CN109748969B - Dimerization fusion protein and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of genetic engineering pharmacy, and particularly relates to a dimerization fusion protein, a preparation method and application thereof. The dimeric fusion protein comprises 1 Thrombopoietin Mimetic Peptide (TMP) diabody protein domain, 1 human serum albumin third domain (3 DHSA). Wherein, the TMP diad and the 3DHSA are connected by a connecting peptide and a dimerization domain. The dimeric fusion protein of the TMP diad and the third structural domain of the human serum albumin can act with a thrombopoietin receptor c-mpl, has the activity of stimulating the proliferation of a TPO-dependent cell line, and can be expressed in mammalian cells. Can be applied to the preparation of the medicines for promoting the thrombopoietin, and is an effective candidate medicine for promoting the thrombopoietin. The invention also provides a preparation method of the dimerization fusion protein of the TMP diad and the third structural domain of the human serum albumin.

Description

Dimerization fusion protein and preparation method and application thereof

Technical Field

The invention belongs to the field of genetic engineering pharmacy, and particularly relates to a dimeric fusion protein of a thrombopoietin mimetic peptide (Thrombopoietin mimetic peptide, TMP) diad-human serum albumin third structural domain (The third domain ofhuman serum albumin,3 DHSA) and a preparation method and application thereof.

Background

Along with the maturation of DNA recombination technology and the development of biopharmaceutical technology, the recombinant protein medicine has the advantages of clear amino acid sequence and base sequence, high safety and the like, becomes the popular field of current new medicine research, and has wide development prospect. However, many small molecular weight polypeptide protein drugs are easily degraded in vivo or cleared by kidneys due to the small molecular weight and unstable molecular structure, so that the residence time of the drugs in plasma is short (Jin Guangze, etc., 2010). Multiple injections of the drug in a short period of time not only can cause instability of blood drug concentration, but can also increase physical pain and economic stress on the patient (side bud, dan Yifeng, 2009). Therefore, optimizing parameters such as the effectiveness and long-acting property of protein drugs has become an important research direction for biopharmaceuticals (Zhao Hongliang et al, 2005).

Numerous studies have shown that three similar domains together constitute HSA. Based on independent studies of the structure and function of the HSA domains, it was found that each domain contains some sites for binding to the ligand, but the ligands bound are different (Kato Y, et al). Studies have shown that neuroleptic binding sites are on HSA-D3, and that some anti-coagulant binding sites are mostly on HSA-D2, and rarely on HSA-D1. Studies have also demonstrated that the primary ligand binding sites are predominantly on HSA-D2 and HSA-D3 of HSA (Thomas Kjeldsen et al, 1998). The Vania EKen anova et al study demonstrated that the IIIA and IIIB substructures have an important role in the maintenance of albumin stability function (Vania EKen anova etal, 2010), and the Chaudhury et al study demonstrated that HSA-D3 possesses all sites for FcRn binding. (Chaudhury et al, 2006). Recently, a great deal of researches show that 3DHSA is a main part of drug receptor binding, can increase the molecular weight of target protein after being fused with protein, can resist acidic environment and degradation of protease under the action of receptor-mediated endocytosis protection, has similar functions as HSA full-length protein, can reduce the difficulty of fusion into protein purification and can reduce the risk of immune reaction during clinical administration. Zhao Shujiang et al initially demonstrated that the use of pichia pastoris expression systems to express fusion protein 3DHSA and HSA did not exhibit the prior degradation of fusion protein 3DHSA, did not exhibit polymerization during purification, and could avoid kidney filtration with greater strength, enhancing protein stability. The expressed foreign protein 3 DHSA-Nartogarstim is capable of stimulating the proliferation of mouse cells NFS-60 and is concentration dependent (Zhao Shujiang et al, 2013). Therefore, the intensive research of HSA-D3 provides important basis and clues for further research of long-acting fusion protein drugs.

In 1997, AF12505 was screened as a highly active TPO mimetic peptide by Cwirla et al. And shows that AF12505 is a 14 amino acid short peptide and has the function of stimulating the proliferation of rhTPO-dependent cell lines (Shao Bo, 2006). But the activity was very low, only 1/4000 of rhTPO. After dimer modification of AF12505, the activity was significantly improved. Subsequently, AF12505 was subjected to chemical modification and biological modification, respectively, and it was found that the IgG1Fc-TPO fusion protein had a function of promoting the proliferation of TPO-dependent cell lines. 2008, anin corporation fused AF12505 with the Fc fragment of the antibody, developed the product Nplate for the treatment of primary thrombocytopenia. The product has TPO activity, and is different from TPO, and the generation of neutralizing antibodies is avoided, but because the Fc fragment is a non-inert protein, the Fc fragment can mediate various immune reactions in vivo. Then, in order to overcome the defects of the NPlate, the scientific researchers in China connect AF12505 with HSA to construct a fusion protein, but the result of an activity experiment shows that the fusion protein does not have biological activity for promoting the generation of blood platelets.

