Dual-signal peptide for improving secretion level of blood coagulation factor viii and use thereof

Abstract

Provided is a dual-signal peptide. The dual-signal peptide comprises a first signal peptide and a second signal peptide, wherein the first signal peptide comprises an exogenous signal peptide, the second signal peptide comprises an endogenous signal peptide of blood coagulation factor VIII, and the first signal peptide and the second signal peptide are linked in tandem. The dual-signal peptide of the present invention enables substantial improvements in both the secretion effect and coagulation activity of blood coagulation factor VIII, so that the yield of FVIII is greatly increased, and the industrial production efficiency thereof is improved.

Description

A dual-signal peptide for improving the secretion level of coagulation factor VIII and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to a prior application, patent application number 202410097964.6, filed with the State Intellectual Property Office of China on January 23, 2024, entitled “A Dual Signal Peptide for Increasing Factor VIII Secretion and Its Application.” The entire text of this prior application is incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of coagulation factor biosynthesis, and in particular to a dual-signal peptide for improving the secretion level of coagulation factor VIII and applications thereof. Background Art

[0004] Hemophilia A (HA) is an X-linked recessive genetic disease caused by abnormalities in the gene encoding coagulation factor VIII, which leads to the absence or functional defects of coagulation factor VIII. The structure of coagulation factor VIII is composed of several different regions, including 3 A regions, 1 B region and 2 C regions, and the B region is highly glycosylated. The absence of the entire B region structure or most of the B region does not affect the activity of coagulation factor VIII. Studies have shown that whether it is wild-type coagulation factor VIII or recombinantly improved coagulation factor VIII, its molecular structure is generally larger. After the exogenous gene expressing coagulation factor VIII is introduced into the cell, its expression level in the cell is low, far below the actual demand. Therefore, promoting the expression and secretion of coagulation factor VIII in cells is one of the technical problems that those skilled in the art need to solve.

[0005] Signal peptides, also known as leader peptides, signal sequences, transit peptides, and so on, are located at the N-terminus (amino terminus) of nascent proteins and are generally composed of 5-30 amino acids. They are short peptide chains that guide the transport and secretion of newly synthesized proteins. Signal peptides typically consist of three parts: 1. A positively charged N-terminus; 2. A central hydrophobic sequence, often composed primarily of neutral amino acids, which can form a helical structure and is the main functional region of the signal peptide; 3. A negatively charged C-terminus, composed of small amino acids, which serves as the sequence cleavage site for the signal peptide and is part of the signal peptide processing region. Signal peptides are signal recognition elements attached to the endoplasmic reticulum membrane, guiding the nascent protein into the lumen of the endoplasmic reticulum. The signal peptide can then be removed by the action of signal peptidase. For secretory proteins, signal peptides play a crucial role in the transport and secretion of secretory proteins, promoting their transport within the cell and secreting them outside the cell. For secretory proteins expressed from exogenous genes, without a signal peptide, their secretion is poor or non-excreted. However, after attaching a signal peptide, they can be effectively secreted. However, there are many different types of secretory proteins and signal peptides. Different signal peptides can produce different secretion effects for the same secretory protein. Similarly, the same signal peptide can produce different secretion effects when used with different secretory proteins.

[0006] US5880327A discloses a transgenic mammal expressing human coagulation factor VIII. CN109929029A discloses a method for increasing the efficient expression of recombinant human coagulation factor VIII. However, the secretion amount and secretion effect of human coagulation factor VIII in these patents are poor. Summary of the Invention

[0007] Regarding how to promote the expression and secretion of coagulation factor VIII in cells, the inventors of the present application unexpectedly discovered through experiments that when coagulation factor VIII adopts a dual signal peptide, its secretion level in cells can be greatly improved.

[0008] A first aspect of the present invention provides a dual signal peptide, comprising a first signal peptide and a second signal peptide, wherein the first signal peptide comprises an exogenous signal peptide, the second signal peptide comprises an endogenous signal peptide of coagulation factor VIII, and the first signal peptide and the second signal peptide are connected in series.

[0009] In one embodiment of the present invention, the dual signal peptide is used to promote the secretion of coagulation factor VIII and / or enhance the activity of coagulation factor VIII.

[0010] In a preferred embodiment of the present invention, the exogenous signal peptide is one or both of Gaussia luciferase and IgG V.

[0011] In one embodiment of the present invention, the exogenous signal peptide comprises the following amino acid sequence: the amino acid sequence of the exogenous signal peptide is as shown in any one of SEQ ID NO: 4 and SEQ ID NO: 6, or an amino acid sequence that has at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with any one of SEQ ID NO: 4 and SEQ ID NO: 6, and has the same activity.

[0012] In one embodiment of the present invention, the amino acid sequence of the endogenous signal peptide is as shown in SEQ ID NO: 8, or an amino acid sequence that has at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 8 and has the same activity.

[0013] In one embodiment of the present invention, the first signal peptide and the second signal peptide are connected in series, including connecting the C-terminus of the first signal peptide and the N-terminus of the second signal peptide.

[0014] In one embodiment of the present invention, a spacer sequence may or may not be present between the first signal peptide and the second signal peptide. In one embodiment of the present invention, no spacer sequence is present between the first signal peptide and the second signal peptide. In other words, the first signal peptide and the second signal peptide are covalently bonded via a single peptide bond. In another embodiment, a spacer sequence of one or more amino acids is present between the first signal peptide and the second signal peptide. In principle, there is no limitation on the size of the spacer sequence. In one embodiment of the present invention, the spacer sequence between the first and second signal peptides has 2-14 amino acids.

