Recombinant erythropoietin protein for livestock as well as preparation method and application of recombinant erythropoietin protein

Abstract

The invention relates to veterinary recombinant erythropoietin protein as well as a preparation method and application thereof, and belongs to the technical field of biological pharmaceutical engineering. The veterinary recombinant erythropoietin protein contains veterinary erythropoietin protein and an additional peptide fragment, the animal erythropoietin protein comprises dog erythropoietin or cat erythropoietin; the additional peptide fragment comprises a glycosylated rich peptide fragment and / or a basic amino acid peptide fragment. According to the method, the expression quantity of the target protein in host cells is obviously improved, the protein expression quantity is more than 200mg / L, and the expression quantity of the target protein and the recombinant protein without basic amino acid is less than 50mg / L. The biological activity of the target protein is obviously improved, the biological activity is larger than 60000 IU / mg, the biological activity of the cat recombinant erythropoietin is larger than 80000 IU / mg, and the biological activity of the cat recombinant erythropoietin is obviously improved compared with that of recombinant protein without basic amino acid. The expression quantity, the stability and the biological activity of the target protein are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical engineering, and in particular to a recombinant erythropoietin protein for animals, a preparation method thereof, and an application thereof. Background Art

[0002] Erythropoietin (EPO) is a glycoprotein hormone, also known as erythrocyte colony-stimulating factor or erythropoietin. It is the main cytokine that regulates erythropoiesis in mammals. At the same time, this protein has the function of stabilizing erythrocyte membranes, improving the antioxidant and anti-apoptotic functions of erythrocytes, and is the most important hormone regulating erythrocyte production.

[0003] In the field of human medicine, recombinant human erythropoietin (rhEPO) is used to treat chronic kidney disease, inflammatory bowel disease (Crohn's disease and ulcerative colitis), anemia caused by myelodysplasia induced by radiotherapy and chemotherapy, and symptomatic treatment of anemia caused by some cancers, and has significant clinical application value.

[0004] Currently, there are no veterinary erythropoietin drugs on the domestic and international markets. The difficulty in developing canine and feline erythropoietin products lies in the low in vitro expression level of the protein. The reason is that the C-terminus of the protein is unstable and easily degraded by a variety of proteases. Online analysis of potential enzyme cleavage sites on the protein shows that the proteases that can degrade canine and feline erythropoietin from the C-terminus of the protein include Arg-C proteinase (clostripain), Asp-N endopeptidase (aspartic acid protease), Asp-N endopeptidase+N-terminal Glu (N-terminal glutamic acid) and Trypsin (trypsin).

[0005] The lack of erythropoietin (EPO) for dogs and cats has necessitated the use of human EPO for the treatment of anemia. Research data indicates that after three to six months of treatment with human EPO, approximately 50% of dogs and cats develop antibodies to the EPO, leading to a gradual loss of efficacy in subsequent treatment. This is due to the low conservatism of EPO across species, and the immunogenicity of human EPO in dogs and cats, which leads to the development of anti-Drug antibodies with repeated use. Therefore, the development of veterinary EPO has significant clinical significance, with dogs and cats experiencing the most pressing clinical need.

[0006] The biggest challenges in using recombinant proteins for disease treatment are their degradation in the blood, and their clearance by the kidneys and liver. Metabolic studies in humans indicate that the half-life of recombinant erythropoietin after subcutaneous administration is between 6 and 12 hours. Due to this short half-life, daily dosing is required to maintain effective blood concentrations.

[0007] In recent years, there have been great advances in the technology for extending the half-life of protein drugs. The main methods are: (1) mutating one or more sites in the protein sequence to improve the resistance of the protein to proteases; (2) linking the target protein to a high molecular weight polymer, such as PEG (polyethylene glycol) modification; (3) carrier-mediated in vivo delivery; (4) by fusing the target protein to the end of the peptide or functional protein that does not affect the activity of the protein and has no significant biological function, thereby increasing the molecular weight of the protein and reducing the protein, such as the Fc fragment of the fusion antibody or albumin. Among them, the fourth method only needs to be completed and verified at the protein expression vector design stage, does not require additional process steps, and is relatively more economical and feasible.

[0008] Currently, there is a need to design and prepare recombinant erythropoietin with higher protein expression and stability so that it is suitable for use in animals such as cats and dogs. Summary of the Invention

[0009] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a veterinary recombinant erythropoietin protein and its preparation method and application, and further improve the expression level, biological activity and stability of the recombinant protein.