Regarding the study of TMP-HSA long-acting medicine, the TMP monomer is a short peptide composed of 14 amino acids, and the amino acid sequence is IEGPTLRQWLAARA (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-LeU-Ala-Ala-Arg-Ala). This amino acid sequence has no sequence homology to TPO and therefore does not cause self cross-infection (Shao Bo, 2006). Theoretically, when TMP exists in the form of dimers, it is capable of binding to two closely spaced thrombopoietin receptors c-mpl, respectively, conducting tyrosine phosphorylation signals into the nucleus, activating synthesis of proteins on the ribosome, promoting maturation of macrophage growth factors, and promoting thrombogenesis (Drachman, J.G et al, 1999). Computer modeling showed that protein linker (L) generally exists in four forms, amino acid sequences SG, GGGSG, GGPSG and GGGGSGGGSG, respectively. The present laboratory preliminary study constructs TMP diads by using different L as the connecting peptide of the TMP. Then, through extensive literature investigation, a short linker segment (selected from immunoglobulin hinge regions containing cysteines, which are prone to disulfide bond formation) was designed between the HSA and TMP nucleic acid sequences, making the HSA and TMP segments spatially independent. And successfully constructs a recombinant vector, then integrates with a yeast chromosome, successfully induces and expresses target protein, and utilizes a 20 liter fermentation tank to ferment and produce a target protein sample (Chinese patent 201310084035, 2013), thus establishing a TMP fusion protein in vitro activity detection system (Chinese patent 201310084212,2013).

Researchers find that HSA fusion proteins can have some common problems in the expression production process, for example, HSA fusion proteins can be polymerized or degraded in the expression and storage processes, in vitro biological activities can be reduced to different degrees, expression products are nonuniform, expression levels are low, and the like (cordies AAet al 2012,2012; liu Wenhui, 2015), and the phenomenon not only increases the difficulty of protein purification, but also increases the risk of immune reaction in the clinical application process.

In recent years, it has been found that the HSA third domain (HSAD 3,23 kDa) located at amino acid residues 381-585 is a key site for binding of HSA to FcRn receptor, is resistant to acidic environment and degradation by proteases under receptor-mediated endocytosis protection, ensures a longer half-life in HSA, and assumes the main function of HSA protein (Chaudhury Cet al,2006,2003).

Sadaharu Matsushita et al (sadaharu et al 2004) studied the biological function of the 3 domains of HSA. The results show that HSA III retains 45% of the esterase-like activity of HSA and exhibits weak enolase-like activity in phosphate at pH 7.4. The 3 DHSA-Nartogarstim fusion protein expression vector is constructed by Sheqiang Zhao et al (Sheqiang Zhao et al, 2013; zhao Shujiang, etc. 2013) by utilizing HSAD3, and the target protein is successfully secreted and expressed by taking Pichia pastoris GS115 as an expression host, wherein the expression yield is 86mg/L. Furthermore, no degradation of HSA fusion protein was found during fermentation of the engineering yeast, and no polymerization or degradation occurred during purification of the fusion protein, and in vitro activity experiments showed that 3 DHSA-Nartogarstim had a similar biological function as G-CSF (HSA-Nartogarstim), i.e. a dose-dependent effect in stimulating cell proliferation in NFS-60 mice.

The fusion protein of bispecific antibody-HSA DIII (T84.66 Db-HSAD 3) is constructed by Vania E.Ken anova et al (Vania E et al 2010) through fusion protein technology, and the target protein with the purity of about 98% and stable structure is obtained. In vivo bioactivity experiments show that the direct radioactive count (% ID/g) of Db-HSAD III in vivo is 50 times that of Db, and the average residence time (MRT) of blood is 20 times that of Db, so that the HSAD III has good effects of improving the stability of small molecule protein drugs and prolonging the half-life.

Previously, T84.66Db-HSA (. About.90 kDa) constructed by Paul J.Yazaki (Paul et al., 2008) et al had only 2.79% ID/g for blood retention activity 48h after injection, while T84.66Db-HSAD class (dimer form molecular weight 101 kDa) had 4.00% ID/g for blood retention activity 51h after injection, indicating that under certain conditions HSAD3 is more advantageous as a protein fusion partner than HSA.

