A recombinant human follicle-stimulating hormone Fc fusion protein and preparation method thereof

Abstract

The present invention discloses a recombinant human follicle-stimulating hormone Fc fusion protein and a preparation method. The present invention provides the use of a signal peptide shown in SEQID No.5 or its related biomaterials in any of the following: A1) preparing FSH protein or its fusion protein; A2) increasing the expression level or yield of FSH protein or its fusion protein; A3) promoting the secretion of FSH protein or its fusion protein outside the host cell; the present invention uses three signal peptides of FSH-Fc fusion protein to guide the secretory expression of FSH-Fc fusion protein eukaryotic cells, and the secretory expression of the artificially modified signal peptide SP1 is significantly better than that of the antibody light chain signal peptide SP2 and the luciferase signal peptide SP3, which is more suitable for large-scale industrial production and reduces production costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant human follicle-stimulating hormone Fc fusion protein and a preparation method thereof. Background Art

[0002] Follicle-stimulating hormone (FSH) is secreted by the pituitary gland. It is a glycoprotein with a molecular weight of about 42kDa. It is composed of α-chain and β-chain through non-covalent bonding. It was named because of its early discovery of its role in promoting the maturation of female follicles. Later studies found that FSH is one of the most important hormones in both men and women, regulating a series of physiological processes related to development, growth, puberty and reproduction. The function of FSH in women is to promote the development and maturation of follicles, and to cooperate with luteinizing hormone (LH) to promote the secretion of estrogen and ovulation of mature follicles, and participate in the formation of normal menstruation; the function of FSH in men is to promote the maturation of testicular seminiferous tubules and sperm production. Therefore, FSH can be used alone or in combination with LH to treat infertility.

[0003] FSH can be isolated and extracted from the pituitary gland or urine or prepared using genetic recombination technology. Recombinant FSH products have high purity, good safety, controllable quality, and comparable activity to extracted products, and are highly favored by the market. Clinically, FSH needs to be injected subcutaneously for 8-10 consecutive days to stimulate the development of female follicles, which brings a lot of inconvenience and pain to patients. Therefore, the development of long-acting recombinant FSH products is a future trend. Summary of the invention

[0004] One object of the present invention is to provide FSH fusion protein.

[0005] The FSH fusion protein provided by the present invention is as follows B1) or B2):

[0006] B1) the FSH fusion protein, wherein the A chain is the α chain of FSH, and the B chain is the β chain of FSH connected to a tag;

[0007] B2) the FSH fusion protein, wherein the A chain is the α chain of FSH connected to the SP1 signal peptide, and the B chain is the β chain of FSH connected to the SP1 signal peptide and the tag;

[0008] The amino acid sequence of the SP1 signal peptide is shown in SEQ ID No.5.

[0009] The tag can be any one of Fc, HSA, CTP or XTEN or any combination of two thereof, and the preferred tag is an Fc tag.

[0010] Preferably, the FSH fusion protein shown in B1) is a FSH-Fc fusion protein, and the FSH is human FSH. Specifically, the Fc tag is fused to the carbon terminus of the β chain of the human FSH molecule to form the B chain of FSH-Fc, and the B chain forms a homodimer through the disulfide bond of the Fc structure; the α chain of the FSH molecule (i.e., the A chain of FSH-Fc) is bound to the B chain through a non-covalent bond to form a divalent FSH molecule.

[0011] Preferably, the FSH fusion protein shown in B2) is an SP1-FSH-Fc fusion protein, and FSH is human FSH. Specifically, the Fc tag is fused to the carbon terminus of the β chain of the human FSH molecule, and the SP1 signal peptide is connected to the nitrogen terminus of the β chain of the human FSH molecule to form the B chain of SP1-FSH-Fc, and the B chain forms a homodimer through the disulfide bond of the Fc structure; the SP1 signal peptide is connected to the nitrogen terminus of the α chain of the human FSH molecule to form the A chain of SP1-FSH-Fc, and the A chain of SP1-FSH-Fc is bound to the B chain through a non-covalent bond to form a divalent FSH molecule.

[0012] Further preferably, in the above-mentioned FSH-Fc fusion protein, the above-mentioned Fc is from IgG1-Fc, and the above-mentioned FSH is human FSH. Fc is IgG1-Fc, specifically, the CH2 and CH3 regions of human IgG1-Fc are used; and the Lys (K) amino acid at the carbon terminal is removed to reduce the charge heterogeneity of the molecule.

[0013] The above-mentioned FSH fusion protein is formed by non-covalent binding of the α chain of FSH and the β chain of FSH connected to an Fc tag;

[0014] The FSH fusion protein is formed by non-covalently binding the α chain of FSH connected to the SP1 signal peptide and the β chain of FSH connected to the SP1 signal peptide and the Fc tag.

