is not limited to these embodiments.
Description of the drawings：
Fig. 1：PcDNA3.1-HSA carriers inserted with HSA molecular genes, the fusion as structure thrombopoietin
The template of protein expression vector.
Fig. 2：The fusion protein expression vector collection of illustrative plates of thrombopoietin
Specific implementation mode
Major experimental instrument：
Liquid-transfering gun, superclean bench (safe and sound), magnetic stirring apparatus, micro-wave oven, high-temp steam sterilizing pot, -80 DEG C of Low-temperature Ices
Case (Forma), ultra-pure water instrument (Millipore), ice machine, centrifuge (Hitachi), HDB-PLUS types constant-temperature metal bath,
HZQ-F16OA types constant-temperature shaking incubator (Shanghai one is permanent), PCR instrument (Applied Biosystems), tabletop refrigerated centrifuge
(Thermo), DYY-8B types electrophoresis apparatus (Bole), 300 type gel imagers (GE) of Image Quant etc..
Major experimental material：
1. restriction endonuclease KpnI, EcoRI, SacI, AflII (NEB Products, the U.S.)
2. small put forward plasmid kit, PCR purification kits, DNA plastic recovery kits (Sheng Gong companies, China)
3.T4DNA connections enzyme reagent kit (Takara Products, DaLian, China)
4. carrier pPink α-HC, thermophilic pichia methanolica bacterial strain (Invitrogen Products, the U.S.)
5. Escherichia coli TOP10 (TIANGEN Biotech (Beijing) Co., Ltd.)
6. yeast extract, peptone (Oxford Products, the U.S.)
7.LB culture mediums
Yeast extract 5g, peptone 10g, NaCl 10g, is dissolved in 1000ml deionized waters, the NaOH of 1mol/L is used in combination
PH value is adjusted to 7.0, autoclaving.
8.YPD culture mediums
Yeast extract 10g, tryptone 20g, Agar 20g, is dissolved in 900ml deionized waters, high pressure sterilization, cooling
20% dextroses of the 100ml after filter degerming is added afterwards.
9.YPDS culture mediums
Yeast extract 10g, peptone 20g, D-sorbite 182.2g are dissolved in 900ml deionized waters, high pressure sterilization,
20% dextroses of the 100ml after filter degerming is added after cooling.
10.BMGY fluid nutrient mediums
Yeast extract 10g, peptone 20g, no amino acid yeast nitrogen 13.4g, glycerine 10g, potassium phosphate 26.631g,
It is dissolved in the sterilizing of 1000ml distilled water mesohighs, is cooled to room temperature, adjusts pH to 6.0,4 DEG C save backup.
The configuration of 11.1% Ago-Gel
According to dosage, the TAE buffer solutions per 100ml are added 1g agaroses, are boiled using microwave stove heat, make agarose
Melt completely, a small amount of ethidium bromide (EB) is added dropwise in room temperature when being cooled to non-scald on hand, be poured into after mixing and be well placed comb in advance
Glue groove in, until room temperature is cooled to after solidification completely, to take out comb i.e. usable.
The construction and expression of embodiment 1HSA-TMP fusion protein Yeast expression carriers
One, the acquisition of p29-simple-TMP sequences
1. first according to thermophilic pichia methanolica preference codon to coding (TMP)2Gene order optimize.
2. entrusting (TMP) after Dalian Takara companies synthesis optimizing2Gene, (TMP)2DNA sequence dna such as SEQ ID NO：
Shown in 1, and it is loaded into p29-simple (offer of p29-simple plasmid vectors Dalian Takara companies), obtains carrier
p29-simple-(TMP)2。
3. having included L in wherein p29-simple-TMP, that is, peptide is connected, LDNA sequences are
GGCGGCGGCGGTTCCGGACTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCGAATTCGGTGGTGG
CGGCAGC, amino acid sequence are GGGGSGLEPKSCDKTHTCPPCEF GGGGS.