[0010] To achieve the above object, the technical solution adopted by the present invention is:

[0011] In a first aspect, the present invention provides a recombinant erythropoietin protein for veterinary use, wherein the recombinant erythropoietin protein for veterinary use comprises an animal erythropoietin protein and an additional peptide segment;

[0012] The animal erythropoietin protein includes canine erythropoietin or feline erythropoietin, the amino acid sequence of the canine erythropoietin is shown in SEQ ID NO: 1, and the amino acid sequence of the feline erythropoietin is shown in SEQ ID NO: 83;

[0013] The additional peptide segments include glycosylation-rich peptide segments and / or basic amino acid peptide segments,

[0014] The glycosylation-rich peptide segment contains 24 to 35 amino acids and contains one or more O-glycosylation sites or N-glycosylation modification sites, and the total number of threonine and serine in the glycosylation-rich peptide segment accounts for more than 20%;

[0015] The basic amino acid peptide segment contains 1 to 10 basic amino acids, and the basic amino acids include at least one of arginine, lysine and histidine.

[0016] The present invention directly adds an appropriate number of basic amino acids to the C-terminus of canine or feline erythropoietin to stabilize the protein structure. Compared with the in vitro expression of the native protein, the expression of the recombinant protein with the addition of basic amino acids at the C-terminus is significantly improved. The present invention further improves the expression, biological activity, and stability of the recombinant protein by first adding a glycosylated polypeptide to the C-terminus of canine or feline erythropoietin and then adding an appropriate number of basic amino acids.

[0017] Proteins with a high content of basic amino acids at the C-terminus, such as ribosomal proteins and histones, have better protein stability. The C-terminus of ribosomal proteins is rich in arginine, which interacts with the RNA phosphate backbone through salt bridges to stabilize the overall structure. The C-terminus of histones is rich in lysine, which binds to the DNA phosphate backbone to stabilize and maintain chromatin structure. From the perspective of protein structural biology, the principles involved mainly involve the following mechanisms:

[0018] (1) Charge interaction: basic amino acids neutralize the negative charge at the C-terminus: the C-terminus of proteins usually has a free carboxyl group (COO - ), negatively charged at physiological pH. The side chains of basic amino acids are positively charged (such as -NH3 of lysine). + The guanidine group of arginine (such as glutamic acid and aspartic acid) can interact with the negative charge of the C-terminus through electrostatic attraction, forming a salt bridge (ionic bond), thereby reducing the electrostatic repulsion at the C-terminus and enhancing the stability of the local structure. Shielding electrostatic repulsion: If other negatively charged residues (such as glutamic acid and aspartic acid) are present near the C-terminus, the positive charge of the basic amino acid can neutralize these repulsive forces and prevent the structure from becoming loose.

[0019] (2) Hydrogen bond formation: The side chains of basic amino acids (such as the guanidine group of arginine and the imidazole group of histidine) or the main chain amino groups can form hydrogen bonds with the carboxyl oxygen atom at the C-terminus, further fixing the C-terminal conformation.

[0020] (3) Steric hindrance effect: The side chains of basic amino acids are usually large (such as the guanidine group of arginine), which can form a tight stack with the residues near the C-terminus through hydrophobic interaction or van der Waals force, limiting the flexibility of the C-terminus, reducing conformational entropy, and thus stabilizing the structure.

[0021] (4) Synergistic effect with metal ions: In some proteins, basic amino acids may help bind metal ions, such as Ca 2+ and Mg 2+ , these ions can further bridge the negative charge at the C-terminus and the basic residues to form a stable complex.

[0022] (5) Regulating protein degradation: C-terminal stability is related to protein half-life. Basic amino acids may indirectly maintain structural integrity by masking C-terminal degradation signals (such as degradation-related hydrophobic residues) and reducing proteasome recognition.

[0023] Furthermore, the C-terminus of the animal erythropoietin protein is connected to the N-terminus of the additional peptide segment.

[0024] Furthermore, when the additional peptide segment includes a basic amino acid peptide segment, the N-terminus of the basic amino acid peptide segment is connected to the C-terminus of the animal erythropoietin protein.

[0025] Furthermore, when the additional peptide segment includes a glycosylation-rich peptide segment and a basic amino acid peptide segment, the N-terminus of the glycosylation-rich peptide segment is connected to the C-terminus of the animal erythropoietin protein, and the C-terminus of the glycosylation-rich peptide segment is connected to the N-terminus of the basic amino acid peptide segment.