[0015] The Fc tag is at the C-terminus of the α chain of FSH;

[0016] The SP1 signal peptide is connected to the N-terminus of the α chain or β chain of FSH.

[0017] In the above-mentioned FSH fusion protein, in B1), the amino acid sequence of the A chain of the FSH fusion protein is shown as SEQ ID No.1; the amino acid sequence of the B chain of the FSH fusion protein is shown as SEQ ID No.2;

[0018] Or, in B2), the amino acid sequence of the A chain of the FSH fusion protein is shown as SEQ ID No.7; the amino acid sequence of the B chain of the FSH fusion protein is shown as SEQ ID No.8.

[0019] The biological materials related to the above fusion protein are also within the scope of protection of the present invention.

[0020] The biological material is the coding gene of the fusion protein or an expression box, a recombinant vector, a recombinant bacterium or a recombinant cell line containing the coding gene.

[0021] The coding gene of the above fusion protein is any one of the following C1-C4:

[0022] C1: In B1), the nucleotide sequence of the gene encoding the A chain of the FSH fusion protein is shown in SEQ ID No.3, and the nucleotide sequence of the gene encoding the B chain of the FSH fusion protein is shown in SEQ ID No.4;

[0023] C2: In B2), the nucleotide sequence of the A chain encoding gene of the FSH fusion protein is shown as SEQ ID No.9, and the nucleotide sequence of the B chain encoding gene of the FSH fusion protein is shown as SEQ ID No.10;

[0024] C3: a DNA molecule that hybridizes with any of the DNA molecules defined in C1 or C2 under stringent conditions and encodes the above-mentioned fusion protein;

[0025] C4: A DNA molecule that has a DNA sequence identity of more than 75% with the DNA sequence defined by any one of the DNA molecules defined in C1 or C2 and encodes the above-mentioned fusion protein.

[0026] In the above nucleic acid molecules or coding genes, identity refers to the identity of nucleotide sequences. The identity of nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, searching for the identity of a pair of nucleotide sequences, performing calculations, and then obtaining the value (%) of identity.

[0027] In the above nucleic acid molecules or encoding genes, the above 90% identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0028] In a specific embodiment of the present invention, the recombinant expression plasmid pCGS3-SP1-FSH-Fc is a recombinant expression plasmid obtained by inserting the DNA fragment shown in SEQ ID No.9 into the multiple cloning site (such as HindIII and XhoI) of the pCGS3 vector, and inserting the DNA fragment shown in SEQ ID No.10 into the multiple cloning site (such as BstBI and PacI) of the pCGS3 vector.

[0029] The expression cassette refers to a DNA capable of expressing the fusion protein described in the first aspect in a host cell, and the DNA includes not only a promoter for initiating transcription of the target gene, but also a terminator for terminating transcription of the target gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0030] Wherein, the host cell is a eukaryotic host cell.

[0031] Furthermore, the eukaryotic host cell may be HEK293 cell, CHO cell, yeast cell, insect cell, etc. In a specific embodiment of the present invention, the host cell is specifically Expi293F cell.

[0032] The application of the above-mentioned biomaterial in any of the following A1)-A3) is also within the scope of protection of the present invention:

[0033] A1) preparing FSH protein or its fusion protein;

[0034] A2) increasing the expression level or yield of FSH protein or its fusion protein;

[0035] A3) Promote the secretion of FSH protein or its fusion protein outside the host cell.

[0036] Another object of the present invention is to provide a method for preparing FSH protein.

[0037] The method provided by the present invention comprises the following steps: expressing the coding gene of the fusion protein in a host bacterium or a host cell to obtain FSH protein.

[0038] The above method comprises the following steps:

[0039] 1) Introducing the coding genes of the A chain and the B chain in the above fusion protein into the host bacteria to obtain recombinant bacteria;

[0040] 2) Cultivating the recombinant bacteria to obtain FSH protein.

[0041] The coding genes of the A chain and the B chain in the above fusion protein are introduced into the host bacteria through the recombinant plasmid pCGS3-SP1-FSH-Fc;

[0042] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0043] In the above step 2), the culturing is terminated when the cell viability drops to 65% to 75%.

[0044] After step 2), the method further comprises step 3): obtaining FSH-Fc fusion protein from the supernatant of the culture product after culture: collecting the culture and centrifuging at 3500 g for 30 min, collecting the supernatant for ultrafiltration concentration, Protein A 4FF filler affinity chromatography and ultrafiltration replacement.

[0045] The use of SP1 signal peptide or related biological materials in any of the following A1)-A3) is also within the scope of protection of the present invention:

[0046] A1) preparing FSH protein or its fusion protein;

[0047] A2) increasing the expression level or yield of FSH protein or its fusion protein;

[0048] A3) promoting the secretion of FSH protein or its fusion protein outside the host cell;

[0049] The amino acid sequence of the SP1 signal peptide is shown in SEQ ID No.5.