Two, the clone of the segment of HSA cDNA：
Obtain from plasmid pcDNA3.1-fip-HSA clone (HSA complete genome sequences are synthesized by precious biotech firm's full genome,
PcDNA3.1 plasmids are purchased from Invitrogen)
Three, the acquisition of HSA molecular sequences
1. designing synthetic pcr primer object：
P1:GGTACCTCATAAGCCTAAGGCAGCTTG
P2:TCCGGAGATGCACACAAGAGTGAG
2.PCR is expanded：Using the DNA of carrier pcDNA3.1-HSA as masterplate, using P1 and P2 as upstream and downstream primer,
Carry out PCR amplification.Reaction condition is as follows：1. being denaturalized：94 DEG C, 5min；2. being denaturalized：94 DEG C, 1min；3. renaturation：55 DEG C, 30S；④
Extend：72 DEG C, 2min；5. return to step " 2. ", 35 cycles；6. extending：72 DEG C, 5min, global cycle number is 30 times.By PCR
Product carries out 1% agarose gel electrophoresis, and as a result display amplifies the DNA bands of the HSA molecules of about 1.8kb sizes.
Four, pPink α-HC-HSA- (TMP)2The structure of fusion protein Yeast expression carrier
1. extracting p29-simple- (TMP)2Carrier, KpnI and EcoRI double digestion plasmids, glue recycle corresponding TMP
(KpnI/EcoRI) DNA fragmentation, DNA sequence dna such as SEQ ID NO：Shown in 1, amino acid sequence such as SEQ ID NO：Shown in 2；
2. by product EcoRI and SacI double digestion of the HSA molecules after PCR amplification, glue recycles corresponding HSA (EcoRI/
SacI) DNA fragmentation, DNA sequence dna such as SEQ ID NO：Shown in 3, amino acid sequence such as SEQ ID NO：Shown in 4；
3. simultaneously, the DNA of SacI and KpnI double digestions carrier pPink α-HC (Invitrogen Products), glue recycling
PPink α-HC (SacI/KpnI) carrier segments；
4.T4DNA enzymes connect TMP (KpnI/EcoRI) DNA fragmentation, HSA (EcoRI/SacI) DNA fragmentations and pPink α-HC
(SacI/KpnI) carrier segments, transformed competence colibacillus Escherichia coli TOP10 are coated on ammonia benzyl resistance LB 37 DEG C of overnight incubations of plate, sieve
Select positive colony.Institute's clone is sent to Invitrogen for sequencing, and the correct clone designation of sequence is pPINK α-HC-HSA-
(TMP)2。
Five, expression of the HSA-TMP fusion proteins in yeast
Correct carrier pPink α-HC-HSA- (TMP) will be sequenced2DNA with AflII digestions recycle after obtain pPink α-
HC-HSA-(TMP)2, transformed yeast competent cell.Then transformed bacteria solution is inoculated in PAD tablets, 30 DEG C are cultivated 3-4 days, are chosen
Take positive colony.It will obtain positive colony and be inoculated with BMGY fluid nutrient mediums respectively, 30 DEG C are cultivated 48 hours, and BMMY is then forwarded to
Induced expression in culture medium, after continuing 96 hours, 1500rpm low-temperature centrifugations 15 minutes take supernatant, SDS-PAGE electrophoresis detection eggs
White expression, molecular weight about 70kD protein bands are HSA-TMP fusion proteins, and the amino acid sequence of the fusion protein is such as
SEQ ID NO：6, the DNA sequence dna such as SEQ ID NO of the amino acid sequence of encoding said fusion protein：Shown in 5.Selection expression water
Highest bacterial strain is put down as engineering bacteria, is frozen in -80 DEG C of conservations
Embodiment 2HSA- (TMP)2The bioactivity of fusion protein detects
1, experimentation
Plating cells, c-mpl, c-fos, pAdVAntage, Renilla, four plasmid co-transfections.48h after transfection adds respectively
Enter positive control, blank control and given the test agent, detects firefly luciferase vigor and renilla luciferase vigor, meter respectively
Calculate ratio fluorescent.
Positive control：Spy has than Australia (recombination human thrombopoietin injection) purchase from Shenyang three lives pharmacy than Australia spy
Limit responsible company
Blank control：Serum
Ratio fluorescent=firefly fluorescent value/sea pansy fluorescent value.
Specific steps refer to Chinese patent (CN201310084212).