[0026] Furthermore, when the animal erythropoietin protein includes canine erythropoietin, the glycosylation-rich peptide segment includes at least one of a glycosylation-rich peptide segment in canine mucin 5B, a carboxyl-terminal peptide of human chorionic gonadotropin, and a glycosylation-rich peptide segment in canine IgG D heavy chain Fc; the amino acid sequence of the glycosylation-rich peptide segment in canine mucin 5B is shown in SEQ ID NO: 2, the amino acid sequence of the carboxyl-terminal peptide of human chorionic gonadotropin is shown in SEQ ID NO: 3, and the amino acid sequence of the glycosylation-rich peptide segment in canine IgG D heavy chain Fc is shown in SEQ ID NO: 4;

[0027] When the animal erythropoietin protein includes feline erythropoietin, the glycosylation-rich peptide segment includes at least one of the glycosylation-rich peptide segment in feline fibronectin III, the carboxyl-terminal peptide of human chorionic gonadotropin, and the glycosylation-rich peptide segment in the Fc of a feline antibody; the amino acid sequence of the glycosylation-rich peptide segment in the feline fibronectin III is shown in SEQ ID NO: 84, and the amino acid sequence of the glycosylation-rich peptide segment in the Fc of the feline antibody is shown in SEQ ID NO: 85.

[0028] Furthermore, the amino acid sequence of the basic amino acid peptide segment is shown in SEQ ID NO: 5 to SEQ ID NO: 16, which correspond to basic amino acids C1 to C12 in sequence.

[0029] Preferably, the basic amino acid peptide segment contains arginine and histidine, or contains lysine and histidine.

[0030] More preferably, the amino acid sequence of the basic amino acid peptide segment is as shown in SEQ ID NO: 13 to SEQ ID NO: 16, corresponding to amino acids C9 to C12 in sequence.

[0031] In a specific embodiment of the present invention, the amino acid sequence of the recombinant erythropoietin protein for veterinary use is shown as SEQ ID NO: 17 to SEQ ID NO: 66 or SEQ ID NO: 86 to SEQ ID NO: 135.

[0032] Preferably, when the animal erythropoietin protein includes canine erythropoietin, the amino acid sequence of the recombinant veterinary erythropoietin protein is as shown in SEQ ID NO: 19 to SEQ ID NO: 64. More preferably, the amino acid sequence of the recombinant veterinary erythropoietin protein is as shown in SEQ ID NO: 25 to SEQ ID NO: 28, SEQ ID NO: 37 to SEQ ID NO: 40, SEQ ID NO: 49 to SEQ ID NO: 50, or SEQ ID NO: 61 to SEQ ID NO: 64.

[0033] Preferably, when the animal erythropoietin protein includes feline erythropoietin, the amino acid sequence of the recombinant veterinary erythropoietin protein is shown in SEQ ID NO: 88 to SEQ ID NO: 133. More preferably, the amino acid sequence of the recombinant veterinary erythropoietin protein is shown in SEQ ID NO: 94 to SEQ ID NO: 97, SEQ ID NO: 106 to SEQ ID NO: 109, SEQ ID NO: 118 to SEQ ID NO: 121, or SEQ ID NO: 130 to SEQ ID NO: 133.

[0034] In a second aspect, the present invention provides the use of the recombinant erythropoietin protein for veterinary use in the preparation of a medicament for preventing and treating anemia in animals.

[0035] Furthermore, the veterinary recombinant erythropoietin protein is used in the preparation of a medicament for treating anemia symptoms caused by endogenous erythropoietin deficiency in companion animals; the anemia is caused by at least one of chronic kidney disease, viral infection, inflammatory bowel disease, and myelodysplasia; and the companion animals include dogs or cats. When the veterinary erythropoietin protein includes canine erythropoietin, the amino acid sequence of the veterinary recombinant erythropoietin protein is as shown in SEQ ID NO: 17 to SEQ ID NO: 66, and the veterinary erythropoietin protein is used to prepare a medicament for canine anemia; when the veterinary erythropoietin protein includes feline erythropoietin, the amino acid sequence of the veterinary recombinant erythropoietin protein is as shown in SEQ ID NO: 86 to SEQ ID NO: 135, and the veterinary erythropoietin protein is used to prepare a medicament for feline anemia.

[0036] In a third aspect, the present invention provides a drug for preventing and treating anemia in animals, wherein the drug contains the recombinant erythropoietin protein for animal use.