[0050] The above-mentioned FSH protein is composed of the FSH chain and the FSH β chain non-covalently bound;

[0051] In the above application, the A chain of the FSH fusion protein is the α chain of FSH, and the B chain is the β chain of FSH connected to a tag; the tag can be any one of Fc, HSA, CTP or XTEN or any combination of two thereof, and the preferred tag is the Fc tag.

[0052] The β chain of FSH to which a tag is attached is a β chain of FSH to which a tag is attached at the C-terminus.

[0053] In the above application, the biological material related to the SP1 signal peptide is the coding gene of the SP1 signal peptide or an expression cassette, recombinant vector, recombinant bacteria or recombinant cell line containing the coding gene.

[0054] The present invention designs a kind of FSH-Fc fusion protein, for the candidate molecule of long-acting FSH, and carries out signal peptide optimization experiment on this basis.It is found that artificially modified signal peptide SP1 (SEQ ID No.5) guides the secretory expression of FSH-Fc fusion protein eukaryotic cells.But signal peptide SP1 can not improve secretory expression for all proteins (the different protein expression experience accumulated in this laboratory shows that the CD19 protein expression amount guided by SP1 is extremely low).Therefore, artificially modified signal peptide SP1 is significantly better than antibody light chain signal peptide SP2 and luciferase signal peptide SP3 for guiding the secretory expression amount of FSH-Fc fusion protein eukaryotic cells, and is more suitable for large-scale industrial production, reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is the prediction map of secretion expression of artificially modified signal peptide SP1. SP(Sec / SPI) is the probability of prediction as signal peptide, CS is the predicted signal peptidase cleavage site, and OTHERS represents the probability of non-signal peptide. The ordinate is the probability of amino acid sequence as predicted structure, and the abscissa is the protein amino acid sequence. Among them, A is the prediction map of FSH-Fc fusion protein A chain, and B is the prediction map of FSH-Fc fusion protein B chain.

[0056] Figure 2 The enzyme digestion identification diagram for the FSH-Fc fusion protein recombinant expression plasmid was constructed. The A chain was digested with HindIII and XhoI, and the B chain was digested with BstBI and PacI. 1 is pCGS3-SP1-FSH-Fc (artificially modified signal peptide SP1 expression plasmid), 2 is pCGS3-SP2-FSH-Fc (antibody light chain signal peptide SP2 expression plasmid), and 3 is pCGS3-SP3-FSH-Fc (luciferase signal peptide SP3 expression plasmid).

[0057] Figure 3 The non-reducing SDS-PAGE identification diagram of the supernatant of FSH-Fc fusion protein cell secretion. Among them, 1 is pCGS3-SP1-FSH-Fc / 293, 2 is pCGS3-SP2-FSH-Fc / 293, 3 is pCGS3-SP3-FSH-Fc / 293, and 4 is a negative control group without plasmid transfection.

[0058] Figure 4 The grayscale analysis of the supernatant secreted by the FSH-Fc fusion protein cells. The peak indicated by the arrow is the FSH-Fc fusion protein peak (coinciding with the negative background protein band), A is pCGS3-SP1-FSH-Fc / 293, B is pCGS3-SP2-FSH-Fc / 293, C is pCGS3-SP3-FSH-Fc / 293, and D is the negative control group without plasmid transfection.

[0059] Figure 5 The purification and identification of FSH-Fc fusion protein by SDS-PAGE, where NR is non-reducing SDS-PAGE, R is reducing SDS-PAGE, 1 is SP1-FSH-Fc, 2 is SP2-FSH-Fc, and 3 is SP3-FSH-Fc. DETAILED DESCRIPTION

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0061] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0062] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0063] The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0064] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0065] The main reagents and their manufacturers in the following examples are as follows:

[0066] pCGS3 expression vector: Merck;

[0067] HindIII:NEB;

[0068] XhoI:NEB Corporation;

[0069] BstBI: New England Biolabs Inc.

[0070] PacI:NEB Corporation;

[0071] GXL Premix: TAKARA Corporation;

[0072] DNA Ligation Kit Ver.2.1: TAKARA;

[0073] Expi293F TM Cells: Thermo Fisher Scientific.

