CN108623690A - A kind of fusion protein of thrombopoietin and its preparation method and application - Google Patents

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

A fusion protein of thrombopoietin and its preparation method and application

technical field

The invention belongs to the field of genetic engineering pharmaceuticals, and relates to a fusion protein of thrombopoietin and its preparation method and application, in particular to a thrombopoietin mimetic peptide (Thrombopoietin Mimetic Peptide, TMP) duplex encoded by a yeast preferred codon Fusion protein with human serum albumin molecule (Human serum Albumin, HSA).

Background technique

In clinical practice, primary and secondary thrombocytopenia caused by various reasons are often encountered, such as primary thrombocytopenic purpura, aplastic anemia, and thrombocytopenia caused by tumor chemotherapy/radiotherapy. For such diseases, it is more suitable to use long-acting platelet-increasing drugs for treatment. On the one hand, it can reduce the number of medications and reduce the pain of acupuncture for patients; on the other hand, it can reduce the dosage of drugs and reduce treatment costs.

Platelets are produced by megakaryocytes, and promoting the proliferation and differentiation of megakaryocytes is the main means to increase the level of platelets. Thrombopoietin (TMP), also known as megakaryocyte growth and development factor (MGDF), is the most important regulator of bone marrow megakaryocyte proliferation, differentiation and platelet production. Shortly after the first successful cloning of TMP gene lacking annotation in 1994, people developed a polyethylene glycol (PEG) recombinant functional TMP genetic engineering product - PEG-rHuMGDF. The results of animal experiments show that PEG-rHuMGDF not only has outstanding platelet-promoting activity, but also has a long half-life in vivo, and a single application can significantly increase platelets. However, in the course of clinical trials, it was found that the antibodies produced by some healthy subjects after receiving the drug treatment had a cross-neutralization reaction with endogenous TMP, resulting in thrombocytopenia in the subjects, so that PEG-rHuMGDF and even The development of recombinant human TMP was stopped by the US FDA in 1998. Affected by it, recombinant human TMP has not been approved for clinical use in the United States, Europe, Japan and other countries.

Human serum albumin molecule (Albumin Human, HSA) is the main protein component in plasma, accounting for about 50-60% of total plasma protein. HSA molecule is a non-glycosylated single-chain protein consisting of 585 amino acid residues, its molecular weight is 66.5kDa (A.Dugaiczyk et al., PNAS, 1982, 79:71-75), and its plasma half-life is as long as More than two weeks. HSA molecules are synthesized in the form of a propeptide in host cells, which contains a signal peptide and a propeptide consisting of 24 amino acid residues, which are excised during transport and secretion. HSA molecule is a stable inert protein that can be used as a drug carrier. The following HSA molecules have been successfully expressed in multiple hosts (EP330451 and EP361991), especially in yeast cells, which can achieve a higher level of stable expression of HSA molecules. When the target polypeptide is fused with HSA molecule, it can not only improve the stability of the polypeptide drug, but also increase the half-life of the polypeptide drug in vivo. The preparation and application of the dimerization fusion protein of patent number CN201310084212.8 discloses the preparation and application of the dimerization thrombopoietin (TPO) mimetic peptide TMP duplex-human serum albumin molecular fusion protein, but It has the problem that the expression effect of the fusion protein is not ideal. In order to improve the expression yield and enhance the biological activity of the fusion protein, the present invention adopts the strategy of coding the TMP duplex of the thrombopoietin mimetic peptide with yeast preferred codons. Based on the above research status, the present invention now provides a fusion protein of thrombopoietin, the TMP duplex of the thrombopoietin mimetic peptide is encoded by yeast preferred codons, and the fusion protein can be expressed in yeast High-level stable expression in vivo can be applied to industrial production; at the same time, the fusion protein has the property of significantly promoting platelet production and has a longer half-life in the human body. The invention also provides the preparation method and application of the fusion protein.

The heterologous expression of recombinant proteins is affected by the sequence characteristics of the gene itself, such as codon preference, GC content, mRNA secondary structure, mRNA stability and other factors. Studies have shown that the replacement of synonymous codons can affect the rate of protein translation elongation [1-2], indicating that codon optimization may have a counterproductive effect on the expression of recombinant proteins. On the other hand, the expression of recombinant proteins is also affected by factors such as hosts, culture conditions, secretion pathways, and promoters. The current gene optimization theory and design methods are still immature, and there are various limitations. Gene optimization strategies It is a necessary condition, but not a sufficient condition, to increase the expression of recombinant protein. To sum up, the strategy of improving the expression level of genetically engineered strains through modification of yeast preferred codons has great uncertainty in actual production and application, and the final result of modification for different target genes is unpredictable.

references:

[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 pairuse within the (GGGGS)3linker sequence results in enhanced protein expression. Mol Immunol, 2004, 40(10): 717-722.

