CN114989307A - Recombinant human blood coagulation factor VIII-Fc fusion protein and preparation method thereof - Google Patents

Abstract

The invention discloses a recombinant human blood coagulation factor VIII-Fc fusion protein and a preparation method thereof. The fusion protein is obtained by fusing coagulation factor VIII and Fc together; the Fc polypeptide comprises two peptide chains, wherein one peptide chain is a fusion polypeptide formed by fusing coagulation factor VIII and Fc of an antibody, and the other peptide chain is an Fc polypeptide comprising Fc, and the Fc of the two peptide chains contains different amino acid sequences and forms heterodimers through disulfide bonds. Experiments prove that the activity of the fusion protein prepared by the invention is 11.43-89.47 IU/mL, which is far greater than the reported yield in the prior art, the fusion protein is suitable for large-scale industrial production, can effectively solve the problem of insufficient blood source FVIII, and fills up the short plate lacking of hemophilia A treatment raw materials.

Description

Recombinant human blood coagulation factor VIII-Fc fusion protein and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant human blood coagulation factor VIII-Fc fusion protein and a preparation method thereof.

Background

Blood coagulation Factor viii (Factor viii, fviii) is a glycoprotein synthesized by hepatic sinus endothelial cells and vascular endothelial cells in the liver, and plays a vital role in hemostasis. Hemophilia caused by a deficiency of F viii is called Hemophilia A (HA), the most clinically prevalent type of hemophilia, accounting for more than about 80% of the total number of hemophiliacs. The severity of the disease of hemophilia A patients is closely related to the deficiency degree of F VIII, wherein the content of the F VIII in severe hemophilia A patients is less than 1% of that of normal people, and the severe hemophilia A patients clinically shows that spontaneous bleeding of joints, muscles, internal organs, deep tissues and the like or bleeding after external injury is not easy to coagulate, the bleeding amount is higher, and even the lives of the patients are threatened in severe cases.

The main treatment for hemophilia a is fviii replacement therapy, i.e. the patient with hemophilia a is infused with fviii periodically, maintaining the fviii content of the patient at not less than 1% of the normal content. However, endogenous FVIII has a short half-life of about 12 hours, and is shorter in children, requiring patients to be treated by intravenous injection 3 times a day or week. In addition, the traditional preparation method of FVIII is to extract and purify from human blood, the blood source is limited, the cost is high, and the traditional preparation method is easy to be polluted by virus, so that the safety of the traditional product has potential risks. Therefore, the production of recombinant FVIII by genetic engineering is an urgent problem to be solved.

Disclosure of Invention

The technical problem to be solved by the invention is how to prepare the recombinant human coagulation factor FVIII-Fc fusion protein and/or how to improve the artificial synthesis efficiency of the human coagulation factor FVIII protein and/or how to improve the stability of the artificial human coagulation factor FVIII protein.

In order to solve the above technical problems, the present invention provides a fusion protein, in the first place. The fusion protein may be a fusion protein obtained by fusing coagulation factor VIII and Fc together. The fusion protein can comprise two peptide chains, wherein one peptide chain is a fusion polypeptide formed by fusing coagulation factor VIII and Fc of an antibody, and the other peptide chain is an Fc polypeptide containing Fc. The Fc of the fusion polypeptide and the Fc of the Fc polypeptide contain different amino acid sequences. The Fc of the fusion polypeptide and the Fc of the Fc polypeptide may form a heterodimer via a disulfide bond.

In the fusion protein described above, the Fc of the fusion polypeptide may comprise the hinge region of IgG1, CH2, and CH 3. The Fc of the Fc polypeptide may also comprise the hinge region of IgG1, CH2, and CH 3. The Fc of the fusion polypeptide introduced S354C, D356E, L358M, T366W and N434A mutations; the Fc of the Fc polypeptide introduced Y349C, D356E, L358M, T366S, L368A, Y407V, and N434A mutations. Both the Fc of the fusion polypeptide and the Fc of the Fc polypeptide can be derived from human IgG 1. The positions of the above mutations are all Eu numbering scheme positions of the antibody.

In the above fusion protein, the Fc of the fusion polypeptide may be any one of the following polypeptides:

A1) the amino acid sequence is the polypeptide of amino acid residue 1463-1689 of SEQ ID No.2 in the sequence table;

A2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown in A1) to obtain a polypeptide with more than 90% of identity with A1);

A3) a fusion polypeptide obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in A1) or A2).

The Fc of the Fc polypeptide can be any one of the following:

B1) the amino acid sequence is the polypeptide of 21 st-247 th amino acid residue of SEQ ID No.4 in the sequence table;

B2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown in B1) to obtain a polypeptide with more than 90% of identity with B1);

B3) a fusion polypeptide obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in B1) or B2).

In the above fusion protein, the human coagulation factor VIII may be any one of the following proteins:

C1) protein with amino acid sequence of amino acid residues 20-1457 of SEQ ID No.2 of the sequence table;

C2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown by C1) to obtain a protein with more than 90% of identity with C1);

C3) a fusion polyprotein obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in C1) or C2).

