CN109748969A - A kind of Dimerized fusion protein and its preparation method and application - Google Patents

Abstract

The invention belongs to genetic engineering pharmaceutical fields, and in particular to a kind of Dimerized fusion protein and its preparation method and application.The Dimerized fusion protein includes 1 thrombopoietin simulating peptide (TMP) dyad protein structure domain, 1 human serum albumins third structural domain (3DHSA).Wherein, it is connected between TMP dyad, between TMP dyad and 3DHSA by link peptide and Dimerized structural domain.The Dimerized fusion protein of TMP dyad of the present invention and human serum albumins third structural domain can be acted on thrombopoietin receptor c-mpl, and the activity that there is stimulation TPO dependent cell system to be proliferated can be expressed in mammalian cells.It can be applied in the drug for preparing thrombopoietin, be effective drug candidate of thrombopoietin.The present invention also provides the preparation methods of the TMP dyad and the Dimerized fusion protein of human serum albumins third structural domain.

Description

A kind of Dimerized fusion protein and its preparation method and application

Technical field

The invention belongs to genetic engineering pharmaceutical fields, and in particular to a kind of thrombopoietin simulating peptide (Thrombopoietin mimetic peptide, TMP) dyad-human serum albumins third structural domain (The third Domain ofhuman serum albumin, 3DHSA) Dimerized fusion protein and its preparation method and application.

Background technique

With the maturation of DNA recombinant technique and the development of medicine biological technique, recombinant protein medicine is because of its amino acid sequence Popular domain of the advantages that column and base sequence understand, highly-safe as current new drug research, there is vast potential for future development. However having many small-molecular-weight polypeptide protein drugs since molecular weight is smaller, the unstable reason of molecular structure is easy in vivo It is degraded or is removed by kidney, cause such drug retention time in blood plasma short (golden light pool etc., 2010).Drug is in the short time Interior multiple injection, not only will cause the unstable of blood concentration, and can increase on body pain and economically to patient Pressure (Bian Lei, Shi Yifeng, 2009).Therefore, optimize the parameters such as validity and the long-term effect of pharmaceutical grade protein and have become biological system The important research direction (Zhao Hongliang etc., 2005) of medicine.

A large number of studies show that three similar structural domains together constitute HSA.According to each structural domain of independent studies HSA Structure and function obtain on each structural domain of HSA all containing some sites closed with cooperation base junction, but the ligand combined is respectively not Identical (Kato Y, et al.).Studies have shown that stabilize class drug binding site on HSA-D3, some anticoagulant binding sites In major part in HSA-D2, only a few is on HSA-D1.Research also confirms that main ligand binding site is mainly HSA's On HSA-D2 and HSA-D3 (Thomas Kjeldsen et al, 1998).The researchs such as Vania EKen anova have shown that IIIA and IIIB minor structure plays a significant role (Vania EKen anova etal, 2010) in the maintenance of albumin stability feature, The researchs such as Chaudhury have shown that all sites for possessing FcRn combination on HSA-D3.(Chaudhury et al,2006).Recently Numerous studies discovery, 3DHSA are the main portions that drug receptor combines, with the molecule that can increase destination protein after protein fusion Amount, is resistant to the degradation of acidic environment and protease under receptor-mediated endocytosis protective effect, has phase with HSA full-length proteins As function, and can reduce merge into protein purification difficulty and reduce clinical application when there is the risk being immunoreacted.Zhao Stating strong et al. Preliminary Study proves that, using pichia yeast expression system expressed fusion protein 3DHSA and HSA, the two is compared, melted Hop protein 3DHSA do not occur before signs of degradation, also do not occur polymerism in purification process, and can greater strength avoid kidney The dirty worry effect of crossing, enhances the stability of albumen.Expressed foreign protein 3DHSA-Nartograstim can stimulate mouse thin Born of the same parents NFS-60 increment, and at concentration-dependant (Zhao Shu strong etc., 2013).Therefore, the further investigation of HSA-D3 is further to grind Study carefully long-acting fusion protein drug and provides important evidence and clue.

1997, AF12505 was screened as high activity TPO simulating peptide by Cwirla etc..And prove that AF12505 is 14 The small peptide of a amino acid has the stimulation value-added function of rhTPO dependent cell strain (Shao Bo, 2006).But activity is very low, only It is the 1/4000 of rhTPO.After carrying out dimer modification to AF12505, activity is significantly improved.Then, AF12505 is carried out respectively Chemical modification and bio-modification, discovery IgG1Fc-TPO fusion protein have the increment function for promoting TPO dependent form cell strain. 2008, the Fc segment composition of AF12505 and antibody are developed product Nplate by Amgen, small for treating primary blood Plate reduces disease.The product has TPO activity, and not homologous with TPO, avoids the generation of neutrality antibody, but due to Fc piece Section is non-inert albumen, it can mediate panimmunity to react in vivo.Then, China's researcher is to overcome lacking for Nplate Point is connected AF12505 with HSA, has constructed fusion protein, but activity experiment is not the results show that this fusion protein has Have and promotes thrombopoietic bioactivity.