Contents of the invention

The purpose of the present invention is to provide a fusion protein of thrombopoietin, which has a long half-life and can be stably expressed at a high level in a host.

Another object of the present invention is to provide a method for preparing the above fusion protein.

Another object of the present invention is to provide the application of the above fusion protein.

The above-mentioned purpose described in the present invention is achieved through the following technical solutions:

A fusion protein of thrombopoietin disclosed by the present invention comprises human serum albumin HSA molecule and thrombopoietin mimetic peptide TMP duplex, and the amino acid sequence of said human serum albumin HSA molecule is shown as SEQ ID NO: 4, or a sequence having the same function after the amino acid sequence is mutated, the DNA sequence encoding the amino acid sequence of the HSA molecule is shown in SEQ ID NO: 3, and the thrombopoietin mimetic peptide TMP duplex The amino acid sequence of the amino acid sequence is shown in SEQID NO: 2, or a sequence having the same function after the amino acid sequence is mutated. The thrombopoietin mimetic peptide TMP duplex is encoded by a yeast-preferred codon, encoding the promoting The DNA sequence of the amino acid sequence of the thrombopoietin mimetic peptide TMP duplex is shown in SEQ ID NO: 1, and the fusion protein also includes a peptide segment that can form a dimerization domain.

In the fusion protein of thrombopoietin disclosed by the present invention, the peptide segment that can form the dimerization domain contains at least two cysteines.

In the fusion protein of thrombopoietin disclosed by the present invention, the peptide segment that can form a dimerization domain contains two cysteines.

In the fusion protein of thrombopoietin disclosed by the present invention, the peptide that can form the dimerization domain is H peptide, and its amino acid sequence is shown in Seq ID No:8.

A fusion protein of thrombopoietin disclosed by the present invention, the peptide segment that can form a dimerization domain is an H peptide segment and a peptide segment selected from the fip domain, and the amino acid sequence of the H peptide segment is as Seq ID No: 8, the amino acid sequence of the peptide selected from the fip domain is shown in Seq ID No:10.

A fusion protein of thrombopoietin disclosed by the present invention, wherein the HSA molecule is connected to the C-terminus of the fusion protein, the structural formula is expressed as HSA-L3-H-L4-TMP-L1-TMP, and L1, L3, and L4 represent peptides Linker, the amino acid sequence of L1 is GGPPSG, the amino acid sequence of L3 is GGGGSGL, the amino acid sequence of L4 is EFGGGGS, and H represents the H peptide that can form the dimerization domain.

A fusion protein of thrombopoietin disclosed by the present invention, wherein the HSA molecule is located at the N-terminal of the fusion protein, the structural formula is expressed as TMP-L1-TMP-L2-H-fip-HSA, L1 and L2 represent peptide linkers, L1 The amino acid sequence of L2 is GGPPG, the amino acid sequence of L2 is GGGGSRS, H represents the H peptide segment that can form a dimerization domain, and fip represents a peptide segment selected from the fip domain; the amino acid sequence of the fusion protein is as shown in SEQ ID NO : shown in 6, the DNA sequence encoding the nucleotide sequence of the fusion protein is shown in SEQ ID NO: 5.

The invention discloses a dimerized fusion protein formed by fusion protein of thrombopoietin.

The invention discloses a fusion protein of thrombopoietin, which comprises human serum albumin HSA molecule and thrombopoietin mimetic peptide TMP duplex, and the amino acid sequence of said human serum albumin HSA molecule is as shown in SEQ ID NO: 4 shown, or the sequence having the same function after the amino acid sequence is mutated, the DNA sequence encoding the amino acid sequence of the HSA molecule is shown in SEQ ID NO: 3, and the thrombopoietin mimic peptide TMP duplex The amino acid sequence is shown in SEQ ID NO: 2, or a sequence having the same function after the amino acid sequence is mutated. The TMP duplex of the thrombopoietin mimetic peptide is encoded by a yeast preferred codon, encoding the The DNA sequence of the amino acid sequence of the thrombopoietin mimetic peptide TMP duplex is shown in SEQ ID NO: 1, and the fusion protein also includes a dimerization domain.