In the above fusion protein, the Fc of the fusion polypeptide and the factor VIII may be linked directly or through a flexible polypeptide.

In the above fusion protein, the fusion polypeptide may be any one of the following polypeptides:

D1) the amino acid sequence is the polypeptide of SEQ ID No.2 in the sequence table;

D2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown by D1) to obtain a polypeptide with more than 90% of identity with D1);

D3) a fusion polypeptide obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in D1) or D2).

The Fc polypeptide may be any one of the following:

E1) the amino acid sequence is the polypeptide of SEQ ID No.4 in the sequence table;

E2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown in E1) to obtain a polypeptide with more than 90% of identity with E1);

E3) a fusion polypeptide obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in E1) or E2).

In the above-mentioned nucleic acid molecules or encoding genes, identity means identity of nucleotide sequences. The identity of the nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, the identity of a pair of nucleotide sequences can be searched, calculation can be performed, and then the value (%) of identity can be obtained.

The 90% or greater identity in the nucleic acid molecule or encoding gene may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above-mentioned nucleic acid molecule or encoding gene, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; it can also be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In order to solve the above technical problems, the present invention also provides a biomaterial related to the above fusion protein. The biological material may be at least one of:

m1) a nucleic acid molecule encoding a fusion protein as described above;

m2) an expression cassette comprising the nucleic acid molecule described in M1);

m3) a recombinant vector containing the nucleic acid molecule of M1) or a recombinant vector containing the expression cassette of M2);

m4) a recombinant microorganism containing M1) the nucleic acid molecule, or a recombinant microorganism containing M2) the expression cassette, or a recombinant microorganism containing M3) the recombinant vector;

m5) a transgenic cell line containing M1) the nucleic acid molecule, or a transgenic cell line containing M2) the expression cassette, or a transgenic cell line containing M3) the recombinant vector.

In the biological material described above, the coding sequence of the nucleic acid molecule may be SEQ ID No.1 and SEQ ID No. 3.

In order to solve the above technical problems, the present invention also provides a method for preparing a fusion protein. The method comprises the following steps: expressing a gene encoding any of the above fusion proteins in a cell line to obtain the fusion protein; the cell line is a eukaryotic cell line.

The eukaryotic cell line can be CHOZN cell, HEK293 cell, CHO cell, yeast cell, insect cell, etc.

Products containing any of the above-described fusion proteins and/or the above-described biological materials are also within the scope of the present invention. The product may be any of the following:

f1, products for the treatment of hemophilia a;

f2, recombinant human factor VIII related product;

f3, product for treating hemorrhagic diseases.

Any of the following uses of any of the fusion proteins described above and/or the biomaterials described above also fall within the scope of the present invention:

p1, for use in the preparation of a product for the prevention and/or treatment of haemophilia a;

p2, in the preparation of products related to the recombinant human blood coagulation factor VIII;

p3, and application thereof in preparing products for preventing and/or treating hemorrhagic diseases.

In the above application, the product may be a medicament.

The invention provides a design idea of FVIII-Fc and explains a screening method of a cell strain stably expressing FVIII-Fc. The invention adopts artificially synthesized human blood coagulation FVIII with a deleted B structural domain to be connected with an Fc structural domain to form recombinant FVIII-Fc fusion protein, and the recombinant FVIII-Fc fusion protein is secreted and expressed in eukaryotic cells, wherein the recombinant FVIII-Fc fusion protein stabilizes Fc two chains by introducing a pestle and mortar structure, so that the yield of target protein is greatly improved, the activity of the culture supernatant is 11.43-89.47 IU/mL, which is far greater than the reported yield in the prior art, the invention is suitable for large-scale industrial production, can effectively solve the problem of insufficient blood source FVIII, and fills up short plates lacking in hemophilia A treatment raw materials.

Drawings

FIG. 1 is an enzyme digestion identification diagram for construction of an FVIII-Fc expression plasmid. Wherein, 1 is an FVIII-Fc expression plasmid, 2 is an FVIII-Fc expression plasmid Hind III and XhoI double enzyme digestion, and 3 is an FVIII-Fc expression plasmid BstBI and PacI double enzyme digestion.

FIG. 2 is the curves of viable cell density of the selected monoclonal cell strain in shake flask Batch (top), FB1 (bottom left), FB2 (bottom right). The abscissa is the incubation time (days) and the ordinate is the viable cell density (number of cells/mL).

FIG. 3 is a graph of the cell viability of the screening monoclonal cell lines in shake flasks Batch (top), FB1 (bottom left), FB2 (bottom right). The abscissa represents the culture time (days), and the ordinate represents the cell viability (%).