[0037] Furthermore, the medicine also contains a pharmaceutically acceptable carrier.

[0038] Furthermore, the carrier includes at least one of a diluent, an adjuvant and a sustained-release agent.

[0039] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the recombinant erythropoietin protein for veterinary use.

[0040] Furthermore, in the nucleic acid molecule, the nucleotide sequence of the gene encoding the canine erythropoietin is shown as SEQ ID NO: 67, the nucleotide sequence of the gene encoding the cat erythropoietin is shown as SEQ ID NO: 136, the nucleotide sequence of the gene encoding the glycosylation-rich peptide segment in the canine mucin 5B is shown as SEQ ID NO: 68, the nucleotide sequence of the gene encoding the carboxyl-terminal peptide of human chorionic gonadotropin is shown as SEQ ID NO: 69, the nucleotide sequence of the gene encoding the glycosylation-rich peptide segment in the Fc of the canine IgG D heavy chain is shown as SEQ ID NO: 70, the nucleotide sequence of the gene encoding the glycosylation-rich peptide segment in the cat fibronectin III is shown as SEQ ID NO: 137, the nucleotide sequence of the gene encoding the glycosylation-rich peptide segment in the Fc of the cat antibody is shown as SEQ ID NO: 138, the nucleotide sequence of the gene encoding the basic amino acid C1 is shown as SEQ ID NO: 71, and the nucleotide sequence of the gene encoding the basic amino acid C2 is shown as SEQ ID NO: The nucleotide sequence of the gene encoding the basic amino acid C3 is shown in SEQ ID NO:72, the nucleotide sequence of the gene encoding the basic amino acid C3 is shown in SEQ ID NO:73, the nucleotide sequence of the gene encoding the basic amino acid C4 is shown in SEQ ID NO:74, the nucleotide sequence of the gene encoding the basic amino acid C5 is shown in SEQ ID NO:75, the nucleotide sequence of the gene encoding the basic amino acid C6 is shown in SEQ ID NO:76, the nucleotide sequence of the gene encoding the basic amino acid C7 is shown in SEQ ID NO:77, the nucleotide sequence of the gene encoding the basic amino acid C8 is shown in SEQ ID NO:78, the nucleotide sequence of the gene encoding the basic amino acid C9 is shown in SEQ ID NO:79, the nucleotide sequence of the gene encoding the basic amino acid C10 is shown in SEQ ID NO:80, the nucleotide sequence of the gene encoding the basic amino acid C11 is shown in SEQ ID NO:81, and the nucleotide sequence of the gene encoding the basic amino acid C12 is shown in SEQ ID NO:82.

[0041] In a fifth aspect, the present invention provides a recombinant expression vector containing the nucleic acid molecule.

[0042] Furthermore, the backbone vector of the recombinant expression vector includes pCDNA3.1, pCDNA 3.1-GS, pEE12.4, pIRES, pTT5, pCEP4 or pCDNA 3.4.

[0043] In a sixth aspect, the present invention provides a recombinant cell containing the recombinant expression vector.

[0044] Furthermore, the host cells of the recombinant cells include CHO cells, COS cells, NS0 cells, BHK cells, HEK-293 cells, PER-C6, CRFK or MDCK cells.

[0045] In a seventh aspect, the present invention provides a method for preparing the recombinant erythropoietin protein for veterinary use, culturing the recombinant cells, and expressing the recombinant erythropoietin protein for veterinary use.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] Significantly increases the expression of the target protein in host cells, reaching >200 mg / L, compared to less than 50 mg / L for recombinant proteins without the addition of basic amino acids. Significantly increases the biological activity of the target protein, reaching >60,000 IU / mg. The biological activity of feline recombinant erythropoietin exceeds 80,000 IU / mg, significantly higher than that of recombinant proteins without the addition of basic amino acids. This approach enhances the expression, stability, and biological activity of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Design of a recombinant erythropoietin protein for veterinary use. A is erythropoietin protein + basic amino acid peptide; B is erythropoietin protein + glycosylation-rich peptide + basic amino acid peptide. DETAILED DESCRIPTION

[0049] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0050] Example 1 Design of different recombinant canine erythropoietin proteins, cell pool screening, and protein preparation

[0051] 1. Design of recombinant canine erythropoietin protein sequence and control recombinant canine erythropoietin protein sequence

[0052] (1) The protein sequence of canine erythropoietin (cEPO) was searched on NCBI, and the sequence with NCBI accession number BAM65821.1 was selected. Its amino acid sequence is shown in SEQ ID NO: 1 (sequence number A1): MGACECPALFLLLSLLLLPLGLPVLGAPPRLICDSRVLER YILEAREAENVTMGCAQGCSFSENITVPDTKVNFYTWKRMDVGQQALEVWQGLALLSEAILRGQALLA NASQPSETPQLHVDKAVSSLRSLTSLLRALGAQKEAMSLPEEASPAPLRTFTVDTLCKLFRIYSNFLRGKLTLYTGEACRRGDR.