The invention discloses a fusion protein of thrombopoietin, wherein the dimerization domain dimers the fusion protein of thrombopoietin through the covalent action of disulfide bonds.

In the fusion protein of thrombopoietin disclosed by the present invention, the dimerization domain contains at least two disulfide bonds formed by cysteine.

In the fusion protein of thrombopoietin disclosed by the invention, the dimerization domain contains two disulfide bonds formed by cysteine.

In the fusion protein of thrombopoietin disclosed by the present invention, the dimerization domain includes an H peptide segment, and the amino acid sequence of the H peptide segment is shown in Seq ID No:8.

In a fusion protein of thrombopoietin disclosed by the present invention, the dimerization domain includes an H peptide segment and a peptide segment selected from the fip domain, and the amino acid sequence of the H peptide segment is shown in Seq ID No: 8, selected from The amino acid sequence of the peptide from the fip domain is shown in Seq ID No:10.

In the fusion protein of thrombopoietin disclosed by the present invention, the HSA molecule is connected to the C-terminus of the fusion protein, and the structural formula is expressed as HSA-L3-H-L4-TMP-L1-TMP, and L1, L3, and L4 represent peptide linkers , the amino acid sequence of L1 is GGPPSG, the amino acid sequence of L3 is GGGGSGL, the amino acid sequence of L4 is EFGGGGS, and H represents the H peptide that can form the dimerization domain.

A fusion protein of thrombopoietin disclosed by the present invention, the HSA molecule is located at the N-terminal of the fusion protein, the structural formula is expressed as TMP-L1-TMP-L2-H-fip-HSA, L1 and L2 represent peptide linkers, The amino acid sequence of L1 is GGPPG, the amino acid sequence of L2 is GGGGSRS, H represents the H peptide segment that can form a dimerization domain, and fip represents a peptide segment selected from the fip domain; the amino acid sequence of the fusion protein is as shown in SEQ ID As shown in NO:6, the DNA sequence encoding the nucleotide sequence of the fusion protein is shown in SEQ ID NO:5.

The invention discloses a dimerization fusion protein formed by fusion protein of thrombopoietin.

The invention discloses a fusion protein of thrombopoietin, which is expressed and prepared by yeast cells.

The invention discloses a fusion protein of thrombopoietin, and the yeast is Pichia methanolophilus.

The application of a fusion protein of thrombopoietin disclosed by the invention in the preparation of medicine for treating primary or secondary thrombocytopenia.

A preparation method of a fusion protein of thrombopoietin disclosed by the present invention, the steps are as follows:

1) Whole-gene synthesis of the TMP duplex sequence encoded by yeast preferred codons;

2) Obtain the HSA molecular sequence by PCR amplification;

3) Digesting with restriction endonucleases, ligating and transforming Escherichia coli to obtain the DNA sequence encoding the fusion protein of the thrombopoietin mimetic peptide duplex encoded by the yeast preferred codon and the human serum albumin molecule Recombinant expression vector;

4) Transform the recombinant expression vector described in step 3) into competent Escherichia coli TOP10, and then transform into a host expression system for expression to obtain the fusion protein.

A recombinant expression vector containing the gene encoding the fusion protein of the above-mentioned thrombopoietin. The expression vectors that can be used to carry the gene encoding the fusion protein of the present invention include, but are not limited to, plasmids commonly used in prokaryotic and eukaryotic expression systems.

A host expression system containing the above-mentioned recombinant expression vector. The host can be bacteria, yeast, mammalian cells, etc., wherein yeast is preferred, and Pichia methanolophilus is more preferred; the fusion protein of the present invention expressed recombinantly can be extracted from the corresponding cell culture by various methods , Purification, these methods include techniques such as centrifugation, ultrafiltration and liquid chromatography, among which liquid chromatography includes chromatography techniques such as ion exchange, hydrophobicity and molecular sieves.