In summary, in order to further optimize the activity of the TMP fusion protein and the expression amount of the heterologous fusion protein in the host protein, the 3DHSA domain is used as a fusion partner of TMP to construct the fusion protein of the dimeric thrombopoietin mimetic peptide TMP diad and the third domain of human serum albumin, and the design can reduce the degradation and polymerization of the fusion protein and enhance the half-life period and in vivo stability of the fusion protein. The invention provides a preparation method and application of a fusion protein of a dimeric thrombopoietin mimetic peptide TMP diad and a third structural domain of human serum albumin. The fusion protein gene is cloned and inserted into a mammalian expression vector, so that the recombinant fusion protein is efficiently expressed in mammalian cells. The expression product has significant c-mpl receptor dependent cellular level bioactivity.

Reference to the literature

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 them, etc. Human serum albumin fusion technology development [ J ]. Pharmaceutical developments 2015,39 (3): 199-203. Zhao Shujiang, zhang Yu, tian, etc. Recombinant human serum albumin 3 domain-Nartogarstim fusion protein in pichia pastoris

Expression and preliminary studies of (C) J. University of Chinese medical science, university of medical science, 2013,46 (6): 577-582.

Disclosure of Invention

The invention aims to provide preparation and application of a dimeric thrombopoietin mimetic peptide TMP diad-human serum albumin third domain fusion protein.

The dimerization fusion protein consists of a thrombopoietin mimetic peptide (Thrombopoietin mimetic peptide, TMP) diad, a connecting peptide and a third structural domain of human serum albumin. The dimerized fusion protein also contains a dimerization domain.

The dimeric fusion protein of the thrombopoietin mimetic peptide diad-human serum albumin third structural domain is formed by covalent action of disulfide bonds between the fusion proteins of the thrombopoietin mimetic peptide diad-human serum albumin third structural domain.

The dimerization domain of the invention is selected from peptide segment H and peptide segment fip of the fip domain.

The gene sequence of the peptide fragment H is shown as SEQ ID No. 7, and the amino acid sequence is shown as SEQ ID No. 8.

The gene sequence selected from fip domains is shown as SEQ ID No. 9, and the amino acid sequence is shown as SEQ ID No. 10.

The third structural domain 3DHSA gene sequence of the human serum albumin is shown as a Seq ID No. 3, and the amino acid sequence is shown as a Seq ID No. 4.

The fusion protein TMP-L1-TMP-L2-H-fip-3DHSA has a gene sequence shown in Seq ID No. 5 and an amino acid sequence shown in Seq ID No. 6.

The fusion protein TMP-L1-TMP-L2-H-fip-3DHSA is prepared by expressing Chinese hamster ovary suspension cells CHO-S. The method comprises the following steps:

(1) Constructing a target fusion protein expression vector by an infusion technology or an enzyme digestion connection method;

(2) Preparing a large amount of fusion protein expression vectors without heat source through extraction, and carrying out transfection;

(3) Resuscitates CHO-S,37 ℃,8% CO ₂ Culturing at 170rpm for 6-9d;

(4) The day before transfection, cells were passaged at a passaging density of 5X 10 ⁵ cells/mL；

(5) On the day of transfection, the cell density was adjusted to 1X 10 ⁶ Preparing a transfection complex by cells/mL, and carrying out transfection, wherein PEI in the transfection complex is DNAmass ratio=3:1-5:1;

(6) 6h after transfection, the incubator temperature was adjusted to 32℃and CO ₂ The concentration is adjusted to 7%;

(7) Sampling every other day after transfection, calculating cell activity until the cell activity is reduced to below 60%, and collecting samples.

The fusion protein TMP-L1-TMP-L2-H-fip-3DHSA gene sequences are respectively inserted into the mammalian cell expression vectors pCHO1.0 and pcDNA3, so that two fusion protein TMP-L1-TMP-L2-H-fip-3DHSA mammalian cell expression vectors are constructed.

The fusion protein TMP-L1-TMP-L2-H-fip-3DHSA expresses the signal peptide adopting the fusion protein signal peptide as Fc or the signal peptide of HSA in mammalian cells. The amino acid sequence of the Fc signal peptide is shown as SEQ ID No. 12, and the gene sequence is shown as SEQ ID No. 11; the amino acid sequence of the HSA signal peptide is shown as SEQ ID No. 14, and the gene sequence is shown as SEQ ID No. 13.

The dimeric fusion protein of the thrombopoietin mimetic peptide diad-human serum albumin third domain has the biological activity of c-mpl receptor dependence on the cellular level.

The dimerization fusion egg can be applied to preparation of thrombopoietin-promoting medicines.