About the research of TMP-HSA depot drug product, on molecular structure, TMP monomer be by the small peptide of 14 Amino acid profiles, Amino acid sequence is IEGPTLRQWLAARA (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-LeU-Ala-Ala- Arg-Ala).This amino acid sequence and TPO do not have sequence homology, therefore crossover will not be caused to infect (Shao Bo, 2006). Theoretically, in the presence of TMP is in the form of dimer, the thrombopoietin receptor c-mpl that can be closer with two Respectively in connection with conduction tyrosine phosphorylation signal enters nucleus, activates the synthesis of albumen on ribosomes, promotes macrophage raw The maturation of the long factor promotes thrombocytopoiesis (Drachman, J.G et al., 1999).The connection of computer simulation show albumen Generally there are four types of existence forms by peptide linker (L), and amino acid sequence is respectively SG, GGGSG, GGPSG and GGGGSGGGSG.This reality Room early-stage study is tested by constructing TMP dyad using different L as the link peptide of TMP.Then, pass through lot of documents tune Grind, devised between HSA and TMP nucleic acid sequence one section of very short junction fragment (this segment be selected from immunoglobulin hinge region, Wherein contain cysteine, easily formation disulfide bond), keep HSA and TMP segment spatially relatively independent.And successfully construct weight Group carrier, is then integrated with yeast chromosomal, and success inducing expression goes out target protein, and is produced using 20 liters of ferment tanks Destination protein sample (Chinese patent 201310084035,2013) is gone out, has established TMP fusion protein external activity detection system (Chinese patent 201310084212,2013).

Researcher has found that HSA fusion protein can generate some common problems in Expression product process, for example, HSA merges egg White to occur to polymerize or degrade during expression, preservation, Bioactivity can reduce in various degree, and expression product is uneven One, expression it is low etc. (Cordes AAet al., 2012,2012；Liu Wenhui, 2015), it is pure that this phenomenon had both increased albumen The difficulty of change also makes the risk being immunoreacted increase occur during clinical application.

Recent study discovery, positioned at the HSA third structural domain (HSAD3,23kDa) of 381-585 amino acids residue It is key position of the HSA in conjunction with FcRn receptor, acidic environment and protease is resistant under receptor-mediated endocytosis protective effect Degradation, ensure that longer half-life period in HSA body, assume responsibility for HSA albumen major function (Chaudhury Cet al., 2006,2003)。

Biology of the Sadaharu Matsushita et al. (Sadaharuet al., 2004) to 3 structural domains of HSA Function is studied.The results show that HSAD III remains the class esterase active of HSA45%, in the phosphate of pH7.4, table Faint class enol enzymatic activity is showed.Shuqiang Zhao et al. (ShuQiang Zhaoet al., 2013；Zhao Shu is strong etc., 2013) 3DHSA-Nartograstim fusion protein expression vector is constructed using HSAD3, is expression with Pichia pastoris GS115 Host, successful secretion have simultaneously given expression to destination protein, and expression yield is 86mg/L.Moreover, not having in Engineering Yeast fermentation process It is found the signs of degradation that HSA fusion protein often occurs, during fusion protein purification, does not also occur to polymerize and degrade, body Outer activity experiment shows that 3DHSA-Nartograstim has the biology function similar with G-CSF (HSA-Nartograstim) Can, i.e. the effect of the cell Proliferation of dose-dependant sexual stimulus NFS-60 mouse.

Vania E.Ken anova et al. (Vania E et al., 2010) constructs double spies by fusion protein technology Xenoantibody-HSA D III (T84.66Db-HSAD3) fusion protein obtains purity about 98%, the destination protein of stable structure.In vivo Biological activity test shows that the direct radiocounting (%ID/g) of Db-HSAD III in vivo is 50 times of Db, and blood is average stagnant Staying the time (MRT) is 20 times of Db, illustrates that HSAD Ш has good increase small molecular protein medicine stability, extends half-life period Effect.

Before this, the T84.66Db-HSA (~90kDa) of Paul J.Yazaki (Paul et al., 2008) et al. building exists 48h after injection, blood retains vigor and only has 2.79%ID/g, and T84.66Db-HSAD Ш (dimeric forms molecular weight 101kDa) the blood reservation vigor of 51h has and still has 4.00%ID/g after injection, illustrates that under given conditions, HSAD3 is as egg White fusion partner ratio HSA is advantageously.

In conclusion in order to advanced optimize the activity of TMP fusion protein and heterologous fusion proteins in host protein 3DHSA structural domain is constructed Dimerized thrombopoietin simulating peptide by expression quantity, the present invention TMP dyad and human serum albumins third domain fusion protein, the design can reduce the degradation and polymerization of fusion protein, together The half-life period of Shi Zengqiang fusion protein and stability in vivo.The present invention provides Dimerized thrombopoietin simulating peptide The preparation method and application of TMP dyad and human serum albumins third domain fusion protein.We have cloned fusion protein Gene, and it is inserted into mammalian expression vector, make recombination fusion protein high efficient expression in mammalian cells.Expression produces Object has the bioactivity of the cellular level of significant c-mpl receptor-independent.

Bibliography

Cordes AA,Carpenter JF,Randolph TW.Selective domain stabilization as a strategy to reduce human serum albumin-human granulocyte colony stimulating factor aggregation rate[J].J Pharm Sci.,2012,101(6):2009-2016.

Cordes AA,Platt CW,Carpenter JF,et al.Selective domain stabilization as a strategy to reduce fusionprotein aggregation[J].JPharm Sci.,2012,101(4): 1400-1409.

Chaudhury C,Brooks CL,Carter DC,et al.Albuminbinding to FcRn:distinct from the FcRn-IgG interaction[J].Biochemistry,2006,45(15):4983-4990.

Chaudhury C,Mehnaz S,Robinson JM,et al.The major histocompatibility complex-related Fc receptor for IgG(FcRn)binds albumin andprolongs ints lifepan[J].J.Exp.Med.,2003,197(3):315-322.

S Matsushita,Y Isima,V T G Chuang,et al.Functional Analysis of Recombinant Human Sesum Albumin Domains for Pharmaceutial Applications[J] .Pharmaceitical Research,2004,21(10):1924-1931.