The fusion protein of the present invention can be used together with pharmaceutical carriers to form pharmaceutical preparations, and these pharmaceutical carriers include water, saline, sugars, alcohols, amino acids and the like. The pharmaceutical preparation made of the fusion protein of the present invention is preferably a lyophilized preparation with a water content of less than 3% or no water, and the administration methods include intravenous infusion, injection (including subcutaneous and intramuscular injection), intranasal, respiratory tract, etc., wherein Subcutaneous or intramuscular injection is preferred.

The fusion protein can be stably and efficiently expressed in yeast, and can be applied to industrial production; at the same time, the fusion protein has the characteristic of significantly promoting platelet production, has a long half-life in the human body, and can be used to prepare and treat various primary diseases. Or drugs for diseases such as secondary thrombocytopenia.

Specific examples are provided below to realize a thrombopoietin fusion protein of the present invention and its preparation method and application. But not limited to these examples.

Description of drawings:

Figure 1: The pcDNA3.1-HSA vector inserted with the HSA molecular gene is used as a template for constructing a fusion protein expression vector of thrombopoietin.

Figure 2: The fusion protein expression vector map of thrombopoietin

Detailed ways

Main experimental instruments:

Pipette gun, ultra-clean bench (Antai), magnetic stirrer, microwave oven, high-temperature steam sterilizer, -80°C low-temperature refrigerator (Forma), ultra-pure water instrument (Millipore), ice maker, centrifuge (Hitachi) , HDB-PLUS constant temperature metal bath, HZQ-F16OA constant temperature shaking incubator (Shanghai Yiheng), PCR instrument (Applied Biosystems), desktop refrigerated centrifuge (Thermo), DYY-8B electrophoresis instrument (Bole), Image Quant 300 gel imager (GE), etc.

Main experimental materials:

1. Restriction endonucleases KpnI, EcoRI, SacI, AflII (products of NEB Company, USA)

2. Small extraction plasmid kit, PCR purification kit, DNA gel recovery kit (Shenggong Company, China)

3. T4 DNA Ligase Kit (product of Takara Company, Dalian, China)

4. Vector pPinkα-HC, Pichia methanolophilus strain (product of Invitrogen, USA)

5. Escherichia coli TOP10 (Tiangen Biochemical Technology (Beijing) Co., Ltd.)

6. Yeast extract, peptone (product of Oxford, USA)

7. LB medium

5 g of yeast extract, 10 g of peptone, and 10 g of NaCl were dissolved in 1000 ml of deionized water, adjusted to pH 7.0 with 1 mol/L NaOH, and sterilized by autoclaving.

8. YPD medium

10 g of yeast extract, 20 g of tryptone, and 20 g of Agar were dissolved in 900 ml of deionized water, sterilized by high pressure, and after cooling, 100 ml of 20% dextrose sterilized by a filter were added.

9. YPDS medium

10 g of yeast extract, 20 g of peptone, and 182.2 g of sorbitol were dissolved in 900 ml of deionized water, sterilized by high pressure, and 100 ml of 20% dextrose sterilized by a filter were added after cooling.

10. BMGY liquid medium

Yeast extract 10g, peptone 20g, amino acid-free yeast nitrogen source 13.4g, glycerol 10g, potassium phosphate 26.631g, dissolve in 1000ml double distilled water, autoclave, cool to room temperature, adjust pH to 6.0, store at 4°C for later use.

11.1% Agarose gel configuration

According to the dosage, for every 100ml of TAE buffer, add 1g of agarose, heat and boil in a microwave oven to completely melt the agarose, drop a small amount of ethidium bromide (EB) when it is cooled at room temperature until it is not hot, mix well and pour it into Place the comb in the glue tank in advance, wait until it cools at room temperature until it is completely solidified, then pull out the comb and use it.

Construction and expression of embodiment 1 HSA-TMP fusion protein yeast expression vector

1. Acquisition of p29-simple-TMP sequence

1. First, optimize the gene sequence encoding (TMP) ₂ according to the preferred codons of Pichia methanolophilus.

2. Entrust Dalian Takara Company to synthesize the optimized (TMP) ₂ gene, the DNA sequence of (TMP) ₂ is shown in SEQ ID NO: 1, and load it into p29-simple (p29-simple plasmid vector provided by Dalian Takara Company ) to obtain the vector p29-simple-(TMP) ₂ .

3. The p29-simple-TMP already contains L, namely the connecting peptide, the LDNA sequence is GGCGGCGGCGGTTCCGGACTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCGAATTCGGTGGTGGCGGCAGC, and the amino acid sequence is GGGGSGLEPKSCDKTHTCPPCEF GGGGS.