[0074] Expi293 TM Expression Medium: Thermo Fisher Scientific;

[0075] ExpiFectamine TM 293 Transfection Kit: Thermo Fisher;

[0076] Opti-MEM TM I Reduced Serum Medium: Thermo Fisher Company;

[0077] PageRuler TM Prestained protein molecular weight standard, 10 to 180 kDa: Thermo Fisher;

[0078] Protein A 4FF filler purification kit: Sangon Biotech (Shanghai) Co., Ltd.;

[0079] Amicon Ultra-15 centrifugal filter device: Millipore;

[0080] Amicon Ultra-0.5 centrifugal filter device: Millipore;

[0081] PBS pH 7.4 (1×): Gibco;

[0082] Gel imaging system: Protein Simple;

[0083] Cell counter: Roche;

[0084] Clean bench: Suzhou Antai Air Technology Co., Ltd.;

[0085] Electric constant temperature water bath: Fisher Scientific;

[0086] CO2 constant temperature shaker: CRYSTAL;

[0087] HYG-A fully constant temperature bottle shaking cabinet: Taicang Experimental Equipment Factory;

[0088] DYY-6C electrophoresis apparatus: Beijing Liuyi Instrument Factory;

[0089] DYCP-31DN horizontal electrophoresis tank: Beijing Liuyi Instrument Factory;

[0090] Micropipette: Eppendorf.

[0091] Example 1: Signal peptide prediction

[0092] SignalP-5.0 online software was used to predict the efficiency of artificially modified signal peptide SP1: the A chain amino acid sequence (SEQ ID No.7) and B chain amino acid sequence (SEQ ID No.8) of SP1-FSH-Fc fusion protein were respectively input into the URL https: / / services.healthtech.dtu.dk / service.php? SignalP-5.0, eukaryotic organisms were selected, and the server was submitted to predict the cleavage position of SP1 signal peptide. The prediction results are as follows Figure 1 As shown, the probability of the SP1 signal peptide structure of the A chain and B chain of FSH-Fc was 99.95%, and the probability of signal peptidase cleavage was 94.97% and 85.57%, respectively. The cleavage site was located between the 19th and 20th amino acids, which was in line with expectations.

[0093] In summary, the signal peptide SP1 has high cleavage efficiency, which is conducive to the secretory expression of FSH-Fc fusion protein ( Figure 1 ).

[0094] The amino acid sequence of the signal peptide SP1 is SEQ ID No.5, and the nucleotide sequence of the gene encoding it is SEQ ID No.6.

[0095] Example 2: Construction of recombinant expression plasmid

[0096] 1. SP1-FSH-Fc gene synthesis

[0097] Bio-Pharma was commissioned to optimize the A chain (SEQ ID No. 7) and B chain (SEQ ID No. 8) codons of the SP1-FSH-Fc (SP1 signal peptide fused FSH-Fc) fusion protein. The optimized A chain and B chain nucleotide sequences were SEQ ID No. 9 and SEQ ID No. 10, respectively. The delivered cloning plasmids were named pUC57-SP1-FSHA and pUC57-SP1-FSHB-Fc, respectively.

[0098] 2. SP2-FSH-Fc fragment amplification

[0099] The above-synthesized pUC57-SP1-FSHA and pUC57-SP1-FSHB-Fc were used as templates to amplify the A chain nucleotide sequence of SP2-FSH-Fc (SP2-FSHA, SEQ ID No. 11) and the B chain nucleotide sequence of SP2-FSHB-Fc (SEQ ID No. 12), respectively. The details are as follows:

[0100] Using pUC57-SP1-FSHA as template, primers 1 and 5 for the first round of amplification, fragment 1 was obtained; using fragment 1 as template, primers 3 and 5 for the second round of amplification, fragment 2 was obtained; fragment 2 was the target fragment of SP2-FSHA (denoted as the A chain of SP2-FSH-Fc).

[0101] Using pUC57-SP1-FSHB-Fc as template, primers 2 and 6 were used for the first round of amplification to obtain fragment 3; using fragment 3 as template, primers 4 and 6 were used for the second round of amplification to obtain fragment 4; fragment 4 was the target fragment of SP2-FSHB-Fc (denoted as B chain of SP2-FSH-Fc).

[0102] The primer sequences used in the above PCR amplification are as follows:

[0103] Primer 1: 5'-CTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCGCCCCCGACGTGCAGGACTG-3';

[0104] Primer 2: 5'-TGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCAACAGCTGCGAGCTGACCAAC-3';

[0105] Primer 3: 5'-CCCAAGCTTGCCGCCACCATGAGCGTGCCAACCCAGGTGCTGGGCCTGCTGCTGCT-3';

[0106] Primer 4: 5'-ACGAAGTTCGAAGCCGCCACCATGAGCGTGCCAACCCAGGTGCTGGGCCTGCTGCTGCT-3';

[0107] Primer 5: 5′-CCCGCTCGAGTTAGCTCTTGTGGTAGTAGCAGGTG-3′;

[0108] Primer 6: 5'-CCTTAATTAATTAGCCGGGGCTCAGGCTCAGG-3'.