ShuQiang Zhao,Yu Zhang,Hong Tian,et al.Extending the Serum Half-Life of G-CSF via Fusion with the DomainⅢof Human Serum Albumin[J].BioMed Research International,2013,2013(6):107238-107238.

Vania E K,Tove O,Felix BS,etal.Tuning the serum persistence ofhuman serum albumin domain Ⅲ:diabody fusion proteins[J].Protein Engineering, Design&Selection,2010,23(10):789-798.

P J Yazaki,TKassa,CCheung,et al.Bioditribution and tumor imaging ofan anti-CEA single-chain antibody-albumin fusionprotein[J].NuclearMedicine andBiology,2008,35:151-158.

Liu Wenhui, Wu Min, Shen its etc..Human serum albumins integration technology progress [J].Pharmacy progress, 2015,39 (3): 199-203. Zhao Shuqiang, Zhang Yu, field surging etc..The 3rd structural domain-Nartograstim fusion protein of recombination human serum albumin In Pichia pastoris

Expression and preliminary Journal of Sex Research [J].China Medicine University's journal, 2013,46 (6): 577-582.

Summary of the invention

The purpose of invention is, provides a kind of Dimerized thrombopoietin simulating peptide TMP dyad-human seralbumin The preparation and application of albumen third domain fusion protein.

Dimerized fusion protein of the present invention is by thrombopoietin simulating peptide (Thrombopoietin Mimetic peptide, TMP) dyad, link peptide and human serum albumins third structural domain composition.The Dimerized fusion Albumen also contains Dimerized structural domain.

Thrombopoietin simulating peptide dyad of the present invention-human serum albumins third structural domain dimer Changing fusion protein is by between thrombopoietin simulating peptide dyad-human serum albumins third structural domain fusion protein What the covalent effect of disulfide bond was formed.

Dimerized structural domain of the present invention is selected from the peptide fragment fip of peptide fragment H and fip structural domain.

The gene order of peptide fragment H of the present invention is as shown in Seq ID No:7, amino acid sequence such as Seq ID No:8 It is shown.

Gene order of the present invention selected from fip structural domain is as shown in Seq ID No:9, amino acid sequence such as Seq Shown in ID No:10.

Human serum albumins third structural domain 3DHSA gene order of the present invention is as shown in Seq ID No:3, amino Acid sequence such as Seq ID No:4.

Fusion protein TMP-L1-TMP-L2-H-fip-3DHSA of the present invention, gene order such as Seq ID No:5 institute Show, amino acid sequence is as shown in Seq ID No:6.

Fusion protein TMP-L1-TMP-L2-H-fip-3DHSA of the present invention is by Chinese Hamster Ovary suspension cell CHO-S expression preparation.Steps are as follows:

(1) purpose fusion protein expression vector is constructed by infusion technology or digestion connection method；

(2) a large amount of extract prepares apyrogenic fusion protein expression vector, to be transfected；

(3) recovery CHO-S, 37 DEG C, 8%CO₂, 170rpm culture 6-9d；

(4) day before transfection, cell passage, passes on density 5 × 10⁵cells/mL；

(5) on the transfection same day, cell density is adjusted to 1 × 10⁶Cells/mL prepares transfection composite, is transfected, transfection PEI:DNAmass ratio=3:1-5:1 in compound；

(6) 6h after transfecting, is adjusted to 32 DEG C for incubator temperature, CO₂Concentration is adjusted to 7%；

(7) next day after transfecting, samples, and calculates cell viability, until cell viability is down to 60% hereinafter, receiving sample.

Fusion protein TMP-L1-TMP-L2-H-fip-3DHSA gene order of the present invention is inserted into mammal respectively In fibrocyte expression vector pCHO1.0 and pcDNA3, so that two kinds of fusion protein TMP-L1-TMP-L2-H-fip-3DHSA of building are fed Newborn animal cell expression vectors.

Fusion protein TMP-L1-TMP-L2-H-fip-3DHSA of the present invention expresses use in mammalian cells Fusion protein signal peptide is the signal peptide of Fc or the signal peptide of HSA.The amino acid sequence of Fc signal peptide such as Seq ID No: Shown in 12, gene order is as shown in Seq ID No:11；The amino acid sequence of HSA signal peptide is as shown in Seq ID No:14, base Because sequence is as shown in Seq ID No:13.

Thrombopoietin simulating peptide dyad of the present invention-human serum albumins third structural domain dimer Change the bioactivity that fusion egg has c-mpl receptor-independent on a cellular level.

Dimerized fusion egg of the present invention can be applied in the drug for preparing thrombopoietin.

Detailed description of the invention

The Dimerized thrombopoietin simulating peptide TMP dyad-human serum albumin fusion proteins lactation of Fig. 1 is dynamic The building of object fibrocyte expression vector pcDNA3-TMP-L1-TMP-L2-3DHSAF and pcDNA3-TMP-L1-TMP-L2-3DHSAH.

The Dimerized thrombopoietin simulating peptide TMP dyad-human serum albumin fusion proteins lactation of Fig. 2 is dynamic The structure of object fibrocyte expression vector pCHO1.0-TMP-L1-TMP-L2-3DHSAF and pCHO1.0-TMP-L1-TMP-L2-3DHSAH It builds.

Specific embodiment

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), ultrapure water instrument (Millipore), ice machine, centrifuge (Hitachi), HDB-PLUS type constant-temperature metal bath, HZQ-F16OA type constant-temperature shaking incubator (Shanghai one is permanent), PCR instrument (Applied Biosystems), tabletop refrigerated centrifuge (Thermo), DYY-8B type electrophoresis apparatus (Bole), 300 type gel imager (GE) of Image Quant etc., CO₂Incubator (Thermo Science), clean work station (SW-CJ-ID type, the star of famous brand), inverted microscope (Olmpus, Japan).