2. Cloning of fragments of HSA cDNA:

Obtained from plasmid pcDNA3.1-fip-HSA clone (HSA complete gene sequence was synthesized by Bao Biological Company, pcDNA3.1 plasmid was purchased from Invitrogen)

3. Obtaining the molecular sequence of HSA

1. Design and synthesize PCR primers:

P1: GGTACCTCATAAGCCTAAGGCAGCTTG

P2:TCCGGAGATGCACACAAGAGTGAG

2. PCR amplification: The DNA of the carrier pcDNA3.1-HSA was used as a template, and P1 and P2 were used as upstream and downstream primers respectively to carry out PCR amplification. The reaction conditions are as follows: ① Denaturation: 94°C, 5min; ② Denaturation: 94°C, 1min; ③ Renaturation: 55°C, 30S; ④ Extension: 72°C, 2min; ⑤ Return to step "②", 35 cycles; ⑥ Extend: 72°C, 5min, the total number of cycles is 30 times. The PCR product was subjected to 1% agarose gel electrophoresis, and the result showed that a DNA band of about 1.8 kb in size was amplified.

4. Construction of pPinkα-HC-HSA-(TMP) ₂ fusion protein yeast expression vector

1. Extract p29-simple-(TMP) ₂ vector, KpnI and EcoRI double digestion plasmid, gel recovery corresponding TMP (KpnI/EcoRI) DNA fragment, the DNA sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: as shown in 2;

2. Digest the HSA molecule amplified by PCR with EcoRI and SacI, and recover the corresponding HSA (EcoRI/SacI) DNA fragment from the gel. The DNA sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4 shown;

3. At the same time, SacI and KpnI double-enzyme-cut the DNA of the carrier pPinkα-HC (product of Invitrogen Company), and gel recovered the pPinkα-HC (SacI/KpnI) carrier fragment;

4. T4 DNA enzyme ligated TMP (KpnI/EcoRI) DNA fragments, HSA (EcoRI/SacI) DNA fragments and pPinkα-HC (SacI/KpnI) vector fragments, transformed into competent Escherichia coli TOP10, and coated on ampicillin-resistant LB plate 37 Cultivate overnight at ℃, and select positive clones. The obtained clone was sent to Invitrogen Company for sequencing, and the clone with the correct sequence was named pPINKα-HC-HSA-(TMP)2.

5. Expression of HSA-TMP fusion protein in yeast

DNA of vector pPinkα-HC-HSA-(TMP) ₂ sequenced correctly was digested with AflII and recovered to obtain pPinkα-HC-HSA-(TMP) ₂ , which was transformed into competent yeast cells. Then the transformed bacteria liquid was inoculated on the PAD plate, cultured at 30°C for 3-4 days, and positive clones were picked. The positive clones were inoculated into BMGY liquid medium, cultured at 30°C for 48 hours, and then transferred to BMMY medium to induce expression. After 96 hours, centrifuged at 1500rpm for 15 minutes at low temperature, took the supernatant, and detected protein expression by SDS-PAGE electrophoresis In this case, the protein band with a molecular weight of about 70kD is the HSA-TMP fusion protein, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 6, and the DNA sequence encoding the amino acid sequence of the fusion protein is shown in SEQ ID NO: 5. Select the strain with the highest expression level as the engineering strain, and freeze it at -80°C for preservation

The biological activity detection of embodiment 2HSA-(TMP) ₂ fusion protein

1. Experimental process

Cell plating, c-mpl, c-fos, pAdVAntage, Renilla, four plasmid co-transfection. 48 hours after transfection, the positive control, blank control and test samples were added to detect firefly luciferase activity and Renilla luciferase activity, respectively, and calculate the fluorescence ratio.

Positive control: TPIAO TPIAO (recombinant human thrombopoietin injection) purchased from Shenyang Sansheng Pharmaceutical Co., Ltd.

Blank control: serum

Fluorescence ratio = firefly fluorescence value/renilla fluorescence value.

For specific steps, see Chinese patent (CN201310084212).