[0109] 3. SP3-FSH-Fc fragment amplification

[0110] The above two synthesized pUC57-SP1-FSHA and pUC57-SP1-FSHB-Fc were used as templates to amplify the A chain nucleotide sequence (SP3-FSHA, SEQ ID No. 13) and the B chain nucleotide sequence (SP3-FSHB-Fc, SEQ ID No. 14) of SP3-FSH-Fc, respectively. The details are as follows:

[0111] Using pUC57-SP1-FSHA as a template, primers 5 and 7 were used for the first round of amplification to obtain fragment 5; using fragment 5 as a template, primers 5 and 9 were used for the second round of amplification to obtain fragment 6; fragment 6 is the target fragment of SP3-FSHA (denoted as the A chain of SP3-FSH-Fc).

[0112] Using pUC57-SP1-FSHB-Fc as template, primers 6 and 8 were used for the first round of amplification to obtain fragment 7; using fragment 7 as template, primers 6 and 10 were used for the second round of amplification to obtain fragment 8; fragment 8 was the target fragment of SP3-FSHB-Fc (referred to as B chain of SP3-FSH-Fc).

[0113] The primer sequences used in the above PCR amplification are as follows:

[0114] Primer 5: 5′-CCCGCTCGAGTTAGCTCTTGTGGTAGTAGCAGGTG-3′;

[0115] Primer 6: 5'-CCTTAATTAATTAGCCGGGGCTCAGGCTCAGG-3';

[0116] Primer 7: 5'-GTTTGCTCTGATTTGTATTGCCGTGGCTGAGGCCGCCCCCGACGTGCAGGACTG-3';

[0117] Primer 8: 5'-GTGTTGTTGCTCTGATTTGTATTGCCGTGGCTGAGGCCAACAGCTGCGAGCTG-3';

[0118] Primer 9: 5'-CCCAAGCTTGCCGCCACCATGGGGGTGAAGGTGTTGTTGCTCTGATTTGTATTGC-3';

[0119] Primer 10: 5'-ACGAAGTTCGAAGCCGCCACCATGGGGGTGAAGGTGTTGTTTGCTCTGATTTGTATTGC-3'.

[0120] IV. Construction of FSH-Fc fusion protein recombinant expression plasmid

[0121] The A chains of FSH-Fc fused with different connecting peptides synthesized or amplified by PCR: SP1-FSH-Fc A chain, SP2-FSH-Fc A chain, SP3-FSH-Fc A chain and pCGS3 were double-digested with HindIII and XhoI and cloned into the pCGS3 expression vector using a DNA ligation kit (TAKARA), and were named pCGS3-SP1-FSHA, pCGS3-SP2-FSHA, and pCGS3-SP3-FSHA intermediate vectors, respectively.

[0122] The FSH-Fc B chains fused with different connecting peptides synthesized or PCR amplified from the above-mentioned one, two and three genes: SP1-FSH-Fc B chain, SP2-FSH-Fc B chain, SP3-FSH-Fc B chain and the pCGS3-SP1-FSHA, pCGS3-SP2-FSHA, pCGS3-SP3-FSHA intermediate vectors constructed above were double-digested with BstBI and PacI and cloned into three intermediate vectors using a DNA ligation kit (TAKARA), and were named pCGS3-SP1-FSH1-Fc, pCGS3-SP2-FSH1-Fc, pCGS3-SP3-FSH1-Fc recombinant expression plasmids, respectively.

[0123] The results of enzyme digestion identification of the three recombinant expression plasmids are as follows: Figure 2 As shown in the figure, it can be seen that 1 is pCGS3-SP1-FSH-Fc, 2 is pCGS3-SP2-FSH-Fc, and 3 is pCGS3-SP3-FSH-Fc. The sizes of the enzyme-cut bands are consistent with expectations.

[0124] The structure of the recombinant expression plasmid pCGS3-SP1-FSH-Fc is described as follows: the DNA fragment shown in SEQ ID No.9 is inserted between the restriction sites HindIII and XhoI of the pCGS3 vector, wherein positions 1-6 of SEQ ID No.9 are HindIII restriction sites, positions 7-15 are Kozak sequences, positions 16-72 are the coding gene for the natural SP1 signal peptide, positions 73-348 are the A chain coding gene for FSH-Fc (only the α chain coding gene for FSH), positions 349-351 are stop codons, and positions 352-357 are XhoI restriction sites; and the DNA fragment shown in SEQ ID No.10 is inserted between the restriction sites BstBI and PacI of the pCGS3 vector, wherein positions 1-6 of SEQ ID No.9 are HindIII restriction sites, positions 7-15 are Kozak sequences, positions 16-72 are the coding gene for the natural SP1 signal peptide, positions 73-348 are the A chain coding gene for FSH-Fc (only the α chain coding gene for FSH), positions 349-351 are stop codons, and positions 352-357 are XhoI restriction sites. Positions 1-6 of No.10 are BstBI restriction site, positions 7-15 are Kozak sequence, positions 16-72 are the coding gene of natural SP1 signal peptide, positions 73-1083 are the coding gene of FSH-Fc B chain (positions 73-405 are FSH β chain, positions 406-1083 are Fc structure), positions 1084-1086 are stop codon, and positions 1087-1094 are PacI restriction site.