Main experimental materials:

1. restriction endonuclease SacI, XbaI, EcoRI, AflII, Avr II, BstZ I, I-HF of Pst, I-HF of Nco (NEB Products, the U.S.)

2.DNA polymerase:Super fidelity dna polymerase (NEB Products, the U.S.), TAKARA TaqTM (TAKARA, the U.S.)

HD Cloning Kit is TaKaRa ClonTech Products, Ligation-Free Cloning Kit is abm Products

4. kit/reagent: PureLinkTM HiPure Plasmid Maxiprep Kit (K210007, Invitrogen)、 Kit(A13696-01,Life Technology)、FreeStyleTM MAX Reagent(16447750,Life Technology)、PEI 40kDa(24765,Polysicence),L-glutamine (Life Technology), CD FortiCHOTM Medium (Life Technology), dehydrated alcohol, isopropanol etc..

5. small propose plasmid kit, PCR purification kit, DNA plastic recovery kit (Sheng Gong company, China)

6.T4DNA connection enzyme reagent kit (Takara Products, DaLian, China)

5.pCHO1.0 and pDNA3 purchase punctures bacterium and powder in invitrogen company, pUC57-Amp/HSA ss-dTMP End is synthesized and is cloned by Law Firm Suzhou Jiangsu Jin Weizhi Biotechnology Co., Ltd full genome.

7. Escherichia coli TOP10 is bought in TIANGEN Biotech (Beijing) Co., Ltd.).

8. yeast extract, peptone (Oxford Products, the U.S.).

9.LB culture medium

Yeast extract 5g, peptone 10g, NaCl 10g, is dissolved in 1000mL deionized water, and with the NaOH of 1mol/L PH value is adjusted to 7.0, autoclaving.

The configuration of 10.1% Ago-Gel

According to dosage, the TAE buffer of every 100mL is added 1g agarose, is boiled using microwave stove heating, 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 building of 1 TMP-L1-TMP-L2-3DHSA fusion protein mammalian expression vector of embodiment

The building of one .pcDNA3-TMP-L1-TMP-L2-3DHSAF expression vector

The acquisition of ss (Fc)-TMP-L1-TMP-L2-H-fip gene order: it is closed in precious bioengineering (Dalian) Co., Ltd At gene order ss-TMP-L-TMP, (ss is the signal peptide sequence of Fc segment, and TMP is simulating peptide.With plasmid pMD19-T-ss- TMP-L1-TMP is template, with primer P1 (ctggatggcctccatcagctcgtccctgctcacgcacggtgggcatgtgtgagttt tg) and P2 (gaagaagctttgctatggagacagacacactcct) the progress first round expands to obtain ss-TMP-L1-TMP, then with the PCR Product is template, with primer P3 (gaagaagctttgctatggagacagacacactcct) and P4 (gaagctgcagcctgaagttgatctcctcctgcttctggatggcctccatcagctcg tc) amplification obtains ss (Fc)- TMP-L1-TMP-L2-H-fip genetic fragment, and double digestion is carried out with restriction enzyme HindIII and PstI, it is recycled with glue Kit recycles above-mentioned digestion products, obtains ss (Fc)-TMP-L1-TMP-L2-H-fip segment of purifying.

The acquisition of 3DHSA gene order: buying plasmid pcDNA3.1 from Invitrogen company, will by precious biotech firm HSA complete genome sequence is synthesized on pcDNA3.1, obtains pcDNA3.1-HSA carrier.3DHSA base is obtained by two-wheeled PCR amplification Because (first round PCR primer is P5:aatgctatgccaaagtg and P6:cttggtcatctcctccc；Second, which takes turns PCR primer, is P7:atggagaccaaccccagcatcgtggaagagcctcagaatt and P8: cccgctcgagtcagttggtggtggtgtaagcctaaggcagcttgac).Purification is carried out to PCR product, is then used The processing of PstI/XhoI double digestion, digestion products utilize plastic recovery kit purification and recovery.

The clone of pcDNA3-TMP-L1-TMP-L2-3DHSAF expression vector and identification: restriction enzyme is used first Hind III-HF and XhoI carries out double digestion to the pcDNA3 carrier extracted in a small amount, and glue receives purifying pcDNA3 (III-HF/ of Hind XhoI) segment；By ready pcDNA3 (III-HF/XhoI of Hind), ss (Fc)-TMP-L1-TMP-L2-H-fip and 3DHSA T4 ligase reaction system is added, 16 DEG C of connections are overnight.Then connection product is transformed into Escherichia coli TOP10 competent cell In.From 20 single colonies of picking on the solid LB plate of the Amp resistance after conversion, it is added and contains 800 μ LAmp⁺Liquid LB's In 1.5mL-tube, 37 DEG C, 220rpm, 6-7h is cultivated, with primer P9:agaacccactgcttactg and P10: Aaaggacagtgggagtg carries out bacterium solution PCR identification, and III-HF/XhoI double digestion of positive colony further progress Hind is reflected It is fixed, bacterium solution PCR and double digestion are finally identified that correct positive colony is sent to Jin Weizhi Science and Technology Ltd. and is sequenced, by core Acid sequence and the completely the same clone of objective gene sequence are stored in -80 DEG C.