Drawings

FIG. 1 construction of a dimeric thrombopoietin mimetic peptide TMP diad-human serum albumin fusion protein mammalian cell expression vector pcDNA3-TMP-L1-TMP-L2-3DHSAF and pcDNA3-TMP-L1-TMP-L2-3 DHSAH.

FIG. 2 construction of the dimeric thrombopoietin mimetic peptide TMP diad-human serum albumin fusion protein mammalian cell expression vector pCHO1.0-TMP-L1-TMP-L2-3DHSAF and pCHO1.0-TMP-L1-TMP-L2-3 DHSAH.

Detailed Description

Main experimental instrument:

pipetting gun, ultra clean bench (Antai), magnetic stirrer, microwave oven, high temperature steam sterilizing pot, -80 ℃ 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 Heng), PCR instrument (Applied Biosystems), desk-top refrigerated centrifuge (Thermo), DYY-8B type electrophoresis instrument (Bere), image Quant 300 type gel imager (GE) and the like, CO ₂ Incubator (Thermo Science), purge table (SW-CJ-ID, famous brand star), inverted microscope (Olmpus, japan).

Main experimental materials:

1. restriction endonucleases SacI, xbaI, ecoRI, aflII, avr II, bstZ I, pstI-HF, ncoI-HF (NEB Co., ltd., U.S.)

DNA polymerase:super fidelity DNA polymerase (NEB Co., product, USA), TAKARA Taq (TAKARA, USA)

HD Cloning Kit is a product of TaKaRa ClonTech company, and Ligation-Free Cloning Kit is a product of abm company

4. Kit/reagent: pureLinkTM HiPure Plasmid Maxiprep Kit (K210007, invitrogen), Kit (A13696-01,Life Technology), freeStyleTM MAX Reagent (16447750,Life Technology), PEI 40kDa (24765, polyscience), L-glutamine (Life Technology), CD FortiCHOTM Medium (Life Technology), absolute ethanol, isopropanol, etc.

5. Small plasmid kit, PCR purification kit and DNA gel recovery kit (Biotechnology Co., china)

T4DNA ligase kit (Takara Co., ltd., dalian China)

pCHO1.0 and pDNA3 were purchased from Invitrogen, pUC57-Amp/HSA ss-dTMP puncture and powder, and were total genetically synthesized and cloned by Jiangsu Suzhou Jin Weizhi Biotech Co.

7. Coli TOP10 was purchased from Tiangen Biochemical technologies (Beijing) Inc.).

8. Yeast extract, peptone (product of Oxford company, usa).

LB medium

Yeast extract 5g, peptone 10g, naCl 10g, dissolved in 1000mL deionized water, and pH adjusted to 7.0 with 1mol/L NaOH, and autoclaved.

10.1% agarose gel configuration

According to the dosage, 1g of agarose is added into every 100mL of TAE buffer solution, the mixture is heated and boiled by a microwave oven, so that the agarose is completely dissolved, a small amount of Ethidium Bromide (EB) is dripped when the mixture is cooled to room temperature and does not scald hands, the mixture is poured into a glue groove in which a comb is placed in advance after the mixture is uniformly mixed, and the comb is pulled out after the mixture is cooled to room temperature and is completely solidified.

EXAMPLE 1 construction of mammalian expression vectors for TMP-L1-TMP-L2-3DHSA fusion proteins

Construction of pcDNA3-TMP-L1-TMP-L2-3DHSAF expression vector

ss (Fc) -TMP-L1-TMP-L2-H-fip Gene sequence acquisition: the gene sequence ss-TMP-L-TMP (ss is the signal peptide sequence of Fc fragment, TMP is the mimic peptide) was synthesized in Takara Bio Inc., the first round of amplification was performed with primers P1 (ctggatggcctccatcagctcgtccctgctcacgcacggtgggcatgtgtgagttttg) and P2 (gaagaagctttgctatggagacagacacactcct) using plasmid pMD19-T-ss-TMP-L1-TMP as template, then the PCR product was used as template, the primers P3 (gaagaagctttgctatggagacagacacactcct) and P4 (gaagctgcagcctgaagttgatctcctcctgcttctggatggcctccatcagctcgtc) were used to amplify the ss (Fc) -TMP-L1-TMP-L2-H-fip gene fragment, and the restriction enzymes HindIII and PstI were used for double cleavage, and the cleavage products were recovered with a gel recovery kit to obtain the purified ss (Fc) -TMP-L1-TMP-L2-H-fip fragment.