2. experimental result
Table 1HSA- (TMP)2The bioactivity testing result of fusion protein
| Experimental group | Relative fluorescence ratio |
| Blank control group | 1.00±0.02 |
| Positive controls | 1.44±0.12 |
| 1-1 yeast strains | 1.37±0.05 |
| 1-3 yeast strains | 1.68±0.01 |
| 1-4 yeast strains | 1.70±0.04 |
| 2-1 yeast strains | 1.80±0.16 |
| 2-2 yeast strains | 1.69±0.12 |
| 2-4 yeast strains | 2.01±0.16 |
| 3-2 yeast strains | 1.11±0.03 |
| 3-3 yeast strains | 1.27±0.09 |
| 3-4 yeast strains | 1.37±0.11 |
| 4-1 yeast strains | 2.00±0.05 |
| 4-2 yeast strains | 2.98±0.09 |
| 4-3 yeast strains | 1.97±0.04 |
| 4-4 yeast strains | 1.79±0.06 |
The result shows that all pcDNA3.1-HSA empty carrier each processing groups of the transfection without c-mpl receptors, relative fluorescence ratio
Rate is 1.Reason group data see the above table 1 everywhere in all transfection c-mpl-pcDNA3.1 carriers.13 plants of ferment are detected as shown in Table 1
HSA- (TMP) in female bacterium expression sample2Fusion protein all has the bioactivity of significant c-mpl receptor-independents.Wherein 4-2
Fusion protein activity expressed by number yeast strain is significantly higher than positive control.Te Biao positive drugs and by TMP bigeminy
Body nucleotide sequence carries out the improved 13 plants of HSA- (TMP) of preferred codons2Fusion protein Yeast expression bacterial strain all have by
Bioactivity that body relies on, wherein No. 2-1, No. 2-4, No. 4-1, No. 4-2, No. 4-3, the fusion proteins of 4-4 Yeast expressions
Bioactivity has obtained conspicuousness raising.
It is disclosed in the preparation and application of the Dimerized fusion protein of patent No. CN201310084212.8 Dimerized
The preparation and application of thrombopoietin (TPO) simulating peptide TMP dyads-human serum albumin fusion proteins, in the patent
Dimerized thrombopoietin simulating peptide TMP dyads-human serum albumin fusion proteins c-mpl receptors of Yeast expression
The cellular level bioactivity average value of dependence is 1.635, the fusion protein biology of thrombopoietin disclosed by the invention
Activity determination result average value is 1.749；
Therefore, the present invention improves melting for thrombopoietin by implementing preferred codons transformation to TMP dyads
The mean value of hop protein bioactivity, higher than the fusion not generated to thrombopoietin simulating peptide TMP dyad genetic modifications
The active mean value of albumen.
In addition, it is No. 2 yeast strains expression that fusion protein activity disclosed in CN201310084212.8 patents is highest
Dimer fusion protein, bioactivity are 1.71 ± 0.11, No. 2-1 disclosed by the invention, No. 2-4, No. 4-1, No. 4-2,4-3
Number, No. 4-4 bioactivity be above the dimer fusion protein of above-mentioned No. 2 yeast strains expression.
By implementing preferred codons transformation to TMP dyads, the fusion protein of thrombopoietin is not only increased
The mean value of bioactivity, it is No. 2-1 disclosed by the invention, No. 2-4, No. 4-1, No. 4-2, No. 4-3, No. 4-4, what the yeast generated
The activity of fusion protein is also significantly improved.