FIG. 4 shows the results of cell activity assays of the selected monoclonal cell strains, i.e., the flask Batch (top), the FB1 (bottom left) and the FB2 (bottom right). The abscissa is the culture time (days) and the ordinate is the activity of the recombinant FVIII-Fc fusion protein (IU/mL).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The main reagents and their manufacturer information in the following examples are as follows:

CHOZN GS ^-/- cell: merck corporation;

pCGS3 expression vector: merck corporation;

screening Medium Ex-Cell CD CHO Fusion Medium: SAFC corporation;

growth Medium Ex-Cell CD CHO Fusion Medium (containing 6mM Glutamine): SAFC Corp Ltd

Ex-Cell CHO Cloning Medium: SAFC corporation;

Ex-Cell Advanced CHO Fed-batch Medium: SAFC corporation;

EX-Cell Advanced Feed 1: SAFC corporation;

COATEST SP VIII octafactor detection kit: chromogenix corporation;

DNA Ligation Kit Ver.2.1: baori doctor Tech technology (Beijing) Ltd;

SanPrep column type DNA gel recovery kit: biometrics (Shanghai) Inc.;

cell counting instrument: roche Inc.;

superclean bench: suzhou Antai air technologies, Inc.;

electric heating constant temperature water bath: fisher Scientific Inc.;

CO ₂ constant temperature shaking table: CRYSTAL corporation;

HYG-A full constant temperature shake flask cabinet: taicang City laboratory plant;

model DYY-6C electrophoresis apparatus: six instrument factories in Beijing;

DYCP-31DN type horizontal electrophoresis tank: six instrument factories in Beijing;

a micropipette: eppendorf Ltd.

Example 1 construction of recombinant human factor VIII-Fc fusion protein expression plasmid

1.1 Synthesis of the Gene of interest

A nucleotide sequence (SEQ ID No.1) encoding the FVIII-Fc fusion polypeptide and a nucleotide sequence (SEQ ID No.3) encoding the Fc polypeptide were artificially synthesized by Kinsley Biotechnology Ltd (hereinafter abbreviated as Kinsley), and the delivery plasmids were a cloning plasmid pUC57-simple-H containing the nucleic acid sequence of FVIII-Fc and a cloning plasmid pUC57-simple-L containing the nucleic acid sequence of Fc polypeptide. In two nucleotide sequences, in a coding sequence of the FVIII-Fc fusion polypeptide, namely, the 70 th-4383 th nucleotide of SEQ ID No.1 in the sequence table is a human FVIII coding sequence with a B domain deletion, wherein the 1 st-6 th nucleotide is a BstBI enzyme cutting site, the 7 th-12 th nucleotide is a Kozak site, the 13 th-69 th nucleotide is a signal peptide coding gene, the 70 th-2289 th nucleotide is a Heavy Chain (HC) coding gene, the 2290 th-1231 th nucleotide is a partial B domain coding gene, the 2332 th-4283 th nucleotide is a Light Chain (LC) coding gene, the 4384 th-4398 th nucleotide is a Linker coding gene, the 4399 th-5079 th nucleotide is an Fc region coding gene, the 5080-5082 is a stop codon, and the 5083-5090 is a PacI enzyme cutting site.

The FVIII polypeptide in the FVIII-Fc fusion polypeptide is human FVIII with a deleted B structure domain; wherein the Fc comprises a hinge region-CH 2-CH3 structure of IgG1, five amino acid mutation sites are present (S354C, D356E, L358M, T366W and N434A), wherein the mutation sites D356E and L358M are designed to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) effects, wherein the mutation site T366W forms a "knob" like protuberance in the higher order structure of the Fc region, wherein the mutation site S354C forms a cysteine residue in the "knob" like protuberance in the higher order structure of the Fc region, and wherein the mutation site N434A is designed to increase the half-life of a subsequently prepared recombinant human coagulation factor VIII-Fc fusion protein.

The amino acid sequence of the FVIII-Fc fusion polypeptide is shown as SEQ ID No.2 in the sequence table. The 20-1457 amino acid residues of SEQ ID No.2 are a human FVIII sequence with a deleted B domain, wherein the 1-19 amino acid is a signal peptide sequence, the 20-759 amino acid is a Heavy Chain (HC), the 760-773 amino acid is a partial B domain sequence, the 774-1457 amino acid is a Light Chain (LC), the 1458-1462 amino acid is a Linker (connecting FVIII and Fc), and the 1463-1689 amino acid is an Fc sequence.

In the other Fc polypeptide coding sequence, namely, the 1 st-6 th nucleotide of SEQ ID No.3 in the sequence table is HindIII enzyme cutting site, the 7 th-12 th nucleotide is Kozak site, the 13 th-72 th nucleotide is signal peptide coding gene, the 73 th-753 th nucleotide is Fc region coding gene, the 754 and 756 th nucleotide is stop codon, and the 757 and 762 th nucleotide is XhoI enzyme cutting site.

The amino acid sequence of the Fc polypeptide is shown as SEQ ID No.4 in a sequence table. The 1 st to 20 th amino acids of SEQ ID No.4 are signal peptides, and the 21 st to 247 th amino acids are Fc. The Fc also comprises a hinge region-CH 2-CH3 structure of IgG1, and the Fc of the Fc and the FVIII-Fc fusion polypeptide contains different amino acid sequences and amino acid mutation sites, and the Fc contains seven amino acid mutation sites (Y349C, D356E, L358M, T366S, L368A, Y407V and N434A), wherein the design of the mutation sites D356E and L358M is also used for reducing antibody-dependent cell-mediated cytotoxicity (ADCC) effect, the mutation sites T366S, L368A and Y407V can enable the high-order structure of the Fc to form a 'hole' -like recess, the mutation site Y349C can enable the high-order structure of the Fc region to generate cysteine residues in the 'hole' -like recess, and the mutation site N434A is also used for increasing the half-life of the subsequently prepared recombinant human coagulation factor VIII-Fc fusion protein.