Obtaining the 3DHSA gene sequence: plasmid pcDNA3.1 was purchased from Invitrogen, and the HSA complete gene sequence was synthesized on pcDNA3.1 by Takara Bio Inc., to obtain pcDNA3.1-HSA vector. The 3DHSA gene was obtained by two rounds of PCR amplification (the first round PCR primers were P5: aatgctatgccaaagtg and P6: cttggtcatctcctccc; the second round PCR primers were P7: atggagaccaaccccagcatcgtggaagagcctcagaatt and P8: cccgctcgagtcagttggtggtggtgtaagcctaaggcagcttgac). And purifying the PCR product, and then carrying out double enzyme digestion treatment by using PstI/XhoI, wherein the enzyme digestion product is purified and recovered by using a gel recovery kit.

Cloning and identification of pcDNA3-TMP-L1-TMP-L2-3DHSAF expression vector: firstly, carrying out double enzyme digestion on a small amount of extracted pcDNA3 vector by using restriction enzymes HindIII-HF and XhoI, and carrying out gel collection and purification on pcDNA3 (HindIII-HF/XhoI) fragments; the prepared pcDNA3 (HindIII-HF/XhoI), ss (Fc) -TMP-L1-TMP-L2-H-fip and 3DHSA were added to the T4 ligase reaction system and ligated overnight at 16 ℃. The ligation product was then transformed into E.coli TOP10 competent cells. 20 single colonies were picked from transformed Amp-resistant solid LB plates and added to a solid LB plate containing 800. Mu. LAmp ⁺ 1.5mL-tube of liquid LB was incubated at 37℃and 220rpm for 6-7h with primer P9: agaacccactgcttactg and P10: aaaggacagtgggagtg the bacterial liquid PCR identification is carried out, the positive clone is further subjected to HindIII-HF/XhoI double restriction enzyme identification, finally, the bacterial liquid PCR and the positive clone with correct double restriction enzyme identification are sent to Jin Wei intelligent technology Co for sequencing, and the clone with the nucleic acid sequence completely consistent with the target gene sequence is stored at-80 ℃.

Construction of a pcDNA3-TMP-L1-TMP-L2-3DHSAH expression vector

Ss (HSA) -TMP-L1-TMP-L2-H-fip Gene sequence acquisition: the gene sequence ss-TMP-L1-TMP (ss is the signal peptide sequence of HSA, TMP is the mimetic peptide) was synthesized at Jin Wei, intelligent technologies Co., ltd.) and was synthesized on pUC57-Amp/HSA ss-dTMP vector. The plasmid pUC57-Amp/HSA ss-dTMP was used as a template, and the primers P11 (ccaagcttatgaagtgggtaacctttat) and P12 (aactgcagcctgaagttgatctcct) were used for PCR amplification to obtain ss (HSA) -TMP-L1-TMP-L2-H-fip gene fragment, which was digested with restriction enzymes HindIII and PstI, and the above digested product was recovered with a gel recovery kit to obtain a purified ss (HSA) -TMP-L1-TMP-L2-H-fip fragment.

Cloning and identification of pcDNA3-TMP-L1-TMP-L2-3DHSAH expression vector: the prepared pcDNA3 (HindIII-HF/XhoI), ss (HSA) -TMP-L1-TMP-L2-H-fip and 3DHSA were added to the T4 ligase reaction system and ligated overnight at 16 ℃. The ligation product was then transformed into E.coli TOP10 competent cells. From transformationAfter Amp-resistant solid LB plates were picked 20 single colonies and added with 800. Mu. LAmp ⁺ 1.5mL-tube of liquid LB was incubated at 37℃and 220rpm for 6-7h with primer P9: agaacccactgcttactg and P10: aaaggacagtgggagtg the bacterial liquid PCR identification is carried out, the positive clone is further subjected to HindIII-HF/XhoI double restriction enzyme identification, finally, the bacterial liquid PCR and the positive clone which is correctly identified by double restriction enzyme identification are sent to Jin Weizhi Biotechnology Co-Ltd for sequencing, and the clone with the nucleic acid sequence completely consistent with the target gene sequence is stored at-80 ℃.

Construction of a three.pCHO1.0-TMP-L1-TMP-L2-3 DHSAF expression vector

preparation of pCHO1.0 linearization vector the pCHO1.0 vector was digested with restriction enzymes AvrII and Bsz I, and then subjected to gel recovery and purification for use.