Sequence table (SEQUENCE LISTING)
<110>Lanzhou University
<120>A kind of fusion protein of thrombopoietin
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 99
<212> DNA
<213>The gene order of TMP dyads
<400> 1
attgagggtc caactttgag acaatggttg gctgctagag ccggtggtcc atctggaatc 60
gagggaccta ccctgagaca gtggcttgct gctagagct 99
<210> 2
<211> 33
<212> PRT
<213>The amino acid sequence of TMP dyads
<400> 2
1 Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly Pro Ser Gly Ile
21 Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala
<210> 3
<211> 1755
<212> DNA
<213>The gene order of HSA molecules
<400> 3
gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60
gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120
aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtcagctgaa 180
aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240
cgtgaaacct atggtgaaat ggctgactgt tgtgcaaaac aagaacctga gagaaatgaa 300
tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360
gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420
gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480
tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540
aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaagtgt 600
gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660
cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720
gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780
gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840
aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900
gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960
gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020
tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080
tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140
gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200
tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260
ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320
cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380
tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440
ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500
gagtttaatg ctgaaacgtt caccttccat gcagatatat gcacactttc tgagaaggag 1560
agacaaatca agaaacaaac tgcacttgtt gagcttgtga aacacaagcc caaggcaaca 1620
aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680
gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740
gctgccttag gctta 1755
<210> 4
<211> 585
<212> PRT
<213>The amino acid sequence of HSA molecules
<400> 4
1 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys
21 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val
41 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu
61 Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
81 Arg Glu Thr Tyr Gly Glu MET Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu
101 Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val
121 Asp Val MET Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr
141 Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg
161 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro
181 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys
201 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser
221 Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
241 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu
261 Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu
281 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu MET Pro Ala
301 Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
321 Glu Ala Lys Asp Val Phe Leu Gly MET Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp
341 Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys
361 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu
381 Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu
401 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr
421 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His
441 Pro Glu Ala Lys Arg MET Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
461 Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
481 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys
501 Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu
521 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr
541 Lys Glu Gln Leu Lys Ala Val MET Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys
561 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln
581 Ala Ala Leu Gly Leu
<210> 5
<211> 2010
<212> DNA
<213>The gene order of ss-TMP-L1-TMP-L2-H-fip-HSA fusion proteins
<400> 5
aagtgggtaa cctttatttc ccttcttttt ctctttagct cggcttattc caggggtgtg 60
tttcgtcgag atgcacacaa gagtgaggtt gctcatcggt ttaaagattt gggagaagaa 120
aatttcaaag ccttggtgtt gattgccttt gctcagtatc ttcagcagtg tccatttgaa 180
gatcatgtaa aattagtgaa tgaagtaact gaatttgcaa aaacatgtgt tgctgatgag 240
tcagctgaaa attgtgacaa atcacttcat accctttttg gagacaaatt atgcacagtt 300
gcaactcttc gtgaaaccta tggtgaaatg gctgactgtt gtgcaaaaca agaacctgag 360
agaaatgaat gcttcttgca acacaaagat gacaacccaa acctcccccg attggtgaga 420
ccagaggttg atgtgatgtg cactgctttt catgacaatg aagagacatt tttgaaaaaa 480
tacttatatg aaattgccag aagacatcct tacttttatg ccccggaact ccttttcttt 540
gctaaaaggt ataaagctgc ttttacagaa tgttgccaag ctgctgataa agctgcctgc 600
ctgttgccaa agctcgatga acttcgggat gaagggaagg cttcgtctgc caaacagaga 660
ctcaagtgtg ccagtctcca aaaatttgga gaaagagctt tcaaagcatg ggcagtagct 720
cgcctgagcc agagatttcc caaagctgag tttgcagaag tttccaagtt agtgacagat 780
cttaccaaag tccacacgga atgctgccat ggagatctgc ttgaatgtgc tgatgacagg 840
gcggaccttg ccaagtatat ctgtgaaaat caagattcga tctccagtaa actgaaggaa 900