[0053] (2) Selection of glycosylation-rich peptides

[0054] Canine mucin 5B is a highly glycosylated protein (NCBI accession number: XP_038281459.1). A peptide with relatively rich O-glycosylation (sequence number B1) was selected from it, and its amino acid sequence is shown in SEQ ID NO: 2: SSPGTSQGVTTSTTQTGPSSPGTS; the carboxyl terminal (CTP) sequence of human chorionic gonadotropin (sequence number B2) is shown in SEQ ID NO: 3: SSSSKAPPPSLPSPSRLPGPSDTPIL PQ; antibodies are proteins with rich glycosylation modifications, and a peptide with rich glycosylation (sequence number B3) was selected from canine IgG D heavy chain Fc (GenBank: AAL35304.1). Its amino acid sequence is shown in SEQ ID NO: 4: HQPSVYVLPPSPKELSSSDTVTLT.

[0055] (3) Random combinations of basic amino acids fused to the C-terminus of the protein, 12 of which are selected for illustration, and their sequences are shown in SEQ ID NO: 5 to SEQ ID NO: 16 in Table 1.

[0056] Table 1 Basic amino acid sequence combinations

[0057]

[0058]

[0059] (4) The basic amino acids shown in Table 1 were connected to the C-terminus of canine erythropoietin, and the resulting sequences were shown as D1 to D12 in Table 2.

[0060] (5) The B1 peptide segment was first connected to the C-terminus of canine erythropoietin to obtain an A1+B1 fusion protein. The basic amino acids shown in Table 1 were then connected to the C-terminus of the A1+B1 fusion protein. The resulting sequence is shown in D13 to D24 in Table 2.

[0061] The B2 peptide segment was first connected to the C-terminus of canine erythropoietin to obtain the A1+B2 fusion protein, and the basic amino acids shown in Table 1 were then connected to the C-terminus of the A1+B2 fusion protein. The resulting sequence is shown in D25 to D36 in Table 2.

[0062] The B3 peptide segment was first connected to the C-terminus of canine erythropoietin to obtain the A1+B3 fusion protein, and the basic amino acids shown in Table 1 were then connected to the C-terminus of the A1+B3 fusion protein. The resulting sequence is shown in D37 to D48 in Table 2.

[0063] (6) Control scheme: The wild-type canine erythropoietin protein sequence (A1) and the canine erythropoietin sequence connected only to the B1 or B2 peptide segment without being connected to the two sequences of basic amino acids shown in Table 1 (shown as D49 and D50 in Table 2) were used as control sequences.

[0064] Table 2 Design scheme of recombinant canine erythropoietin protein

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] 2. Design of recombinant canine erythropoietin protein expression vector

[0072] The present invention uses pCDNA3.1(+) as a backbone vector, reverse-translates the D1-D50 and A1 proteins in Table 2 according to the amino acid sequence, and performs codon optimization and overall nucleic acid sequence optimization according to the preference of CHO cells. The optimized nucleic acid sequences encoding the D1-D50 and A1 proteins, and the nucleic acid sequences encoding the A1, B1-B3, and C1-C12 proteins are shown in Table 3. A gene synthesis company uses conventional technical means to perform full gene synthesis and expression vector construction, thereby obtaining 51 corresponding recombinant plasmids.

[0073] Table 3

[0074]

[0075] 3. Transfect the expression vector into CHO-K1 cells for expression verification

[0076] The 51 recombinant plasmids in step 2 were transfected into CHO-K1 cells respectively. CHO-K1 cells were cultured in suspension using KD-CHO medium (Cat. No. K03201) from Zhuhai Kairui Biotechnology Co., Ltd., in a 37°C, 5% carbon dioxide incubator with a shaker speed of 120 rpm. Cells in the logarithmic growth phase of the appropriate generation with a viability greater than 97% were taken and cultured 24 hours before transfection at a rate of 1×10 6 The cells were transferred at a density of 100 cells / mL, and the transfection experiment was performed according to the instructions of PEI 40K transfection reagent (Cat. No.: 40816ES02 -100 mg) of Yisheng Biotechnology Co., Ltd. At the same time, the backbone vector pcDNA3.1(+) was used for transfection as a negative control for expression detection; 48 hours after transfection, the culture supernatant of each group of transfected cells was collected for Western-blot detection, and the culture supernatant of the cells transfected with the backbone vector pcDNA3.1(+) was used as a negative control sample.