[0125] SEQ ID No.9 encodes the A chain of the SP1-FSH-Fc protein shown in SEQ ID No.7. Positions 1-19 of SEQ ID No.7 are the SP1 signal peptide, and positions 20-111 are the A chain of FSH-Fc; SEQ ID No.10 encodes the B chain of the SP1-FSH-Fc protein shown in SEQ ID No.8. Positions 1-19 of SEQ ID No.8 are the SP1 signal peptide, and positions 20-356 are the B chain of FSH-Fc (positions 20-130 are the β chain of FSH, and positions 131-356 are the Fc structure).

[0126] The structure of the recombinant expression plasmid pCGS3-SP2-FSH-Fc is described as follows: the DNA fragment shown in SEQ ID No.11 is inserted between the restriction sites HindIII and XhoI of the pCGS3 vector, wherein positions 1-6 of SEQ ID No.11 are HindIII restriction sites, positions 7-15 are Kozak sequences, positions 16-75 are the coding gene for the natural SP2 signal peptide, positions 76-351 are the A chain coding gene for FSH-Fc (only the α chain coding gene for FSH), positions 352-354 are stop codons, and positions 355-360 are XhoI restriction sites; the DNA fragment shown in SEQ ID No.12 is inserted between the restriction sites BstBI and PacI of the pCGS3 vector, wherein positions 1-6 of SEQ ID No.11 are HindIII restriction sites, positions 7-15 are Kozak sequences, positions 16-75 are the coding gene for the natural SP2 signal peptide, positions 76-351 are the A chain coding gene for FSH-Fc (only the α chain coding gene for FSH), positions 352-354 are stop codons, and positions 355-360 are XhoI restriction sites. Positions 1-6 of No.12 are BstBI restriction site, positions 7-15 are Kozak sequence, positions 16-75 are the coding gene of natural SP2 signal peptide, positions 76-1086 are the coding gene of FSH-Fc B chain (positions 76-408 are FSH β chain, positions 409-1086 are Fc structure), positions 1087-1089 are stop codon, and positions 1090-1097 are PacI restriction site.

[0127] The structure of the recombinant expression plasmid pCGS3-SP3-FSH-Fc is described as follows: the DNA fragment shown in SEQ ID No.13 is inserted between the restriction sites HindIII and XhoI of the pCGS3 vector, wherein positions 1-6 of SEQ ID No.13 are HindIII restriction sites, positions 7-15 are Kozak sequences, positions 16-66 are the coding gene for the natural SP3 signal peptide, positions 67-342 are the A chain coding gene for FSH-Fc (only the α chain coding gene for FSH), positions 343-345 are stop codons, and positions 346-351 are XhoI restriction sites; the DNA fragment shown in SEQ ID No.14 is inserted between the restriction sites BstBI and PacI of the pCGS3 vector, wherein positions 1-6 of SEQ ID No.13 are HindIII restriction sites, positions 7-15 are Kozak sequences, positions 16-66 are the coding gene for the natural SP3 signal peptide, positions 67-342 are the A chain coding gene for FSH-Fc (only the α chain coding gene for FSH), positions 343-345 are stop codons, and positions 346-351 are XhoI restriction sites. Positions 1-6 of No.14 are BstBI restriction site, positions 7-15 are Kozak sequence, positions 16-66 are the coding gene of natural SP3 signal peptide, positions 67-1077 are the B chain coding gene of FSH-Fc (positions 67-399 are FSH β chain, positions 400-1077 are Fc structure), positions 1078-1080 are stop codons, and positions 1081-1088 are PacI restriction site.

[0128] The above-mentioned recombinant expression plasmids pCGS3-SP1-FSH-Fc, pCGS3-SP2-FSH-Fc and pCGS3-SP3-FSH-Fc express fusion protein SP1-FSH-Fc, fusion protein SP2-FSH-Fc and fusion protein SP3-FSH-Fc respectively. These fusion proteins are secreted outside the cells and are all fusion proteins FSH-Fc.

[0129] The amino acid sequence of the A chain of the fusion protein FSH-Fc is SEQ ID No.1, and the amino acid sequence of the B chain of FSH-Fc is SEQ ID No.2; the nucleotide sequence of the gene encoding the A chain of FSH-Fc is SEQ ID No.3, and the nucleotide sequence of the gene encoding the B chain of FSH-Fc is SEQ ID No.4.

[0130] Example 3: Expression of FSH-Fc fusion protein

[0131] 1. Host Cells

[0132] Take the seed bank cells of host cell Expi293F from the liquid nitrogen tank, thaw quickly in a 37°C water bath, and aseptically transfer the thawed cell suspension to a 125ml vial containing 30ml preheated complete growth medium. The shaking incubator is cultured under the following conditions: 37°C, 8% CO2, 120rpm, amplitude 25mm, humidity ≥80%. Take the cell suspension after 15-30min to test the cell density and viability.