tgctgtgaaa aacctctgtt ggaaaaatcc cactgcattg ccgaagtgga aaatgatgag 960
atgcctgctg acttgccttc attagctgct gattttgttg aaagtaagga tgtttgcaaa 1020
aactatgctg aggcaaagga tgtcttcctg ggcatgtttt tgtatgaata tgcaagaagg 1080
catcctgatt actctgtcgt gctgctgctg agacttgcca agacatatga aaccactcta 1140
gagaagtgct gtgccgctgc agatcctcat gaatgctatg ccaaagtgtt cgatgaattt 1200
aaacctcttg tggaagagcc tcagaattta atcaaacaaa attgtgagct ttttgagcag 1260
cttggagagt acaaattcca gaatgcgcta ttagttcgtt acaccaagaa agtaccccaa 1320
gtgtcaactc caactcttgt agaggtctca agaaacctag gaaaagtggg cagcaaatgt 1380
tgtaaacatc ctgaagcaaa aagaatgccc tgtgcagaag actatctatc cgtggtcctg 1440
aaccagttat gtgtgttgca tgagaaaacg ccagtaagtg acagagtcac caaatgctgc 1500
acagaatcct tggtgaacag gcgaccatgc ttttcagctc tggaagtcga tgaaacatac 1560
gttcccaaag agtttaatgc tgaaacgttc accttccatg cagatatatg cacactttct 1620
gagaaggaga gacaaatcaa gaaacaaact gcacttgttg agcttgtgaa acacaagccc 1680
aaggcaacaa aagagcaact gaaagctgtt atggatgatt tcgcagcttt tgtagagaag 1740
tgctgcaagg ctgacgataa ggagacctgc tttgccgagg agggtaaaaa acttgttgct 1800
gcaagtcaag ctgccttagg cttaggcggc ggcggttccg gactggagcc caagagctgc 1860
gacaagaccc acacctgccc tccctgcgaa ttcggtggtg gtggttctat tgagggtcca 1920
actttgagac aatggttggc tgctagagcc ggtggtccat ctggaatcga gggacctacc 1980
ctgagacagt ggcttgctgc tagagcttaa 2010
<210> 6
<211> 670
<212> PRT
<213>The amino acid sequence of ss-TMP-L1-TMP-L2-H-fip-HSA fusion proteins
<400> 6
1 Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val
21 Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu
41 Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu
61 Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu
81 Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val
101 Ala Thr Leu Arg Glu Thr Tyr Gly Glu MET Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu
121 Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg
141 Pro Glu Val Asp Val MET Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys
161 Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe
181 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys
201 Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg
221 Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala
241 Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp
261 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg
281 Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu
301 Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu
321 MET Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys
341 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly MET Phe Leu Tyr Glu Tyr Ala Arg Arg
361 His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu
381 Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe
401 Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln
421 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln
441 Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys
461 Cys Lys His Pro Glu Ala Lys Arg MET Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu
481 Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys
501 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr
521 Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser
541 Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro
561 Lys Ala Thr Lys Glu Gln Leu Lys Ala Val MET Asp Asp Phe Ala Ala Phe Val Glu Lys
581 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala
601 Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Gly Gly Ser Gly Leu Glu Pro Lys Ser Cys
621 Asp Lys Thr His Thr Cys Pro Pro Cys Glu Phe Gly Gly Gly Gly Ser Ile Glu Gly Pro
641 Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly Pro Ser Gly Ile Glu Gly Pro Thr
661 Leu Arg Gln Trp Leu Ala Ala Arg Ala ***
<210> 7
<211> 42
<212> DNA
<213>The gene order of H peptide fragments
<400> 7
gagcccaaga gctgcgacaa gacccacacc tgccctccct gc 42
<210> 8
<211> 14
<212> PRT
<213>The amino acid sequence of H peptide fragments
<400> 8
1 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
<210> 9
<211> 114
<212> DNA
<213>The gene order of peptide fragment selected from fip structural domains
<400> 9
gtgagcaggg acgagctgat ggaggccatc cagaagcagg aggagatcaa cttcaggctg 60
caggactaca tcgacaggat catcgtggcc atcatggaga ccaaccccag catc 114
<210> 10
<211> 38
<212> PRT
<213>The amino acid sequence of peptide fragment selected from fip structural domains
<400> 10
1 Val Ser Arg Asp Glu Leu MET Glu Ala Ile Gln Lys Gln Glu Glu Ile Asn Phe Arg Leu
21 Gln Asp Tyr Ile Asp Arg Ile Ile Val Ala Ile MET Glu Thr Asn Pro Ser Ile
Claims (23)
1. a kind of fusion protein of thrombopoietin, including the simulation of human serum albumin HSA molecule, thrombopoietin
Peptide TMP dyads, the amino acid sequence such as SEQ ID NO of the HSA molecules：Shown in 4 or after the amino acid sequence is mutated
Sequence with said function encodes the DNA sequence dna such as SEQ ID NO of the amino acid sequence of the HSA molecules：It is described shown in 3
Thrombopoietin simulating peptide TMP dyads amino acid sequence such as SEQ ID NO：Shown in 2 or the amino acid sequence passes through
Sequence with said function after mutation, which is characterized in that the thrombopoietin simulating peptide TMP dyads are by one
Made of a yeast preferred codons coding, the amino acid sequence of the thrombopoietin simulating peptide TMP dyads is encoded
DNA sequence dna such as SEQ ID NO：Shown in 1, which further includes the peptide fragment that can form Dimerized structural domain.