Cloning and identification of pCHO1.0-TMP-L1-TMP-L2-3DHSAF expression vector: the pcDNA3-TMP-L1-TMP-L2-3DHSAF plasmid is used as a template, and a primer P13 is used: acggttccgggccgcctaggatggagacagacacac and P14: gtataatatagagtatactcagtggtggtggtggtggtg the ss (Fc) -TMP-L1-TMP-L2-H-fip fragment with the 15bp homologous sequence of the end and the pCHO1.0 linearization carrier band is obtained through PCR amplification reaction, and the PCR amplification product is purified and recovered by using a gel recovery kit. By the principle of In-Fusion seamless cloning technology, according toHD Cloning Kit procedure pCHO1.0 linearization vector was ligated with ss (Fc) -TMP-L1-TMP-L2-H-fip fragment by In-Fusion reaction. Then, 2.5 mu L of In-Fusion reaction product is taken, 50 mu L of top10 competent cell liquid which is just thawed is added, the mixture is gently mixed, the mixture is placed on ice for 30min, and then the mixture is placed on ice for 2min after heat shock for 45s at 42 ℃; finally, 150 mu L of non-resistant LB liquid medium is added into the system, the mixture is resuscitated at 37 ℃ and 150rpm for 1h, and all resuscitated bacterial liquid is uniformly coated on a kana+ resistant plate, and incubated at 37 ℃ for 20h. 20 single colonies were picked, positive clones were identified by two methods of PCR (primers P15: gtctgagcctccttgtcttg and P14: gtataatatagagtatactcagtggtggtggtggtggtg) and avrII and Bsz I, and finally the correct clones were identified by both methods to Jin Weizhi BiotechnologyThe end-point was sequenced and clones with completely identical nucleic acid sequences to the gene sequence of interest were stored at-80 ℃.

Construction of a four.pCHO1.0-TMP-L1-TMP-L2-3 DHSAH expression vector

Ss (HSA) -TMP-L1-TMP-L2-H-fip Gene sequence acquisition: the pUC57-Amp/HSA ss-dTMP plasmid is used as a template, and a ss (HSA) -TMP-L1-TMP-L2-H-fip fragment with 15bp homologous sequences at the tail end of the pCHO1.0 linearization vector and the 3DHSA gene sequence is obtained through PCR amplification reaction (primer P16: acggttccgggccgcctaggatgaagtgggtaa and P17: aactgcagcctgaagttgatctcct) and glue recovery and purification.

Obtaining the 3DHSA gene sequence: the pcDNA3-TMP-L1-TMP-L2-3DHSAF plasmid is used as a template, and a PCR (primer P18: caacttcaggctgcaggactacatcgacaggatcat and P14: gtataatatagagtatactcagtggtggtggtggtggtg) amplification reaction and glue recovery and purification are carried out to obtain a 3DHSA gene fragment with the tail end having 15bp homologous sequences with the pCHO1.0 linearization vector and ss (HSA) -TMP-L1-TMP-L2-H-fip respectively.

Cloning and identification of pCHO1.0-TMP-L1-TMP-L2-3DHSAH expression vector: by the principle of In-Fusion seamless cloning technology, according toHD Cloning Kit procedure the pCHO1.0 linearized vector prepared above, ss (HSA) -TMP-L1-TMP-L2-H-fip and the 3DHSA gene fragment were ligated by an In-Fusion reaction. Then, 2.5 mu L of In-Fusion reaction product is taken, 50 mu L of top10 competent cell liquid which is just thawed is added, the mixture is gently mixed, the mixture is placed on ice for 30min, and then the mixture is placed on ice for 2min after heat shock for 45s at 42 ℃; finally, 150 mu L of non-resistant LB liquid medium is added into the system, the mixture is resuscitated at 37 ℃ and 150rpm for 1h, and all resuscitated bacterial liquid is uniformly coated on a kana+ resistant plate, and incubated at 37 ℃ for 20h. Selecting 20 single colonies, identifying positive clones by two methods of PCR (primer P15: gtctgagcctccttgtcttg and P14: gtataatatagagtatactcagtggtggtggtggtggtg) and avrII and Bsz I, and finally, sending the clones identified correctly by the two methods to Jin Weizhi Biotechnology Co., ltd for sequencing, and preserving the clone with the nucleic acid sequence completely consistent with the target gene sequence at-80 DEG C。

EXAMPLE 2 expression of dimeric thrombopoietin mimetic peptide TMP diad-human serum albumin third domain fusion protein in mammalian cells CHO-S

Chinese hamster ovary suspension cells CHO-S, available from Invitrogen, inc., are wild-type CHO cells that have been acclimatized to a high-density, serum-free suspension culture, and are capable of mass production of secreted recombinant proteins. Since 2012, the laboratory has been working on the study of the expression of fusion proteins by CHO-S cells, creating an optimized transient transfection of fusion protein expression vectors and fusion protein expression systems.