According to design, the two peptide chains of the FVIII-Fc fusion polypeptide and the Fc polypeptide can form the target recombinant human coagulation factor VIII-Fc fusion protein (recombinant FVIII-Fc fusion protein) through Fc dimerization on the two peptide chains respectively. The high-level structure 'knob' -like protrusion of the Fc polypeptide on the F VIII-Fc fusion polypeptide chain can form 'knob-hole' -like depression with the Fc high-level structure 'hole' -like depression on the Fc polypeptide chain to stabilize the combination of two peptide chains in a 'knob-hole' structure, and the cysteine residue at the 'knob' -like protrusion can form a disulfide bond with the cysteine residue at the 'hole' -like depression to stabilize a heterodimer structure, and finally form the recombinant human blood coagulation factor VIII-Fc fusion protein (recombinant F VIII-Fc fusion protein) with stable structure.

1.2 construction of recombinant FVIII-Fc fusion protein expression plasmid

1.2.1 construction of the nucleic acid sequence of the Fc Domain onto the pCGS3 vector to complete the first round of construction

The method comprises the following specific steps: the recombinant vector pUC57-simple-L is subjected to double enzyme digestion by using restriction enzymes HindIII and XhoI to obtain an Fc structural domain nucleic acid sequence fragment, meanwhile, the pCGS3 expression vector is also subjected to double enzyme digestion by using restriction enzymes HindIII and XhoI to obtain a linearized pCGS3 vector, a SanPrep column type DNA glue recovery Kit is used for recovering an Fc structural domain target fragment (SEQ ID No.3) and a linearized pCGS3 vector, then a Ligation Kit (DNA Ligation Kit Ver.2.1) is used for Ligation, the Fc structural domain is completed with the pCGS3 vector, and a recombinant expression vector plasmid pCGS3-Fc plasmid is obtained, wherein the pCGS3-Fc plasmid is a recombinant vector obtained by replacing the nucleic acid sequence of the Fc structural domain between HindIII and XhoI enzyme digestion recognition sites on the pCGS3 vector with the nucleic acid sequence of SEQ ID small piece No.3 in the sequence table, and keeping other sequences on the pCGS3 plasmid unchanged.

1.2.2 construction of the nucleic acid sequence of FVIII onto the pCGS3-Fc plasmid to complete the second round of construction

The method comprises the following specific steps: the recombinant vector pUC57-simple-H was subjected to double digestion with restriction enzymes BstBI and PacI to obtain the target fragment of FVIII-Fc (SEQ ID No.1), meanwhile, the pCGS3-Fc plasmid obtained in the step 1.2.1 is subjected to double enzyme digestion by using restriction enzymes BstBI and PacI to obtain a linearized pCGS3-Fc plasmid, a nucleic acid sequence fragment of the FVIII-Fc and the linearized pCGS3-Fc plasmid are recovered by using a SanPrep column type DNA gel recovery kit, then connecting by using a connecting Kit (DNA Ligation Kit Ver.2.1) to complete the construction of the FVIII-Fc expression vector to obtain a recombinant expression vector plasmid pCGS 3-FVIII + Fc, wherein the pCGS 3-FVIII + Fc plasmid is obtained by replacing a small segment between BstBI and PacI enzyme cutting recognition sites on the pCGS3-Fc plasmid into a nucleic acid sequence of the FVIII-Fc shown by SEQ ID No.1 in a sequence table, and keeping other sequences on the pCGS3-Fc plasmid unchanged.

The enzyme digestion identification result of the obtained recombinant expression vector pCGS 3-FVIII + Fc is shown in FIG. 1. In FIG. 1, lane 1 is the electrophoresis band of pCGS 3-FVIII + Fc plasmid; lane 2 is pCGS 3-FVIII + Fc plasmid Hind III & XhoI double-restriction electrophoresis band (after restriction enzyme, carrier fragment 14578bp, target gene fragment 762bp is SEQ ID No.3 in the sequence table); lane 3 is the electrophoresis band after double enzyme digestion of the pCGS 3-FVIII + Fc expression plasmid BstBI & PacI (after enzyme digestion, the vector is 10250bp, the target gene fragment is 5090bp, namely SEQ ID No.1 in the sequence table), and the size of the enzyme digestion band accords with the expectation.

The 7 th to 12 th nucleotides of SEQ ID No.1 and the 7 th to 12 th nucleotides of SEQ ID No.3 in the sequence table are Kozak sequences for optimized expression; the 5080-5082-th nucleotides of SEQ ID No.1 and the 754-756-th nucleotides of SEQ ID No.3 are stop codon sequences.