2. a kind of fusion protein of thrombopoietin according to claim 1, which is characterized in that described forms
Contain at least two cysteines in the peptide fragment of Dimerized structural domain.
3. a kind of fusion protein of thrombopoietin according to claim 1, which is characterized in that described forms
Containing there are two cysteines in the peptide fragment of Dimerized structural domain.
4. a kind of fusion protein of thrombopoietin according to claim 3, which is characterized in that described forms
The peptide fragment of Dimerized structural domain is H peptide fragments, amino acid sequence such as Seq ID No:Shown in 8.
5. a kind of fusion protein of thrombopoietin according to claim 3, which is characterized in that dimer can be formed
The peptide fragment for changing structural domain is H peptide fragments and the peptide fragment selected from fip structural domains, the amino acid sequence such as Seq ID No of H peptide fragments:8 institutes
Show, the amino acid sequence such as Seq ID No of the peptide fragment selected from fip structural domains:Shown in 10.
6. a kind of fusion protein of thrombopoietin according to claim 4, which is characterized in that the HSA molecules
The ends C- of fusion protein are connected to, structural formula is expressed as HSA-L3-H-L4-TMP-L1-TMP, and L1, L3, L4 indicate peptide linker,
The amino acid sequence of L1 is GGPSG, and the amino acid sequence of L3 is GGGGSGL, and the amino acid sequence of L4 is EFGGGGS, and H expressions can
Form the H peptide fragments of Dimerized structural domain.
7. a kind of fusion protein of thrombopoietin according to claim 5, which is characterized in that the HAS molecules
Positioned at the ends N- of fusion protein, structural formula is expressed as TMP-L1-TMP-L2-H-fip-HSA, L1 and L2 and indicates peptide linker, L1
Amino acid sequence be GGPSG, the amino acid sequence of L2 is GGGGSRS, and H indicates that the H peptide fragments of Dimerized structural domain can be formed,
Fip indicates the peptide fragment selected from fip structural domains；The amino acid sequence of the fusion protein such as SEQ ID NO：Shown in 6, described in coding
The DNA sequence dna of the nucleotide sequence of fusion protein such as SEQ ID NO：Shown in 5.
8. the Dimerized fusion protein that a kind of fusion protein of thrombopoietin described in claim 6 is formed.
9. the Dimerized fusion protein that a kind of fusion protein of thrombopoietin described in claim 7 is formed.
10. a kind of fusion protein of thrombopoietin, including human serum albumins HAS molecules, thrombopoietin mould
Peptidomimetic TMP dyads, the amino acid sequence such as SEQ ID NO of the human serum albumins HAS molecules：Shown in 4 or the amino
The sequence with said function, encodes the DNA sequence dna such as SEQ ID of the amino acid sequence of the HAS molecules after acid sequence is mutated
NO：Shown in 3, the amino acid sequence such as SEQ ID NO of the thrombopoietin simulating peptide TMP dyads：Shown in 2, or
Sequence with said function after the amino acid sequence is mutated, which is characterized in that the thrombopoietin simulating peptide
TMP dyads are made of being encoded by a yeast preferred codons, to encode the thrombopoietin simulating peptide TMP bis-
The DNA sequence dna of conjuncted amino acid sequence such as SEQ ID NO：Shown in 1, which further includes Dimerized structural domain.
11. a kind of fusion protein of thrombopoietin according to claim 10, which is characterized in that the dimerization
Body structural domain is to keep the fusion protein of thrombopoietin Dimerized by the covalent effect of disulfide bond.
12. a kind of fusion protein of thrombopoietin according to claim 11, which is characterized in that the dimerization
The disulfide bond formed by cysteine containing at least two in body structural domain.
13. a kind of fusion protein of thrombopoietin according to claim 12, which is characterized in that the dimerization
Containing there are two the disulfide bond formed by cysteine in body structural domain.
14. a kind of fusion protein of thrombopoietin according to claim 12, which is characterized in that the dimerization
Body structural domain includes H peptide fragments, the amino acid sequence such as Seq ID No of H peptide fragments:Shown in 8.