Preparation of CD-FortiCHOTM complete broth prior to cell resuscitation. 200mM L-glutamine was added to the CD-FortiCHOTM medium to a final concentration of 8mM. Storing in 2-8deg.C in dark place for use, and preheating in 37deg.C water bath before use. The CHO-S cell cryopreservation tube was removed from the liquid nitrogen tank and thawed rapidly (within 1 min) in a 37℃water bath. To 125-mL shaker flash (cell culture specific, polycarbonate Erlenmeyer Flask) was transferred 29mL of CD-FortiCHOTM complete broth, and the cell frozen stock was carefully added to the complete broth with a pipette. Placing shaker flash on a shaking table in a cell incubator at 37deg.C, 8% CO ₂ Culturing at 150 rpm. After the cells are recovered and cultured for 2-3d, the cell density reaches 1 multiplied by 10 ⁶ -2×10 ⁶ cells/mL (cells/mL) were passaged with CD-FortiCHOTM complete medium and passed once for 3-4d, thereby culturing for 6-9d to restore the cells to optimal conditions. The day before cell transfection, cell passaging was performed at a passaging density of 5×10 ⁵ cells/mL. On the day of transfection, the cell density was adjusted to 1X 10 ⁶ The transfection system was 30mL in cells/mL. Extracting plasmids to be transfected by using a heat source-removing mass extraction kit for standby, preparing a transfection complex by using PEI as a transfection reagent according to a reagent specification, wherein the transfection conditions are as follows: PEI DNA mass ratio=3:1, DNA transfection concentration was 50. Mu.g/30 mL system. The prepared transfection complex is dripped into CHO-S suspension cells of a 30mL system, and the culture flask is shaken while dripping, so that the transfection complex is uniformly mixed. The cells were then returned to the shaker of the cell incubator at 37℃with 8% CO ₂ Normal at 150rpmCulturing under culture condition, and culturing at low temperature by adjusting temperature to 32deg.C for 6 hr after transfection. The cell viability was detected by daily sampling until the cell viability was less than 60%, and the culture was collected and centrifuged at 10000rpm for 10min at 4℃to obtain a cell transient expression supernatant containing the target product.

All four clones constructed successfully in example 1 were expressed in CHO-S cells as described above to obtain the target fusion protein with the sequence TMP-L1-TMP-L2-H-fip-3 DHSA.

Example 3 fusion protein Activity assay results

TPO receptor c-mpl is widely present in hematopoietic tissues (including stem cells, megakaryocyte cell line colony-forming cells, etc.), and is a transmembrane protein receptor. TPO or TPO mimetic peptide TMP binds to the extracellular region of the c-mpl receptor, and c-mpl polymerizes to form homodimers, activating various signaling pathways such as intracellular JAK2-STAT5, shc-Ras-MAPK, anti-apoptosis pathways, and the like, leading to increased platelet yield. At the same time, the phosphorylated STAT5 dimer enters the nucleus and binds to c-fos pro, thereby triggering downstream firefly luciferase gene expression. Therefore, the purpose of detecting the activity of the TMP recombinant protein sample to the c-mpl receptor can be achieved by detecting the luminous signal intensity of the firefly luciferase substrate in the cells.

In the experiment, a Renilla constitutive expression vector containing Firefly Luciferase is used as an internal reference, so that errors caused by factors such as cell plating density, experiment operation and the like are eliminated. In the course of the experiment, NIH-3T3 cells were first co-transfected with plasmid pHF443 harboring the c-fos promoter and the Luciferase gene, pRL-SV40Renilla Vector harboring Renilla Luciferase gene, pAdVAntageTM Vector harboring the promoter enhancer gene and plasmid pcDNA3.1/c-mpl harboring the c-mpl gene, and NIH-3T3 cells were allowed to express TPO receptors c-mpl and Firefly Luciferase, renilla Luciferase. Transfected cells were treated with terbium (rhTPO) positive drug and recombinant protein samples, while a blank control group was set. After 6h of cell incubation, the Dua-GloTM Luciferase Reagent kit was used to measure the fluorescence signal intensities of Firefly Luciferase and Renilla Luciferase between the different treatment groups, and the ratio of the fluorescence intensities of Firefly Luciferase and Renilla Luciferase was used to represent the c-mpl receptor dependent bioactivity of the fusion protein cellular level.

The specific experimental process is as follows:

NIH-3T3 cells were plated, pcDNA3.1/c-mpl, pHF443, pAdVAntageTM vector, renilla, four plasmid co-transfection. And respectively adding positive control, blank control and a sample to be tested after transfection for 48 hours, respectively detecting the firefly luciferase activity and the Renilla luciferase activity, calculating the fluorescence ratio, wherein the fluorescence ratio=firefly fluorescence value/Renilla fluorescence value, specifically referring to Chinese patent (CN 201310084212), and finally calculating the specific activity of each fusion protein according to the reference positive control fluorescence ratio.