Example 2 screening of high expression cell lines

1. Host cell

Reviving a CHOZN host cell CHOZN GS ^-/- Cells, after the cells grow to 2 + -1 × 10 ⁶ At cell/mL, according to 0.3X 10 ⁶ cells/mL were inoculated at density and passaged into Ex-Cell CD CHO Fusion (containing 6mM Glutamine) medium. The transfection effect is optimal 6-9 days after the recovery of the CHOZN host cells, and the cells are transfected by 0.5 multiplied by 10 hours before 24 hours ⁶ cells/mL were inoculated into Ex-Cell CD CHO Fusion (containing 6mM Glutamine) medium and used for transfection after 24 hours of culture.

2. Electroporation

CHOZN host cells were transfected by electroporation using a Bio-Rad electrotransfer. Electrotransfer was carried out using a 4mm electric cuvette, and 850uL of electrotransfer solution contained 25. mu.g, 5X 10 of the recombinant vector plasmid pCGS 3-FVIII + Fc plasmid prepared in example 1 ⁶ A CHOZN host cell. Shock parameters 300V, 950 μ F, exponential decay wave. After the electric shock, the cells in the cuvette were transferred in their entirety to a T-25 Cell culture flask containing 5mL of Ex-Cell CD CHO Fusion (containing 6mM Glutamine), and cultured overnight in an incubator (at 37 ℃ C., 5% CO) ₂ 80% humidity) to obtain recombinant CHOZN cells.

Preparation and screening of miniwood

The next day of electrotransformation recombinant CHOZN cells were seeded at 5000 cells/200. mu.L/well to 96WP (20% Ex-Cell CD CHO Fusion + 80% Ex-Cell CHO Cloning Medium) to prepare Minipool (recombinant CHOZN Cell pool). The resulting Minipool 96-well plates (about 80% confluency) were incubated for about 21 days at CHROMOGENIX

SP FVIII assay kit instructions, enzyme reaction kinetics and endpoint method were used to determine recombinant FVIII activity in 96-well plate Minipool supernatants. Minitools with high activity (about Top 30% -40%) were screened and expanded stepwise to 24-well plates, 6-well plates, T25, TPP and shake flasks, and minitools were frozen after cells recovered.

4. Monoclonal screening

Inoculating the cell pool with high expression in the step 3 into a 96-well plate according to the density of 0.5cell/well to screen a monoclonal, and gradually screening and amplifying clones with good growth state and high activity to a 24-well plate, a 6-well plate, T25, TPP and a shake flask for amplification culture according to activity data. High expressing cell pools were seeded in 96-well plates at 0.5cells/well by limiting dilution method, high activity monoclonals were screened and expanded stepwise to 24-well plates, 6-well plates, T25, TPP and shake flasks based on culture supernatant activity assay data, and 24 monoclonals were selected for 8-day TPP Batch evaluation.

Based on the TPP-Batch results, the single clones 1-1, 2-1, 8-1 and 17-1 of Top4 expressing recombinant FVIII-Fc fusion protein were expressed at 0.5X 10 ⁶ cells/mL were inoculated into 250mL shake flasks at 60mL volumes and fed-batch culture was performed according to the protocol in Table 1 (shaker parameters: 120rpm, 5% CO) ₂ At 37 ℃ C.). Timely supplementing glucose and maintaining the content of the glucose at 4g/L, and supplementing a supplemented culture medium with 5% of the original culture volume on odd days.

Taking the culture solution of the recombinant CHOZN cells during the culture process to monitor the viable cell density and the cell viability (figure 2 and figure 3); samples were taken daily from day 3 for activity testing (FIG. 4), and the incubation temperature was lowered to 32 ℃ when the activity reached plateau, until the end of incubation when activity began to decline.

TABLE 1 Top4 monoclonal feeding regimen

Note: batch: culturing in batches; FB 1: fed batch culture protocol 1; FB2 fed batch culture protocol 2

Example 3 Activity assay of FVIII-Fc fusion protein in cell culture supernatant

The activity of FVIII in the recombinant FVIII-Fc fusion protein expressed by the recombinant CHOZN cells transfected with the recombinant expression vector pCGS 3-FVIII + Fc in example 2 was determined using the Chromogenix Coatest SP FVIII kit (Chromogenix, K82408663).

The detection principle is as follows: when activated by thrombin, FVIIIa binds to FIXa in the presence of phospholipids and calcium ions to form an enzyme complex which in turn activates the conversion of factor X to its active form Xa. The activated Xa in turn cleaves the specific chromogenic substrate (S-2765) releasing the chromophoric group pNA. At OD _405nm The amount of pNA produced is determined by knowing the magnitude of the activity of FXa which is directly proportional to its amount, where the levels of FIXa and FX in the system are constant and excessive and the activity of FXa is only somewhat directly related to the level of FVIIa. The activity of the recombinant FVIII-Fc fusion protein in the supernatant of the Top4 monoclonal (1-1, 2-1, 8-1, 17-1) culture expressing the recombinant FVIII-Fc fusion protein obtained in example 2 was determined to be at most 11.43-89.47 IU/mL by a chromophoric method.