15. a kind of fusion protein of thrombopoietin according to claim 12, which is characterized in that the dimerization
Body structural domain includes H peptide fragments and the peptide fragment selected from fip structural domains, the amino acid sequence such as Seq ID No of H peptide fragments:Shown in 8,
The amino acid sequence of peptide fragment selected from fip structural domains such as Seq ID No:Shown in 10.
16. the fusion protein of thrombopoietin according to claim 14, which is characterized in that the HSA molecules connect
The ends C- of fusion protein are connected to, structural formula is expressed as HSA-L3-H-L4-TMP-L1-TMP, and L1, L3, L4 indicate peptide linker, L1
Amino acid sequence be GGPSG, the amino acid sequence of L3 is GGGGSGL, and the amino acid sequence of L4 is EFGGGGS, and H expressions can shape
At the H peptide fragments of Dimerized structural domain.
17. a kind of fusion protein of thrombopoietin according to claim 15, which is characterized in that the HSA points
Son is located at the ends N- of fusion protein, and structural formula is expressed as TMP-L1-TMP-L2-H-fip-HSA, L1 and L2 and indicates peptide linker,
The amino acid sequence of L1 is GGPSG, and the amino acid sequence of L2 is GGGGSRS, and H indicates that the H peptides of Dimerized structural domain can be formed
Section, fip indicate the peptide fragment selected from fip structural domains；The amino acid sequence of the fusion protein such as SEQ ID NO：Shown in 6, coding
The DNA sequence dna of the nucleotide sequence of the fusion protein such as SEQ ID NO：Shown in 5.
18. the Dimerized fusion protein that a kind of fusion protein of thrombopoietin described in claim 16 is formed.
19. the Dimerized fusion protein that a kind of fusion protein of thrombopoietin described in claim 17 is formed.
20. a kind of fusion protein of thrombopoietin according to claim 1 or 10, it is characterised in that the fusion
Albumen is prepared using yeast cell to express.
21. a kind of fusion protein of thrombopoietin according to claim 20, it is characterised in that the yeast
For thermophilic pichia methanolica.
22. a kind of fusion protein of thrombopoietin described in claim 1 to 19 any one is primary in preparation treatment
Application in the drug of property or secondary thrombocytopenia.
23. a kind of preparation method of the fusion protein of thrombopoietin as described in claim 1 or 10, feature exist
In described steps are as follows：
1) the thrombopoietin simulating peptide TMP dyad sequences of full genome synthetic yeast preferred codons coding；
2) HSA molecular sequences are obtained by PCR amplification；
3) by digestion with restriction enzyme, connection and Escherichia coli are converted, obtains and compiles containing encoding the yeast preferred codons
The recombinant expression of the thrombopoietin simulating peptide dyad of code and the DNA sequence dna of the fusion protein of human serum albumins molecule
Carrier；
4) recombinant expression carrier described in step 3) is transformed into competent E.coli TOP10, then is transformed into host expresses system
System is expressed to get the fusion protein.
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| MEIZHU WANG等: "Functional expression of human serum albumin-tandem thrombopoietin mimetic peptide fusion protein as a novel thrombopoietin analog in Pichia pastoris", 《BIOTECHNOLOGY LETTERS》 * |
| 刘朔: "纳豆激酶基因密码子优化设计与合成及在毕赤酵母中的高效表达", 《中国博士学位论文全文数据库》 * |
| 杨洋等: "重组双体化促血小板生成素模拟肽人血清白蛋白融合蛋白(HSA-TMP-TMP)体内生物活性与药效学研究", 《药物生物技术》 * |
| 王海晴等: "促血小板生成素模拟肽二联体连接肽的筛选", 《药物生物技术》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109336974A (en) * | 2018-11-12 | 2019-02-15 | 兰州大学 | A kind of preparation method of anti-TMP rabbit polyclonal antibody |
| CN113527508A (en) * | 2020-04-17 | 2021-10-22 | 上海多米瑞生物技术有限公司 | Preparation method of thrombopoietin peptide-Fc fusion protein |
| CN113527508B (en) * | 2020-04-17 | 2024-01-05 | 上海多米瑞生物技术有限公司 | Preparation method of thrombopoietin peptidomimetic-Fc fusion protein |
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