Experimental results:

table 1 results of detecting the biological Activity of fusion proteins

The results of the double luciferase activity assay in Table 1 show that all of the pcDNA3.1-HSA empty vector treated groups transfected with no mpl receptor, namely, negative control groups, had a relative fluorescence ratio of 1 and a specific activity of 0U/mg.

Table 1 shows: the detected TPO positive medicine and the dimerization fusion protein in the CHO expression sample have obvious mpl receptor dependent cell level biological activity, and the activity is obviously higher than that of the positive medicine.

Therefore, the dimeric fusion protein of the TMP diad and the third structural domain of the human serum albumin can act with a thrombopoietin receptor c-mpl, has the activity of stimulating the proliferation of TPO-dependent cell lines, can be expressed in mammalian cells, and can be applied to the preparation of the thrombopoietin drugs.

Sequence listing

<110> university of Lanzhou

<120> a dimeric fusion protein, method for preparing same and use thereof

<141> 2017-11-08

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atcgaaggcc ccacactgcg gcagtggctg gctgctaggg ccggtggccc ctccggaatc 60

gagggaccca ccctgaggca gtggctggcc gccagagcc 99

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<213> Artificial sequence (Artificial Sequence)

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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

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

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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)

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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

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<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)

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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)

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

1 5 10

<210> 9

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gtgagcaggg acgagctgat ggaggccatc cagaagcagg aggagatcaa cttcaggctg 60

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<210> 10

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

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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)

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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 (5)

1. A dimeric fusion protein, wherein the dimeric fusion protein consists of a Thrombopoietin Mimetic Peptide (TMP) diad, a connecting peptide and a human serum albumin third domain (3 DHSA), wherein the TMP diad comprises 2 TMPs and 1 connecting peptide, and the structural formula is represented as follows: TMP-L1-TMP; the dimerization fusion protein also contains a dimerization domain, wherein the dimerization domain is a peptide segment H and a peptide segment fip selected from fip domains; the TMP duplex is positioned at the N-terminal end of the fusion protein, the 3DHSA is positioned at the C-terminal end of the fusion protein, and the structural formula of the dimerization fusion protein is expressed as follows: TMP-L1-TMP-L2-H-fip-3DHSA; the gene sequence of the peptide fragment H is shown as a Seq ID No. 7, the amino acid sequence is shown as a Seq ID No. 8, the gene sequence of the fip domain is shown as a Seq ID No. 9, and the amino acid sequence is shown as a Seq ID No. 10; the amino acid sequence of the TMP-L1-TMP is shown in SEQ ID NO:2, the gene sequence is shown as SEQ ID NO:1, wherein L1 represents a connecting peptide, the DNA sequence of L1 is GGTGGCCCCTCCGGA, the amino acid sequence of L1 is GGPSG, and the two TMP bodies are connected end to end.

2. A dimerized fusion protein according to claim 1, wherein the 3DHSA has the amino acid sequence of SEQ ID NO:4, and the DNA sequence of the amino acid sequence of the 3DHSA is shown as SEQ ID NO: 3.

3. The dimeric fusion protein of claim 1, wherein the fusion protein has an amino acid sequence set forth in SEQ ID NO:6, the DNA sequence of the amino acid sequence of the fusion protein is shown as SEQ ID NO: shown at 5.

4. A method for preparing a dimerized fusion protein according to any one of claims 1-3, comprising the steps of:

(1) Constructing a target fusion protein expression vector by an infusion technology or an enzyme digestion connection method;

(2) Preparing a large amount of fusion protein expression vectors without heat source through extraction, and carrying out transfection;

(3) Recovery of CHO-S cells, 37 ℃,8% CO ₂ Culturing at 170rpm for 6-9d;

(4) The day before transfection, cells were passaged at a passaging density of 5X 10 ⁵ cells/mL；

(5) On the day of transfection, the cell density was adjusted to 1X 10 ⁶ Preparing transfection complexes by cells/mL, and carrying out transfection, wherein PEI is DNA mass ratio=3:1-5:1 in the transfection complexes;

(6) 6h after transfection, the incubator temperature was adjusted to 32℃and CO ₂ The concentration is adjusted to 7%;

(7) Sampling every other day, calculating cell activity until the cell activity is reduced to below 60%, and collecting samples.

5. Use of a dimerized fusion egg according to claim 1 for the preparation of a medicament for thrombopoietin.