[0077] 4. Screening process of stable CHO-K1 cell pools with various expression vectors

[0078] 24 hours after transfection in step 3, positive transfection cells were screened using KD-CHO medium supplemented with G418 (Geneticin) as follows:

[0079] (1) Cells were collected at 2×10 4 The amount of cells / well was seeded into 24-well plates and cultured in KD-CHO medium supplemented with 400 μg / mL G418 for the first round of screening;

[0080] (2) 4 to 7 days after the first round of screening culture in step (1), the culture medium was replaced for a second round of screening culture, and the concentration of G418 in the KD-CHO culture medium was increased to 600 μg / mL;

[0081] (3) After 4 to 7 days of the second round of screening culture in step (2), the culture medium was replaced for the third round of screening culture. The concentration of G418 in the KD-CHO culture medium was increased to 800 μg / mL, and the cells were cultured until the cell viability increased to greater than 95%. The cell pool screening was completed, and seed cells expressing each recombinant canine erythropoietin protein were obtained.

[0082] 5. Preparation of recombinant canine erythropoietin fusion protein expression samples

[0083] (1) Take the seed cells in the logarithmic growth phase in step 4 and add 1×10 6The cell density was transferred to KD-CHO-CD3 culture medium (Cat. No. K03201-CD3) of Zhuhai Kairui Biotechnology Co., Ltd. for cell expansion and culture. During the process, the cell density was monitored. When the cell density reached 10×10 6 Cells / mL began to supplement nutrients. The feeding plan refers to the instructions for use of CD3 feed K (Cat. No.: K40005K) and CD3 feed C (Cat. No.: K40005C) feeding medium of Zhuhai Kairui Biotechnology Co., Ltd.

[0084] (2) Monitor cell density and viability. When cell viability drops to 90%, stop culturing and collect samples. Centrifuge at 12,000 rpm and 4°C for 15 min. Pass the supernatant through a 0.45 μm filter membrane and perform anion exchange chromatography. Monitor the peak of total protein during the chromatography process using a 280 nm UV monitor. The equilibration solution is 20 mM PB, pH 8.0; the elution solution is 20 mM PB, 0.15 M NaCl, pH 8.0. Collect the elution peak to obtain the purified protein stock solution. Samples collected during the purification process were subjected to SDS-PAGE analysis, and the concentration of the protein stock solution was determined. The yield of canine erythropoietin was calculated. The results are shown in Table 4.

[0085] 6. Biological activity detection of recombinant canine erythropoietin

[0086] Refer to the "Human Erythropoietin In Vivo Biological Activity Assay (Reticulocyte Method)" in Part III of the 2020 edition of the Pharmacopoeia. The national standard for the activity assay of recombinant human erythropoietin (CHO cells) is used as a reference substance to calibrate the activity of the sample. The method is as follows:

[0087] (1) The sample to be tested (protein stock solution) was diluted with sample diluent (prepared with bovine albumin into a sample diluent of 0.1 g / 100 mL using normal saline) to 50 μg / mL, 25 μg / mL, and 12.5 μg / mL as different doses of the sample to be tested.

[0088] (2) Female BALB / c mice aged 6 to 8 weeks were selected, with 4 mice in each group. According to the group, 100 μL of standard or test sample was injected subcutaneously.

[0089] (3) On the fourth day after the injection of step (2), 3 to 4 drops of blood were collected from the mouse orbital cavity and mixed with reticulocyte staining solution at a volume ratio of 1:1. The mixture was allowed to stand at room temperature for 10 minutes. After staining, the reticulocytes showed a blue reticular structure in the cells. The reticulocyte ratio (Ret%) was calculated in at least 1000 red blood cells under a microscope. The in vivo biological activity of the test article was calculated according to the method of parallel lines of the quantitative response of the injected dose to Ret%.