The building of two .pcDNA3-TMP-L1-TMP-L2-3DHSAH expression vectors

The acquisition of ss (HSA)-TMP-L1-TMP-L2-H-fip gene order: base is synthesized in Jin Weizhi Science and Technology Ltd. Because of sequence ss-TMP-L1-TMP (ss is the signal peptide sequence of HSA, and TMP is simulating peptide), which is combined in pUC57- On Amp/HSA ss-dTMP carrier.Using plasmid pUC57-Amp/HSA ss-dTMP as template, with primer P11 (ccaagcttatgaagtgggtaacctttat) it carries out PCR amplification with P12 (aactgcagcctgaagttgatctcct) and obtains Ss (HSA)-TMP-L1-TMP-L2-H-fip genetic fragment, and digested with restriction enzyme HindIII and PstI, it uses Plastic recovery kit recycles above-mentioned digestion products, obtains ss (HSA)-TMP-L1-TMP-L2-H-fip segment of purifying.

The clone of pcDNA3-TMP-L1-TMP-L2-3DHSAH expression vector and identification: by above-mentioned ready pcDNA3 (III-HF/XhoI of Hind), ss (HSA)-TMP-L1-TMP-L2-H-fip and 3DHSA addition T4 ligase reaction system, 16 DEG C Connection is overnight.Then connection product is transformed into Escherichia coli TOP10 competent cell.From consolidating for the Amp resistance after conversion 20 single colonies of picking on body LB plate are added and contain 800 μ LAmp⁺In the 1.5mL-tube of liquid LB, 37 DEG C, 220rpm, training 6-7h is supported, carries out bacterium solution PCR identification with primer P9:agaacccactgcttactg and P10:aaaggacagtgggagtg, and will The identification of III-HF/XhoI double digestion of positive colony further progress Hind, it is finally that bacterium solution PCR and double digestion identification is correctly positive Property clone be sent to Jin Weizhi Biotechnology Co., Ltd and be sequenced, by nucleic acid sequence and completely the same gram of objective gene sequence It is grand to be stored in -80 DEG C.

The building of three .pCHO1.0-TMP-L1-TMP-L2-3DHSAF expression vectors

The preparation of pCHO1.0 linearized vector: pCHO1.0 carrier is passed through into I pair of enzyme of restriction enzyme A vr II and Bsz17 It cuts, it is spare then to carry out glue recovery purifying.

The clone of pCHO1.0-TMP-L1-TMP-L2-3DHSAF expression vector and identification: with pcDNA3-TMP-L1-TMP- L2-3DHSAF plasmid is template, utilizes primer P13:acggttccgggccgcctaggatggagacagacacac and P14: Gtataatatagagtatactcagtggtggtggtggtggtg obtains end by pcr amplification reaction and pCHO1.0 is linearized Ss (the Fc)-TMP-L1-TMP-L2-H-fip segment of carrier with 15bp homologous sequence, pcr amplification product plastic recovery kit Purification and recovery.By the principle of the seamless clone technology of In-Fusion, according toHD Cloning Kit kit behaviour Make step and pCHO1.0 linearized vector is reacted into company by In-Fusion with ss (Fc)-TMP-L1-TMP-L2-H-fip segment It connects.Then 2.5 μ L In-Fusion reaction products are taken, the top10 competent cell liquid that 50 μ L have just thawed is added, mixes gently, 30min is placed on ice, and then 42 DEG C of heat shock 45s, place 2min on ice；150 μ L non-resistant LB liquid are finally added in system Culture medium, 37 DEG C, the bacterium solution after recovery is all spread evenly across on the plate of kana+ resistance by 150rpm recovery 1h, and 37 DEG C incubate Educate 20h.Choose 20 single colonies, by PCR (primer be P15:gtctgagcctccttgtcttg and P14: Gtataatatagagtatactcagtggtggtggtggtggtg) and Avr II and two methods of the identification of I double digestion of Bsz17 it is positive Two methods are finally identified that correctly clone is sent to Jin Weizhi Biotechnology Co., Ltd and are sequenced, by nucleic acid by clone Sequence and the completely the same clone of objective gene sequence are stored in -80 DEG C.

The building of four .pCHO1.0-TMP-L1-TMP-L2-3DHSAH expression vectors

The acquisition of ss (HSA)-TMP-L1-TMP-L2-H-fip gene order: with pUC57-Amp/HSA ss-dTMP plasmid For template, by pcr amplification reaction (primer is P16:acggttccgggccgcctaggatgaagtgggtaa and P17: Aactgcagcctgaagttgatctcct), glue recovery purifying obtains end and pCHO1.0 linearized vector and 3DHSA gene Sequence respectively has ss (HSA)-TMP-L1-TMP-L2-H-fip segment of 15bp homologous sequence.

The acquisition of 3DHSA gene order: using pcDNA3-TMP-L1-TMP-L2-3DHSAF plasmid as template, pass through PCR (primer is P18:caacttcaggctgcaggactacatcgacaggatcat and P14: Gtataatatagagtatactcagtggtggtggtggtggtg) amplified reaction, glue recovery purifying obtain end and pCHO1.0 Linearized vector and ss (HSA)-TMP-L1-TMP-L2-H-fip respectively have the 3DHSA genetic fragment of 15bp homologous sequence.

The clone of pCHO1.0-TMP-L1-TMP-L2-3DHSAH expression vector and identification: pass through seamless gram of In-Fusion The principle of grand technology, according toHD Cloning Kit kit operating procedure is by the pCHO1.0 line of above-mentioned preparation Property carrier, ss (HSA)-TMP-L1-TMP-L2-H-fip and 3DHSA genetic fragment pass through In-Fusion reaction forming.Then 2.5 μ L In-Fusion reaction products are taken, the top10 competent cell liquid that 50 μ L have just thawed is added, mixes gently, places on ice 30min, then 42 DEG C of heat shock 45s, place 2min on ice；Finally it is added 150 μ L non-resistant LB liquid mediums in system, 37 DEG C, the bacterium solution after recovery is all spread evenly across on the plate of kana+ resistance by 150rpm recovery 1h, 37 DEG C of incubation 20h.It chooses Go 20 single colonies, by PCR (primer be P15:gtctgagcctccttgtcttg and P14: Gtataatatagagtatactcagtggtggtggtggtggtg) and Avr II and two methods of the identification of I double digestion of Bsz17 it is positive Two methods are finally identified that correctly clone is sent to Jin Weizhi Biotechnology Co., Ltd and are sequenced, by nucleic acid by clone Sequence and the completely the same clone of objective gene sequence are stored in -80 DEG C.