[0133] When the cell viability recovers to more than 90% and the cell density reaches 5×10 6 cells / ml, calculated as 0.5×10 6 cells / ml for inoculation and amplification.

[0134] 2. Cell Transfection

[0135] pCGS3-SP1-FSH-Fc, pCGS3-SP2-FSH-Fc and pCGS3-SP3-FSH-Fc constructed in Example 2 were transfected into host cells respectively.

[0136] 1. One day before transfection

[0137] 24 h before transfection, cells were plated at 3×10 6 cells / ml and cultured for 24h.

[0138] 2. On the day of transfection

[0139] (1) The cell density should reach 5.5×10 6 cells / ml, the viability should be ≥95%. Dilute the cells to 3×10 cells / ml with fresh pre-warmed complete growth medium. 6cells / ml.

[0140] (2) Prepare transfection reagent and DNA complex

[0141] 1) DNA dilution

[0142] The plasmid (pCGS3-SP1-FSH-Fc, pCGS3-SP2-FSH-Fc or pCGS3-SP3-FSH-Fc constructed in Example 2) was diluted to 1 μg / μl with sterile water. According to the amount of 1 μg of plasmid per 1 ml of cells, the amount of plasmid required for transfecting 50 ml of cells was taken, that is, 50 μl of plasmid was added to 3 ml of Opti-MEM TM I Reduced Serum medium is reserved.

[0143] 2) Dilution of transfection reagent

[0144] Before use, add the transfection reagent ExpiFectamine293 TM Gently invert the Reagent upside down to mix well, and take the amount of transfection reagent required to transfect 50ml of cells, i.e. 160μl ExpiFectamine293 TM The reagent was added to 2.8 ml of Opti-MEMTM I Reduced Serum medium by gently inverting and mixing, and then allowed to stand at room temperature for 5 min.

[0145] 3) Add the diluted transfection reagent to the plasmid and gently mix by inverting up and down. Incubate at room temperature for 20 minutes. Slowly add the mixed transfection reagent and DNA complex to the cell culture. Incubate at 37°C, 8% CO2, 120 rpm, amplitude 25 mm, humidity ≥ 80%.

[0146] 3. First day after transfection

[0147] 22 hours after transfection, add the enhancer in the amount of 50 ml of cells to be transfected. TM 293Transfection Enhancer 1 and 3ml ExpiFectamine TM Mix 293 Transfection Enhancer 2 and slowly add to the cell culture.

[0148] 4. Collection of culture supernatant

[0149] After transfection, the cell viability was monitored every day, and the culture was terminated for 4 days when the viability dropped to 65%-75%. The culture was collected and centrifuged at 3500g for 30 minutes to collect the supernatant, which was named pCGS3-SP1-FSH-Fc / 293 (FSH-Fc fusion protein secreted and expressed by signal peptide SP1), pCGS3-SP2-FSH-Fc / 293 (FSH-Fc fusion protein secreted and expressed by signal peptide SP2) and pCGS3-SP3-FSH-Fc / 293 (FSH-Fc fusion protein secreted and expressed by luciferase signal peptide SP3).

[0150] The supernatant was subjected to SDS-PAGE identification and grayscale analysis.

[0151] Image J software was used for grayscale analysis of SDS protein electrophoresis. The operation steps were Image→Type→32-Bit to grayscale; Process→Subtract Background→OK to remove the background color; Rectangular tool to select lanes→Analyze→Gel→Select First Lane to determine the analysis lane, and repeatedly select multiple lanes for simultaneous analysis; Analyze→Gel→Plot Lane to generate peak area; Linear tool to select the peak graph corresponding to the target band, and Wand tool to calculate the corresponding peak graph area to obtain the proportion of the target protein expression in the total protein.

[0152] The results are as follows Figure 3 and Figure 4 As shown, it can be seen that the expression level of FSH-Fc fusion protein secreted by signal peptide SP1 increased by 6.04 times and 2.58 times compared with light chain signal peptide SP2 and luciferase signal peptide SP3, respectively.

[0153] In summary, compared with the light chain signal peptide SP2 and the luciferase signal peptide SP3, the artificial signal peptide SP1 of the present invention has a higher secretory expression yield of the FSH-Fc fusion protein.

[0154] Example 4: Protein purification

[0155] 1. Ultrafiltration Concentration

[0156] The collected supernatants (i.e., the supernatants obtained after "collecting the culture and centrifuging at 3500 g for 30 min" in step 4 of Example 3): pCGS3-SP1-FSH-Fc / 293 (FSH-Fc fusion protein secreted by signal peptide SP1), pCGS3-SP2-FSH-Fc / 293 (FSH-Fc fusion protein secreted by signal peptide SP2) and pCGS3-SP3-FSH-Fc / 293 (FSH-Fc fusion protein secreted by luciferase signal peptide SP3) were ultrafiltrated and concentrated by centrifugation at 6000 g for 20 min at 4°C using an Amicon Ultra-15 centrifugal filter device (Millipore), and the cell supernatant was finally concentrated to 20 to 30 ml.