Combining the results of the above examples, the present invention successfully prepared the human recombinant FVIII-Fc fusion protein secreted and expressed in eukaryotic cells. The fusion protein can be efficiently expressed in eukaryotic cells, has the activity of 11.43-89.47 IU/mL, is suitable for large-scale industrial production, and has high industrial utilization value.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> Henan Cheng Ming Biotechnology research institute Co., Ltd

<120> recombinant human blood coagulation factor VIII-Fc fusion protein and preparation method thereof

<130> GNCSQ213554

<160> 4

<170> PatentIn version 3.5

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<211> 5090

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acacgggagg ccatccagca cgagtctggc atcctgggac cactgctgta cggagaagtg 1440

ggcgacaccc tgctgatcat ctttaagaac caggctagca ggccctacaa tatctatcct 1500

catggcatca cagatgtgcg gcccctgtac tccaggaggc tgcctaaggg cgtgaagcac 1560

ctgaaggact tcccaatcct gcccggcgag atctttaagt ataagtggac cgtgacagtg 1620

gaggatggcc caaccaagag cgaccccagg tgcctgacac ggtactattc ttccttcgtg 1680

aatatggaga gagatctggc ctctggcctg atcggccctc tgctgatctg ttacaaggag 1740

tctgtggatc agagaggcaa ccagatcatg tccgacaagc gcaatgtgat cctgttcagc 1800

gtgtttgacg agaacaggtc ttggtatctg accgagaaca tccagcggtt cctgcccaat 1860

cctgctggcg tgcagctgga ggatccagag tttcaggcct ccaacatcat gcatagcatc 1920

aatggctacg tgttcgacag cctgcagctg tccgtgtgcc tgcacgaggt ggcttactgg 1980

tatatcctgt ctatcggcgc ccagaccgat ttcctgtccg tgttctttag cggctacacc 2040

ttcaagcata agatggtgta tgaggacacc ctgacactgt tccccttttc cggcgagacc 2100

gtgtttatga gcatggagaa tcctggcctg tggatcctgg gctgccacaa ctccgatttc 2160

aggaatcggg gcatgaccgc tctgctgaag gtgagctctt gtgacaagaa cacaggcgac 2220

tactatgagg attcttacga ggacatctcc gcttatctgc tgagcaagaa caatgccatc 2280

gagccaagga gcttttctca gaatcctcca gtgctgaaga gacaccagcg cgagatcacc 2340

cggaccacac tgcagagcga tcaggaggag atcgactacg acgatacaat ctctgtggag 2400

atgaagaagg aggacttcga tatctatgac gaggatgaga accagagccc caggtctttc 2460

cagaagaaga cccggcatta ctttatcgcc gctgtggagc gcctgtggga ttatggcatg 2520

tccagctctc cacacgtgct gagaaatcgc gcccagtccg gaagcgtgcc acagttcaag 2580

aaggtggtgt tccaggagtt taccgacggc tcctttacac agcctctgta cagaggcgag 2640

ctgaacgagc atctgggcct gctgggccca tatatccgcg ctgaggtgga ggataacatc 2700

atggtgacct tcaggaatca ggcctctcgg ccctactcct tttattccag cctgatcagc 2760

tacgaggagg accagagaca gggcgctgag ccacgcaaga acttcgtgaa gcccaatgag 2820

accaagacat acttttggaa ggtgcagcac catatggccc ctaccaagga cgagttcgat 2880

tgcaaggcct gggcttactt ctccgacgtg gatctggaga aggacgtgca ctctggactg 2940

atcggaccac tgctggtgtg ccataccaac acactgaatc ctgctcacgg caggcaggtc 3000

accgtgcagg agttcgccct gttctttacc atctttgatg agacaaagtc ttggtacttc 3060

acagagaaca tggagaggaa ttgccgggct ccttgtaata tccagatgga ggacccaacc 3120

ttcaaggaga actaccggtt tcatgctatc aatggctata tcatggatac actgccaggc 3180

ctggtcatgg cccaggacca gaggatccgg tggtacctgc tgtccatggg cagcaacgag 3240

aatatccaca gcatccattt ctctggccac gtgtttaccg tgagaaagaa ggaggagtat 3300

aagatggccc tgtacaacct gtatcctggc gtgttcgaga cagtggagat gctgccatcc 3360

aaggctggca tctggagggt ggagtgcctg atcggagagc acctgcatgc cggcatgtct 3420

accctgtttc tggtgtactc caataagtgt cagacaccac tgggcatggc ttctggccat 3480

atcagagatt tccagatcac cgcttccgga cagtacggac agtgggctcc aaagctggct 3540

cgcctgcact attctggctc catcaacgcc tggtccacca aggagccctt ctcctggatc 3600

aaggtggacc tgctggctcc catgatcatc catggcatca agacacaggg cgccaggcag 3660

aagttctctt ccctgtacat cagccagttt atcatcatgt attctctgga tggcaagaag 3720

tggcagacct accggggcaa ttccaccggc acactgatgg tgttctttgg caacgtggac 3780

agctctggca tcaagcacaa catcttcaat ccccctatca tcgctagata catccgcctg 3840

caccctaccc attattctat cagatccaca ctgcgcatgg agctgatggg ctgcgatctg 3900

aacagctgtt ctatgccact gggcatggag tccaaggcca tcagcgacgc tcagatcacc 3960

gcctccagct acttcaccaa tatgtttgct acatggtccc ctagcaaggc caggctgcat 4020

ctgcagggca gatccaacgc ctggcgccct caggtgaaca atccaaagga gtggctgcag 4080

gtggattttc agaagaccat gaaggtgaca ggcgtgacca cacagggcgt gaagtctctg 4140

ctgacctcca tgtatgtgaa ggagttcctg atctcttcca gccaggacgg ccaccagtgg 4200

acactgttct ttcagaacgg caaggtgaag gtgttccagg gcaatcagga ttcctttacc 4260

ccagtggtga acagcctgga cccaccactg ctgacaaggt acctgcggat ccaccctcag 4320

agctgggtgc atcagatcgc tctgagaatg gaggtgctgg gctgcgaggc tcaggatctg 4380

tatggaggag gaggatccga caagacccat acatgccctc catgtccagc tccagagctg 4440

ctgggaggac caagcgtgtt cctgtttcca cctaagccta aggataccct gatgatcagc 4500

cgcaccccag aggtgacatg cgtggtggtg gacgtgtccc atgaggaccc cgaggtgaag 4560

ttcaattggt acgtggacgg cgtggaggtg cacaacgcca agacaaagcc cagggaggag 4620

cagtacaact ctacctatcg ggtggtgtcc gtgctgacag tgctgcacca ggactggctg 4680

aatggcaagg agtataagtg caaggtgtcc aacaaggccc tgcctgctcc aatcgagaag 4740

accatcagca aggctaaggg ccagcctaga gagccacagg tgtacaccct gccaccctgc 4800