The Dimerized thrombopoietin simulating peptide TMP dyad-human serum albumins third structural domain of embodiment 2 melts Expression of the hop protein in mammalian cell CHO-S

Chinese Hamster Ovary suspension cell CHO-S is purchased from U.S. Invitrogen company, is adapted to by domestication culture High density, serum free suspension culture a kind of wild-type CHO cells, be capable of the recombinant protein of mass production secretory.2012 Since, this laboratory has been devoted to expression of the research CHO-S cell to fusion protein, establishes the expressing fusion protein of optimization The transient transfection and expressing fusion protein system of carrier.

The preparation of CD-FortiCHOTM complete culture solution is prepared before cell recovery.It is added in CD-FortiCHOTM culture solution The L-glutamine of 200mM makes final concentration of 8mM.Be kept in dark place in 2-8 DEG C of environment it is spare, using it is preceding in 37 DEG C of water-baths it is pre- Heat.CHO-S cell cryopreservation tube is taken out from liquid nitrogen container, rapid (in~1min) thaws in 37 DEG C of water-baths.Toward 125-mL 29mL CD-FortiCHOTM complete culture solution is moved into shaker Flask (cell culture is dedicated, polycarbonate triangular flask), Carefully cells frozen storing liquid is added in complete culture solution with liquid-transfering gun.Shaker Flask is put into shaking in cell incubator On bed, 37 DEG C, 8%CO₂, 150rpm culture.After cell recovery culture 2-3d, reach 1 × 10 to cell density⁶-2× 10⁶Cells/mL (cells/mL), is passed on CD-FortiCHOTM complete culture solution, and 3-4d is passed once, cultivates 6- with this 9d restores cell to optimum state.On the day before cell transfecting, cell passage is carried out, passes on density 5 × 10⁵cells/mL.Turn On the dye same day, cell density is adjusted to 1 × 10⁶Cells/mL, rotaring redyeing system 30ml.Plasmid to be transfected is a large amount of with heat source is removed Extracts kit extraction is spare, uses PEI as transfection reagent, configures transfection composite, transfection conditions according to reagent specification As follows: PEI:DNA mass ratio=3:1, DNA transfection concentrations are 50 μ g/30mL systems.By configured transfection composite It is added drop-wise in the CHO-S suspension cell of 30mL system, rocks culture bottle when being added dropwise, make to be uniformly mixed.Then cell is put back to On the shaking table of cell incubator, at 37 DEG C, 8%CO₂, cultivate under the regular culture conditions of 150rpm, 6h after transfection, by temperature tune To 32 DEG C of progress Low- temperature cultures.Daily sample detection cell viability, until being collected culture when cell viability is lower than 60% Object, 10000rpm is centrifuged 10min at 4 DEG C, obtains the cell transient expression supernatant containing target product.

Successful four strain clone constructed in embodiment 1 is all expressed in CHO-S cell according to the method described above, Obtain the subject fusion proteins that sequence is TMP-L1-TMP-L2-H-fip-3DHSA.

3 fusion protein of embodiment work Dual-Luciferase Activity determination result

TPO receptor c-mpl is universally present in (including stem cell, megacaryocyte cell line colony formation in hematopoietic tissue Cell etc.), it is a kind of transmembrane protein receptor.TPO TPO simulating peptide TMP is in conjunction with the film exterior domain of c-mpl receptor, c-mpl Aggregate into homodimer, JAK2-STAT5 in active cell, Shc-Ras-MAPK, anti-apoptosis pathways etc. Many A signal pathways cause platelet yield to increase.Meanwhile the STAT5 dimer for being phosphorylated activation enters nucleus, with c- Fos promoter is combined, to cause the firefly luciferase gene expression in downstream.So passing through the light of firefly in detection cell Luciferin zymolyte luminous signal intensity can reach purpose of the detection TMP recombinant protein sample to c-mpl by vitality of subject.

This experiment is to contain the Renilla constitutive expression carrier of Firefly Luciferase as internal reference, to eliminate The factors bring error such as plating cells density, experimental implementation.In experimentation, first with band c-fos promoter and The plasmid pHF443 of Luciferase gene, the pRL-SV40Renilla Vector with Renilla Luciferase gene, The pAdVAntageTM vector of the sub- enhancer gene of tape starting and plasmid pcDNA3.1/c-mpl with c-mpl gene, corotation NIH-3T3 cell is contaminated, NIH-3T3 cell is made to express TPO receptor c-mpl and Firefly Luciferase, Renilla Luciferase.Cell after being transfected with Te Biao (rhTPO) positive drug and recombinant protein sample treatment, while blank is set Control group.After cell incubation 6h, using between Dua-GloTM Luciferase Reagent kit detection different disposal group Firefly Luciferase and Renilla Luciferase fluorescence signal intensity, with Firefly Luciferase and The ratio of Renilla Luciferase fluorescence intensity represents the bioactivity of fusion protein cellular level c-mpl receptor-independent.