2. Experimental results

The biological activity detection result of table 1HSA-(TMP) ₂ fusion protein

test group relative fluorescence ratio Blank control group 1.00±0.02 positive control group 1.44±0.12 Yeast strain No. 1-1 1.37±0.05 Yeast strains 1-3 1.68±0.01 Yeast strains 1-4 1.70±0.04 Yeast strain No. 2-1 1.80±0.16 Yeast strain No. 2-2 1.69±0.12 Yeast strains 2-4 2.01±0.16 Yeast strain No. 3-2 1.11±0.03 Yeast strain No. 3-3 1.27±0.09 Yeast strain No. 3-4 1.37±0.11 Yeast strain No. 4-1 2.00±0.05 Yeast strain No. 4-2 2.98±0.09 Yeast strain No. 4-3 1.97±0.04 Yeast strain No. 4-4 1.79±0.06

The results showed that the relative fluorescence ratio of all treatment groups transfected with pcDNA3.1-HSA empty vector without c-mpl receptor was 1. The data of all treatment groups transfected with the c-mpl-pcDNA3.1 vector are shown in Table 1 above. It can be seen from Table 1 that the HSA-(TMP) ₂ fusion proteins in the 13 tested yeast expression samples all have significant c-mpl receptor-dependent biological activities. Among them, the activity of the fusion protein expressed by No. 4-2 yeast strain was significantly higher than that of the positive control. Tebio-positive drugs and 13 HSA-(TMP) ₂ fusion protein yeast expression strains after modifying the TMP duplex nucleotide sequence with preferred codons all have receptor-dependent biological activities, and No. 2-1 , No. 2-4, No. 4-1, No. 4-2, No. 4-3, and No. 4-4, the biological activity of the fusion protein expressed by the yeast has been significantly improved.

The preparation and application of dimerization fusion protein of patent number CN201310084212.8 discloses the preparation and application of dimerization thrombopoietin (TPO) mimetic peptide TMP duplex-human serum albumin fusion protein, the patent The dimerized thrombopoietin mimetic peptide TMP duplex-human serum albumin fusion protein c-mpl receptor-dependent average biological activity of the dimerized thrombopoietin expressed in yeast is 1.635, and the thrombopoietin disclosed in the present invention has The average value of the bioactivity test results of the fusion protein was 1.749;

Therefore, the present invention improves the mean value of the biological activity of the fusion protein of thrombopoietin by implementing preferred codon modification on the TMP duplex, which is higher than the fusion protein produced without genetic modification of the TMP duplex of the thrombopoietin mimetic peptide activity mean.

In addition, the fusion protein disclosed in the CN201310084212.8 patent has the highest activity of the dimeric fusion protein expressed by No. 2 yeast strain, and its biological activity is 1.71±0.11. -1, 4-2, 4-3, and 4-4 had biological activities higher than the dimer fusion protein expressed by the yeast strain No. 2 above.

By implementing preferred codon modification to the TMP duplex, not only the mean value of the biological activity of the fusion protein of thrombopoietin is improved, but No. 2-1, No. 2-4, No. 4-1 and No. 4-2 disclosed by the present invention , No. 4-3, and No. 4-4, the activity of the fusion protein produced by the yeast has also been significantly improved.

SEQUENCE LISTING

<110> Lanzhou University

<120> A fusion protein of thrombopoietin

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<170> PatentIn version 3.5

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Amino acid sequence of <213> TMP duplex

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gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60

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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

gtccaacacgg 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

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<213> Amino acid sequence of the HSA molecule

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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 Lys Leu Val Ala Ala Ser Gln

581 Ala Ala Leu Gly Leu

<210> 5

<211> 2010

<212>DNA

<213> Gene sequence of ss-TMP-L1-TMP-L2-H-fip-HSA fusion protein

<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 aaacatgtgttgctgatgag 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 ctaggcggc 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> Amino acid sequence of ss-TMP-L1-TMP-L2-H-fip-HSA fusion protein

<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> Gene sequence of H peptide

<400> 7

gagcccaaga gctgcgacaa gacccacacc tgccctccct gc 42

<210> 8

<211> 14

<212> PRT

Amino acid sequence of <213> H peptide

<400> 8

1 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

<210> 9

<211> 114

<212>DNA

<213> Gene sequence selected from peptides of fip domain

<400> 9

gtgagcaggg acgagctgat ggaggccatc cagaagcagg aggagatcaa cttcaggctg 60

caggactaca tcgacaggat catcgtggcc atcatggaga ccaacccccag catc 114

<210> 10

<211> 38

<212> PRT

<213> Amino acid sequence of a peptide selected from the fip domain

<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.