[0157] 2. Affinity chromatography

[0158] The ultrafiltration concentrated supernatant obtained in step 1 was purified using the Protein A 4FF filler purification kit (Sangong). The supernatant and Binding / Wash Buffer were mixed in a volume ratio of 1:1 and allowed to stand for 20 minutes for full incubation. Two column volumes of Binding / Wash Buffer were used to balance the column, and the buffer flowed through the pre-loaded column by gravity. The ultrafiltration concentrated supernatant and the mixed solution of Binding / Wash Buffer were added to the column and flowed through the pre-loaded column by gravity; if there was any remaining sample, it could be loaded again, re-circulated once, and the flow-through was collected into a centrifuge tube. Two column volumes of Binding / wash buffer were used to wash the column and the flow-through was collected until the absorbance of the flow-through at 280 nm was close to the baseline. Two column volumes of Elution Buffer were used to elute the histidine-tagged protein on the column. This step was repeated until the absorbance of the flow-through at 280 nm was close to the baseline. The eluate was collected for purification to obtain the protein solution purified by the nickel column.

[0159] 3. Ultrafiltration Replacement

[0160] The protein solution after nickel column purification was added to Amicon Ultra-15 centrifugal filter device (Millipore), and centrifuged at 10000g for 3min in batches until about 150μl of the solution remained. 300μl PBS buffer (1×, pH7.4) was gently added, and centrifuged at 10000g until 150μl remained, and repeated three times. PBS buffer (1×, pH7.4) was used to elute the ultrafiltration tube to collect the sample, and the final volume was about 2ml to obtain homozygous proteins, which were recorded as SP1-FSH-Fc, SP2-FSH-Fc and SP3-FSH-Fc.

[0161] Reserve 5 μl of the sample for protein concentration determination and SDS-PAGE protein electrophoresis detection.

[0162] The results are as follows Figure 5As shown, the purity of the purified samples of the three signal peptide products SP1, SP2 and SP3 detected by SDS-PAGE was greater than 90%, and the yields of the purified protein products were 18.3 mg / L, 11.74 mg / L and 9.06 mg / L, respectively, which were basically consistent with the grayscale analysis yields in Example 3.

[0163] Based on the results of the above embodiments, the FSH-Fc fusion protein designed by the present invention is subjected to a signal peptide optimization experiment. The study found that the artificially modified signal peptide SP1 (SEQ ID No.5) guides the secretory expression of the FSH-Fc fusion protein in eukaryotic cells. Further confirmation: the secretory expression of the artificially modified signal peptide SP1 is significantly better than that of the antibody light chain signal peptide SP2 and the luciferase signal peptide SP3, and is more suitable for large-scale industrial production, reducing production costs.

[0164] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of FSH fusion protein-related biomaterials in any of the following A1)-A3): A1) Preparation of FSH fusion protein in Expi293F cells; A2) Increase the expression or yield of FSH or its fusion protein in Expi293F cells; A3) promoting the secretion of FSH fusion protein outside the host cell, wherein the host cell is Expi293F cell; The biological material is the coding gene of the FSH fusion protein or an expression cassette or a recombinant vector containing the coding gene; The nucleotide sequence of the gene encoding the A chain of the FSH fusion protein is shown in SEQ ID No.9, and the nucleotide sequence of the gene encoding the B chain of the FSH fusion protein is shown in SEQ ID No.

10.

2. A method for preparing FSH protein, comprising the following steps: expressing the coding gene of the fusion protein described in claim 1 in a host cell to obtain FSH protein; the host cell is Expi293F cell.

3. The method according to claim 2, characterized in that: The method comprises the following steps: 1) introducing the genes encoding the A chain and the B chain of the fusion protein of claim 1 into a host cell to obtain a recombinant cell; 2) Cultivating the recombinant cells to obtain FSH protein.

4. Use of SP1 signal peptide or biological materials related thereto in any of the following A1)-A3): A1) Preparation of FSH fusion protein in Expi293F cells; A2) Increase the expression or production of FSH fusion protein in Expi293F cells; A3) promoting the secretion of FSH protein fusion protein outside the host cell, wherein the host cell is Expi293F cell; The amino acid sequence of the SP1 signal peptide is shown in SEQ ID No.5; The FSH fusion protein has an A chain that is an α chain of FSH and a B chain that is a β chain of FSH connected to a tag; The biological material related to the SP1 signal peptide is the coding gene of the SP1 signal peptide or an expression box, a recombinant vector, a recombinant bacterium or a recombinant cell line containing the coding gene.

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