Specific experiment process is as follows:

NIH-3T3 plating cells, pcDNA3.1/c-mpl, pHF443, pAdVAntageTM vector, Renilla, four Plasmid co-transfection.48h after transfection is separately added into positive control, blank control and given the test agent, detects firefly luciferin respectively Enzyme activity and renilla luciferase vigor, calculating ratio fluorescent, ratio fluorescent=firefly fluorescent value/sea pansy fluorescent value, specifically Step is detailed in Chinese patent (CN201310084212), finally according to reference positive control ratio fluorescent, calculates each fusion egg White ratio is living.

Experimental result:

The bioactivity testing result of 1 fusion protein of table

The Dual-Luciferase Activity determination of table 1 the result shows that, all pcDNA3.1-HSAs of the transfection without mpl receptor are unloaded Body processing group, that is, negative control group, relative fluorescence ratio are 1, than living for 0U/mg.

Table 1 is shown: the Dimerized fusion protein in detected TPO positive drug, CHO expression sample all has significantly The cellular level bioactivity of mpl receptor-independent, and activity is significantly higher than positive drug.

Therefore, the Dimerized fusion protein of TMP dyad of the present invention and human serum albumins third structural domain It can be acted on thrombopoietin receptor c-mpl, the activity that there is stimulation TPO dependent cell system to be proliferated can be in lactation It expresses, can be applied in the drug for preparing thrombopoietin in zooblast.

Sequence table

<110>Lanzhou University

<120>a kind of Dimerized fusion protein and its preparation method and application

<141> 2017-11-08

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 99

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atcgaaggcc ccacactgcg gcagtggctg gctgctaggg ccggtggccc ctccggaatc 60

gagggaccca ccctgaggca gtggctggcc gccagagcc 99

<210> 2

<211> 33

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly

1 5 10 15

Pro Ser Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg

20 25 30

Ala

<210> 3

<211> 615

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 60

tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 120

ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 180

cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 240

tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 300

ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 360

gagtttaatg ctgaaacgtt caccttccat gcagatatat gcacactttc tgagaaggag 420

agacaaatca agaaacaaac tgcacttgtt gagcttgtga aacacaagcc caaggcaaca 480

aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 540

gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 600

gctgccttag gctta 615

<210> 4

<211> 205

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu

1 5 10 15

Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr

20 25 30

Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg

35 40 45

Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys

50 55 60

Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu

65 70 75 80

Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys

85 90 95

Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu

100 105 110

Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr

115 120 125

Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys

130 135 140

Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr

145 150 155 160

Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu

165 170 175

Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly

180 185 190

Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

195 200 205

<210> 5

<211> 909

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

atcgaaggcc ccacactgcg gcagtggctg gctgctaggg ccggtggccc ctccggaatc 60

gagggaccca ccctgaggca gtggctggcc gccagagccg gcggcggcgg aagcagatct 120

gagcccaaga gcagcgacaa aactcacaca tgcccaccgt gcgtgagcag ggacgagctg 180

atggaggcca tccagaagca ggaggagatc aacttcaggc tgcaggacta catcgacagg 240

atcatcgtgg ccatcatgga gaccaacccc agcatcgtgg aagagcctca gaatttaatc 300

aaacaaaatt gtgagctttt tgagcagctt ggagagtaca aattccagaa tgcgctatta 360

gttcgttaca ccaagaaagt accccaagtg tcaactccaa ctcttgtaga ggtctcaaga 420

aacctaggaa aagtgggcag caaatgttgt aaacatcctg aagcaaaaag aatgccctgt 480

gcagaagact atctatccgt ggtcctgaac cagttatgtg tgttgcatga gaaaacgcca 540

gtaagtgaca gagtcaccaa atgctgcaca gaatccttgg tgaacaggcg accatgcttt 600

tcagctctgg aagtcgatga aacatacgtt cccaaagagt ttaatgctga aacgttcacc 660

ttccatgcag atatatgcac actttctgag aaggagagac aaatcaagaa acaaactgca 720

cttgttgagc ttgtgaaaca caagcccaag gcaacaaaag agcaactgaa agctgttatg 780

gatgatttcg cagcttttgt agagaagtgc tgcaaggctg acgataagga gacctgcttt 840

gccgaggagg gtaaaaaact tgttgctgca agtcaagctg ccttaggctt acaccaccac 900

caccaccac 909

<210> 6

<211> 303

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly

1 5 10 15

Pro Ser Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg

20 25 30

Ala Gly Gly Gly Gly Ser Arg Ser Glu Pro Lys Ser Ser Asp Lys Thr

35 40 45

His Thr Cys Pro Pro Cys Val Ser Arg Asp Glu Leu Met Glu Ala Ile

50 55 60

Gln Lys Gln Glu Glu Ile Asn Phe Arg Leu Gln Asp Tyr Ile Asp Arg

65 70 75 80

Ile Ile Val Ala Ile Met Glu Thr Asn Pro Ser Ile Val Glu Glu Pro

85 90 95

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

100 105 110

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

115 120 125

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

130 135 140

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

145 150 155 160

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

165 170 175

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

180 185 190

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

195 200 205

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

210 215 220

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

225 230 235 240

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

245 250 255

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

260 265 270

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

275 280 285

Ala Ala Ser Gln Ala Ala Leu Gly Leu His His His His His His

290 295 300

<210> 7

<211> 42

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

gagcccaaga gcagcgacaa aactcacaca tgcccaccgt gc 42

<210> 8

<211> 14

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

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

1 5 10

<210> 9

<211> 114

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gtgagcaggg acgagctgat ggaggccatc cagaagcagg aggagatcaa cttcaggctg 60

caggactaca tcgacaggat catcgtggcc atcatggaga ccaaccccag catc 114

<210> 10

<211> 38

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

Val Ser Arg Asp Glu Leu Met Glu Ala Ile Gln Lys Gln Glu Glu Ile

1 5 10 15

Asn Phe Arg Leu Gln Asp Tyr Ile Asp Arg Ile Ile Val Ala Ile Met

20 25 30

Glu Thr Asn Pro Ser Ile

35

<210> 11

<211> 63

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

gagacagaca cactcctgct atgggtactg ctgctctggg ttccaggttc cactggtgac 60

ggt 63

<210> 12

<211> 21

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly

1 5 10 15

Ser Thr Gly Asp Gly

20

<210> 13

<211> 69

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

aagtgggtaa cctttatttc ccttcttttt ctctttagct cggcttattc caggggtgtg 60

tttcgtcga 69

<210> 14

<211> 23

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 14

Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr

1 5 10 15

Ser Arg Gly Val Phe Arg Arg

20

Claims (14)