cgcgaggaga tgacaaagaa tcaggtgagc ctgtggtgtc tggtgaaggg cttttatcct 4860

agcgatatcg ccgtggagtg ggagtctaac ggccagccag agaacaatta caagaccaca 4920

cctccagtgc tggacagcga tggctctttc tttctgtatt ctaagctgac cgtggacaag 4980

tccaggtggc agcagggcaa cgtgttctct tgttccgtga tgcacgaggc cctgcacgct 5040

cattacacac agaagagcct gtctctgtcc cctggcaagt aattaattaa 5090

<210> 2

<211> 1689

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu

755 760 765

Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln

770 775 780

Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu

785 790 795 800

Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe

805 810 815

Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp

820 825 830

Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln

835 840 845

Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr

850 855 860

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His

865 870 875 880

Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile

885 890 895

Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser

900 905 910

Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg

915 920 925

Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val

930 935 940

Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp

945 950 955 960

Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu

965 970 975

Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His

980 985 990

Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe

995 1000 1005

Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn

1010 1015 1020

Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys

1025 1030 1035

Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr

1040 1045 1050

Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr

1055 1060 1065

Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe

1070 1075 1080

Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met

1085 1090 1095

Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met

1100 1105 1110

Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly

1115 1120 1125

Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser

1130 1135 1140

Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg

1145 1150 1155

Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro

1160 1165 1170

Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser

1175 1180 1185

Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro

1190 1195 1200

Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe

1205 1210 1215

Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp

1220 1225 1230

Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu

1235 1240 1245

Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn

1250 1255 1260

Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro

1265 1270 1275

Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly

1280 1285 1290

Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys

1295 1300 1305

Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn

1310 1315 1320

Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln

1325 1330 1335

Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu

1340 1345 1350

Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val

1355 1360 1365

Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys

1370 1375 1380

Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu

1385 1390 1395

Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp

1400 1405 1410

Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr

1415 1420 1425

Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala

1430 1435 1440

Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Gly

1445 1450 1455

Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1460 1465 1470

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1475 1480 1485

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

1490 1495 1500

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

1505 1510 1515

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

1520 1525 1530

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

1535 1540 1545

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

1550 1555 1560

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

1565 1570 1575

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

1580 1585 1590

Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp

1595 1600 1605

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

1610 1615 1620

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

1625 1630 1635

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

1640 1645 1650

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

1655 1660 1665

Val Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu

1670 1675 1680

Ser Leu Ser Pro Gly Lys

1685

<210> 3

<211> 762

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aagcttgcca ccatggagac cgacacactg ctgctgtggg tgctgctgct gtgggtgcca 60