[0090] The results of the biological activity of each design scheme are shown in Table 4. Analysis of the protein purification yield and biological activity test results of each scheme showed that the fusion of basic amino acids other than arginine to the C-terminus of canine erythropoietin significantly increased the protein expression and biological activity. First fusing any one of the glycosylated polypeptides B1, B2 or B3 and then adding basic amino acids to the C-terminus of the protein further increased the protein purification yield and biological activity. Among them, the combination of arginine (R) and histidine (H) and the combination of lysine (K) and histidine (H) were the best.

[0091] Table 4 Protein purification yields and biological activity test results of recombinant canine erythropoietin in different schemes

[0092]

[0093]

[0094] In Table 3, “-” indicates low expression level or failure to purify the protein; NA indicates “not detected”.

[0095] Example 2

[0096] 1. Sequence design of recombinant feline erythropoietin protein and control recombinant canine erythropoietin protein

[0097] (1) The protein sequence of feline erythropoietin (fEPO) was searched from NCBI. Its NCBI sequence accession number is NP_001009269.1, and the amino acid sequence is shown in SEQ ID NO: 83 (sequence number A2): MGSCECPALLLLLSLLLLPLGLPVLGAPPRLICDSRVLERY ILGAREAENVTMGCAEGCSFSENITVPDTKVNFYTWKRMDVGQQAVEVWQGLALLSEAILRGQALLAN SSQPSETLQLHVDKAVSSLRSLTSLLRALGAQKEATSLPEATSAAPLRTFTVDTLCKLFRIYSNFLRGKLTLY TGEACRRGDR.

[0098] (2) Selection of glycosylation-rich peptides

[0099] Feline fibronectin III (Fibronectin, NCBI sequence number: XP_019679572.2), from which a peptide containing three potential N-glycosylation sites was selected (sequence number B4), the amino acid sequence of which is shown in SEQ ID NO: 84: VENTTQTYTVDNTSSAYTVDNTSST; the carboxyl terminal (CTP) sequence of human chorionic gonadotropin (human chorionic gonadotropin) (sequence number B2) is shown in SEQ ID NO: 3; the Fc region of feline antibodies is rich in glycosylation modifications, and a glycosylated-rich peptide (sequence number B5) was selected from the antibody sequence with the sequence number GenBank: ATI97577.1, the amino acid sequence of which is shown in SEQ ID NO: 85: QASGLYSLSSMVTVPSSRWLSDTF.

[0100] (3) Random combinations of basic amino acids fused to the C-terminus of the protein, 12 of which are selected for illustration, and their sequences are shown in SEQ ID NO: 5 to SEQ ID NO: 16 in Table 1.

[0101] (4) The basic amino acids shown in Table 1 were connected to the C-terminus of feline erythropoietin, and the resulting sequences were shown as D51 to D62 in Table 5.

[0102] (5) The B4 peptide segment was first connected to the C-terminus of feline erythropoietin to obtain the A2+B4 fusion protein. The basic amino acids shown in Table 1 were then connected to the C-terminus of the A2+B4 fusion protein. The resulting sequence is shown in D63 to D74 in Table 5.

[0103] The B2 peptide segment was first connected to the C-terminus of feline erythropoietin to obtain an A2+B2 fusion protein, and the basic amino acids shown in Table 1 were then connected to the C-terminus of the A2+B2 fusion protein. The resulting sequence is shown in D75 to D86 in Table 5.

[0104] The B5 peptide segment was first connected to the C-terminus of feline erythropoietin to obtain the A2+B5 fusion protein, and the basic amino acids shown in Table 1 were then connected to the C-terminus of the A2+B5 fusion protein. The resulting sequence is shown in D87 to D98 in Table 5.

[0105] (6) Control scheme: The wild-type protein sequence of feline erythropoietin (sequence number A2) and the canine erythropoietin sequence were connected only to the B4 or B2 peptide segment without being connected to the two sequences of basic amino acids shown in Table 1 (shown as D99 and D100 in Table 5) as control sequences.

[0106] Table 5 Recombinant feline erythropoietin protein sequence

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] The design of recombinant feline erythropoietin protein expression vector, expression vector transfection into CHO-K1 cells for expression verification, screening process of each expression vector stably transfected into CHO-K1 cell pool, preparation of recombinant feline erythropoietin fusion protein expression sample, and recombinant feline erythropoietin biological activity detection method are the same as Example 1. The D51-D100 and A2 proteins in Table 5 were reverse translated according to the amino acid sequence and codon optimized and the overall nucleic acid sequence optimized according to the preference of CHO cells. Among the optimized nucleic acid sequences encoding D51-D100 and A2 proteins, the nucleic acid sequences encoding B1-B3 and C1-C12 proteins are shown in Table 3, and the nucleic acid sequences encoding A2, B4 and B5 proteins are shown in Table 6.