1. a kind of Dimerized fusion protein, which is characterized in that the Dimerized fusion protein includes promoting thrombocytopoiesis Plain simulating peptide (TMP) dyad, link peptide and human serum albumins third structural domain (3DHSA), the Dimerized fusion Albumen also contains Dimerized structural domain.

2. a kind of Dimerized fusion protein according to claim 1, it is characterised in that the Dimerized structural domain is Keep fusion protein Dimerized by the covalent effect of disulfide bond.

3. a kind of Dimerized fusion protein according to claim 1, which is characterized in that the Dimerized structural domain is Peptide fragment H and peptide fragment fip selected from fip structural domain.The gene order of the peptide fragment H is as shown in Seq ID No:7, amino acid sequence As shown in Seq ID No:8.The gene order of the fip structural domain is as shown in Seq ID No:9, amino acid sequence such as Seq ID Shown in No:10.

4. a kind of Dimerized fusion protein according to claim 1 to 3, which is characterized in that the TMP includes 2 TMP With 1 link peptide, structural formula is indicated are as follows: TMP-L1-TMP, amino acid sequence is as shown in SEQ ID NO:2, gene order such as SEQ Shown in ID NO:1, wherein L1 indicates link peptide, and the DNA sequence dna of L1 is GGTGGCCCCTCCGGA, amino acid sequence GGPSG, It is to join end to end between TMP dyad.

5. a kind of Dimerized fusion protein according to claim 1, which is characterized in that the 3DHSA has SEQ ID Amino acid sequence shown in NO:4 encodes the DNA sequence dna of the amino acid sequence of the 3DHSA as shown in SEQ ID NO:3；Or Replace, lack or be inserted into the obtained active amino acid with the 3DHSA of amino acid residue in the amino acid sequence Sequence, and the DNA sequence dna of amino acid sequence described in coding.

6. a kind of Dimerized fusion protein according to claim 1 to 3, which is characterized in that the TMP dyad is located at The end N- of fusion protein, 3DHSA are located at the end C- of fusion protein, and structural formula indicates are as follows: TMP-L1-TMP-L2-H-fip- 3DHSA。

7. a kind of Dimerized fusion protein according to claim 6, which is characterized in that the amino acid of the fusion protein Sequence is as shown in SEQ ID NO:6, and the DNA sequence dna of the amino acid sequence of encoding said fusion protein is as shown in SEQ ID NO:5.

8. a kind of thrombopoietin simulating peptide dyad-human seralbumin according to any one of claims 1-7 The Dimerized fusion protein of albumen third structural domain, which is characterized in that the fusion protein uses mammalian cell expression Preparation.

9. a kind of thrombopoietin simulating peptide dyad-human serum albumins third structure according to claim 8 The Dimerized fusion protein in domain, which is characterized in that the mammalian cell is Chinese Hamster Ovary suspension cell CHO- S。

10. a kind of thrombopoietin simulating peptide dyad-human seralbumin according to any one of claims 1-7 The Dimerized fusion protein of albumen third structural domain, it is characterised in that used fusion protein mammalian cell expression carries Body is pCHO1.0 or pcDNA3 or other mammalian cell expression vectors.

11. a kind of thrombopoietin simulating peptide dyad-human seralbumin according to any one of claims 1-7 The Dimerized fusion protein of albumen third structural domain, it is characterised in that used fusion protein signal peptide is the signal peptide of Fc Or the signal peptide of HSA.The amino acid sequence of Fc signal peptide is as shown in Seq ID No:12, gene order such as Seq ID No:11 It is shown；The amino acid sequence of HSA signal peptide is as shown in Seq ID No:14, and gene order is as shown in Seq ID No:13.

12. a kind of thrombopoietin simulating peptide dyad-human serum albumins as described in claim 1-7 any one The preparation method of the Dimerized fusion protein of third structural domain, which is characterized in that the method comprises the steps of:

(1) purpose fusion protein expression vector is constructed by infusion technology or digestion connection method；

(2) a large amount of extract prepares apyrogenic fusion protein expression vector, to be transfected；

(3) recovery CHO-S cell, 37 DEG C, 8%CO2,170rpm cultivate 6-9d；

(4) day before transfection, cell passage, passes on 5 × 105cells/mL of density；

(5) on the transfection same day, cell density is adjusted to 1 × 106cells/mL, prepares transfection composite, is transfected, transfected compound PEI:DNA mass ratio=3:1-5:1 in object；

(6) 6h after transfecting, is adjusted to 32 DEG C for incubator temperature, CO2 concentration is adjusted to 7%；

(7) next day of turning then, samples, and calculates cell viability, until cell viability is down to 60% hereinafter, receiving sample.

13. the biology that a kind of Dimerized fusion egg as described in claim 1 has c-mpl receptor-independent on a cellular level Activity.

14. a kind of Dimerized fusion egg as described in claim 1 is applied in the drug for preparing thrombopoietin.