ggcagcaccg gcgataagac ccacacatgc ccaccttgtc cagctccaga gctgctggga 120

ggaccatccg tgttcctgtt tccacccaag cccaaggaca cactgatgat ctccaggacc 180

cccgaggtga catgcgtggt ggtggacgtg agccacgagg atcctgaggt gaagttcaac 240

tggtacgtgg atggcgtgga ggtgcataat gccaagacca agcccaggga ggagcagtac 300

aactccacct atcgggtggt gagcgtgctg acagtgctgc atcaggactg gctgaacggc 360

aaggagtata agtgtaaggt gtctaataag gccctgcctg ctccaatcga gaagaccatc 420

tccaaggcta agggccagcc cagagagcct caggtgtgca ccctgcctcc atcccgcgag 480

gagatgacaa agaaccaggt gtccctgagc tgtgccgtga agggcttcta ccctagcgac 540

atcgctgtgg agtgggagtc taatggccag ccagagaaca attataagac cacaccccct 600

gtgctggaca gcgatggctc tttctttctg gtgtctaagc tgaccgtgga taagtccaga 660

tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca cgctcattac 720

acacagaaga gcctgtctct gtcccccggc aagtaactcg ag 762

<210> 4

<211> 247

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

20 25 30

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

35 40 45

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

50 55 60

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

65 70 75 80

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

100 105 110

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

115 120 125

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

130 135 140

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

145 150 155 160

Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp

165 170 175

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

180 185 190

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser

195 200 205

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

210 215 220

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser

225 230 235 240

Leu Ser Leu Ser Pro Gly Lys

245

Claims (10)

1. A fusion protein characterized by: the fusion protein is obtained by fusing coagulation factor VIII and Fc together; the fusion protein comprises two peptide chains, wherein one peptide chain is a fusion polypeptide formed by fusing coagulation factor VIII and Fc of an antibody, the other peptide chain is an Fc polypeptide containing Fc, and the Fc of the fusion polypeptide and the Fc of the Fc polypeptide contain different amino acid sequences; the Fc of the fusion polypeptide and the Fc of the Fc polypeptide form a heterodimer via a disulfide bond.

2. The fusion protein of claim 1, wherein: the Fc of the fusion polypeptide is any one of the following polypeptides:

A1) the amino acid sequence is the polypeptide of amino acid residue 1463-1689 of SEQ ID No.2 in the sequence table;

A2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown in A1) to obtain a polypeptide with more than 90% of identity with A1);

A3) a fusion polypeptide obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in A1) or A2);

the Fc of the Fc polypeptide is any one of the following polypeptides:

B1) the amino acid sequence is the polypeptide of 21 st-247 th amino acid residue of SEQ ID No.4 in the sequence table;

B2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown in B1) to obtain a polypeptide with more than 90% of identity with B1);

B3) a fusion polypeptide obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in B1) or B2).

3. The fusion protein of claim 1 or 2, wherein: the human coagulation factor VIII is any one of the following proteins:

C1) protein with amino acid sequence of amino acid residues 20-1457 of SEQ ID No.2 of the sequence table;

C2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown by C1) to obtain a protein with more than 90% of identity with C1);

C3) a fusion polyprotein obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in C1) or C2).

4. The fusion protein of any one of claims 1-3, wherein: the fusion polypeptide is any one of the following polypeptides:

D1) the amino acid sequence is the polypeptide of SEQ ID No.2 in the sequence table;

D2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown by D1) to obtain a polypeptide with more than 90% of identity with D1);

D3) a fusion polypeptide obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in D1) or D2);

the Fc polypeptide is any one of the following polypeptides:

E1) the amino acid sequence is the polypeptide of SEQ ID No.4 in the sequence table;

E2) carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the polypeptide shown in E1) to obtain a polypeptide with more than 90% of identity with E1);

E3) a fusion polypeptide obtained by carboxyl-terminal or/and amino-terminal fusion protein labels of the polypeptide shown in E1) or E2).

5. The biomaterial related to the fusion protein according to any of the claims 1-4, being at least one of:

m1) a nucleic acid molecule encoding the fusion protein of any one of claims 1-4;

m2) an expression cassette containing the nucleic acid molecule of M1);

m3) a recombinant vector containing the nucleic acid molecule of M1) or a recombinant vector containing the expression cassette of M2);

m4) a recombinant microorganism containing M1) the nucleic acid molecule, or a recombinant microorganism containing M2) the expression cassette, or a recombinant microorganism containing M3) the recombinant vector;

m5) a transgenic cell line containing M1) the nucleic acid molecule, or a transgenic cell line containing M2) the expression cassette, or a transgenic cell line containing M3) the recombinant vector.

6. The biomaterial of claim 5, wherein: the coding sequences of the nucleic acid molecules are SEQ ID No.1 and SEQ ID No. 3.

7. A method of making a fusion protein, comprising: expressing a gene encoding the fusion protein of any one of claims 1-4 in a cell line to obtain the fusion protein; the cell line is a eukaryotic cell line.

8. A product comprising the fusion protein of any one of claims 1 to 4 and/or the biological material of claim 5 or 6, said product being any one of:

f1, products for the treatment of hemophilia a;

f2, recombinant human factor VIII related product;

f3, product for treating hemorrhagic diseases.

9. Use of the fusion protein according to any one of claims 1 to 4 and/or the biomaterial according to claim 5 or 6 for any one of the following:

p1, in the preparation of products for the prevention and/or treatment of haemophilia A;

p2, in the preparation of products related to recombinant human coagulation factor VIII;

p3, and application thereof in preparing products for preventing and/or treating hemorrhagic diseases.

10. The product according to claim 8 and/or the use according to claim 9, characterized in that: the product is a medicament.

Images