[0114] Table 6

[0115]

[0116]

[0117] The results of the yield and biological activity of feline erythropoietin are shown in Table 7.

[0118] Analysis of the protein purification yield and biological activity test results of each scheme showed that the fusion of basic amino acids other than arginine at the C-terminus of feline erythropoietin significantly increased the protein expression and biological activity. First fusing any one of the glycosylated polypeptides B2, B4 or B5 and then adding basic amino acids to the C-terminus of the protein further improved the protein purification yield and biological activity. Among them, the combination of arginine and histidine and the combination of lysine and histidine were the best.

[0119] Table 7 Protein purification yield and biological activity test results of recombinant feline erythropoietin in different schemes

[0120]

[0121] In Table 7, “-” indicates low expression level or failure to purify the protein; NA indicates “not detected”.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A recombinant erythropoietin protein for veterinary use, characterized in that: The veterinary recombinant erythropoietin protein contains animal erythropoietin protein and an additional peptide segment; The animal erythropoietin protein includes canine erythropoietin or feline erythropoietin, the amino acid sequence of the canine erythropoietin is shown in SEQ ID NO: 1, and the amino acid sequence of the feline erythropoietin is shown in SEQ ID NO: 83; The additional peptide segments include glycosylation-rich peptide segments and / or basic amino acid peptide segments, The glycosylation-rich peptide segment contains 24 to 35 amino acids and contains one or more O-glycosylation sites or N-glycosylation modification sites, and the total number of threonine and serine in the glycosylation-rich peptide segment accounts for more than 20%; The basic amino acid peptide segment contains 1 to 10 basic amino acids, and the basic amino acids include at least one of arginine, lysine and histidine.

2. The recombinant erythropoietin protein for veterinary use according to claim 1, wherein When the animal erythropoietin protein includes canine erythropoietin, the glycosylation-rich peptide segment includes at least one of a glycosylation-rich peptide segment in canine mucin 5B, a carboxyl-terminal peptide of human chorionic gonadotropin, and a glycosylation-rich peptide segment in canine IgG D heavy chain Fc; the amino acid sequence of the glycosylation-rich peptide segment in canine mucin 5B is shown in SEQ ID NO: 2, the amino acid sequence of the carboxyl-terminal peptide of human chorionic gonadotropin is shown in SEQ ID NO: 3, and the amino acid sequence of the glycosylation-rich peptide segment in canine IgG D heavy chain Fc is shown in SEQ ID NO: 4; When the animal erythropoietin protein includes feline erythropoietin, the glycosylation-rich peptide segment includes at least one of the glycosylation-rich peptide segment in feline fibronectin III, the carboxyl-terminal peptide of human chorionic gonadotropin, and the glycosylation-rich peptide segment in the Fc of a feline antibody; the amino acid sequence of the glycosylation-rich peptide segment in the feline fibronectin III is shown in SEQ ID NO: 84, and the amino acid sequence of the glycosylation-rich peptide segment in the Fc of the feline antibody is shown in SEQ ID NO:

85.

3. The recombinant erythropoietin protein for veterinary use according to claim 1, wherein The amino acid sequences of the basic amino acid peptide segments are shown in SEQ ID NO: 5 to SEQ ID NO:

16.

4. The amino acid sequence of the recombinant erythropoietin protein for veterinary use is shown in SEQ ID NO: 17 to SEQ ID NO: 66 or SEQ ID NO: 86 to SEQ ID NO:

135.

5. Use of the veterinary recombinant erythropoietin protein according to any one of claims 1 to 4 in the preparation of a medicament for preventing and treating anemia in animals.

6. A drug for preventing and treating anemia in animals, characterized in that: The medicine contains the veterinary recombinant erythropoietin protein according to any one of claims 1 to 4.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the recombinant erythropoietin protein for veterinary use according to any one of claims 1 to 4.

8. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 7.

9. A recombinant cell, characterized in that The recombinant cell contains the recombinant expression vector according to claim 8.

10. A method for preparing the recombinant erythropoietin protein for veterinary use according to any one of claims 1 to 4, characterized in that: Cultivate the recombinant cell according to claim 9 to express the veterinary recombinant erythropoietin protein.

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