[0015] In one embodiment of the present invention, the amino acid sequence of the dual signal peptide is as shown in SEQ ID NO: 11, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 11.

[0016] A second aspect of the present invention provides a polypeptide comprising the above-mentioned dual signal peptide and coagulation factor VIII.

[0017] In one embodiment of the present invention, the coagulation factor VIII includes modified or unmodified coagulation factor VIII.

[0018] In one embodiment of the present invention, the coagulation factor VIII is selected from full-length coagulation factor VIII or its variants, coagulation factor VIII with a B region deletion or its variants, including but not limited to BDDF8-CO variant, BDDF8-SQ variant, BDDF8-N222 variant, BDDF8-N6 variant, etc.

[0019] In one embodiment of the present invention, the amino acid sequence of the coagulation factor VIII (excluding the endogenous signal peptide) is as shown in SEQ ID NO: 10, or an amino acid sequence that has at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO: 10 and has the same activity.

[0020] In one embodiment of the present invention, the C-terminus of the dual signal peptide is connected to the N-terminus of coagulation factor VIII.

[0021] In one embodiment of the present invention, a spacer sequence may or may not be present between the dual signal peptide and coagulation factor VIII. In one embodiment of the present invention, no spacer sequence is present between the dual signal peptide and coagulation factor VIII. In other words, the dual signal peptide is covalently bonded to the coagulation factor via a single peptide bond. In another embodiment, a spacer sequence of one or more amino acids is present between the dual signal peptide and coagulation factor VIII. In principle, there is no limit to the size of the spacer sequence. In one embodiment of the present invention, the spacer sequence between the first and second signal peptides has 2-14 amino acids.

[0022] In one embodiment of the present invention, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 12, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 12.

[0023] The third aspect of the present invention provides a polynucleotide encoding the above-mentioned double signal peptide or polypeptide.

[0024] In one embodiment of the present invention, the polynucleotide sequence encoding the first signal peptide is as shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3, or a nucleotide sequence having at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with the polynucleotide sequence encoding the first signal peptide of any one of SEQ ID NO: 1 and SEQ ID NO: 3.

[0025] In one embodiment of the present invention, the polynucleotide sequence encoding the second signal peptide is as shown in SEQ ID NO: 7, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 7.

[0026] In one embodiment of the present invention, the sequence encoding coagulation factor VIII (excluding the endogenous signal peptide) is as shown in SEQ ID NO: 9, or a nucleotide sequence having at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO: 9.

[0027] In one embodiment of the present invention, the polynucleotide sequence encoding the first signal peptide, the polynucleotide sequence encoding the second signal peptide, and the sequence encoding coagulation factor VIII are operably linked.

[0028] In one embodiment of the present invention, the polynucleotide comprises, from the 5' end to the 3' end, a polynucleotide sequence encoding a first signal peptide, a polynucleotide sequence encoding a second signal peptide, and a sequence encoding factor VIII.

[0029] In one embodiment of the present invention, the sequence of the polynucleotide is as shown in SEQ ID NO: 13, or a nucleotide sequence having at least 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 13.

[0030] In one embodiment of the present invention, the polynucleotide further comprises a promoter and a 3' non-translated polynucleotide sequence. In a preferred embodiment of the present invention, the 3' non-translated polynucleotide sequence comprises a Poly A tail.

[0031] In one embodiment of the present invention, the promoter includes a homologous promoter or a heterologous promoter.

[0032] In one embodiment of the present invention, the promoter has tissue-preferred expression.

[0033] In one embodiment of the present invention, the promoter, polynucleotide, and 3' non-translated polynucleotide sequence are operably linked.

[0034] In one embodiment of the present invention, the polynucleotide comprises, from the 5' end to the 3' end, a promoter-a polynucleotide sequence encoding a first signal peptide-a polynucleotide sequence encoding a second signal peptide-a 3' untranslated polynucleotide sequence.

[0035] In one embodiment of the present invention, the polynucleotide comprises from 5' to 3' end a promoter - a polynucleotide sequence encoding a first signal peptide - a polynucleotide sequence encoding a second signal peptide - a sequence encoding factor VIII - a 3' untranslated polynucleotide sequence.

[0036] In one embodiment of the present invention, the polynucleotide comprises, from 5' to 3' end, a CMV promoter - a polynucleotide sequence encoding a first signal peptide - a polynucleotide sequence encoding a second signal peptide - a sequence encoding factor VIII - a PolyA tail.

[0037] The fourth aspect of the present invention provides an expression vector comprising the above-mentioned polynucleotide.

[0038] In one embodiment of the present invention, the expression vector may be a plasmid or a viral vector. In a preferred embodiment, the plasmid comprises an AAV expression plasmid. The viral vector is selected from the group consisting of an AAV vector, a lentiviral vector, a retroviral vector, an adenoviral vector, and a synthetic viral vector.

[0039] In a fifth aspect, the present invention provides a host cell comprising one or more of the above-mentioned dual signal peptides, polypeptides, polynucleotides or expression vectors.

[0040] In one embodiment of the present invention, the host cell includes bacteria, fungi, viruses, animal cells, and / or plant cells.

[0041] In a sixth aspect, the present invention provides a pharmaceutical composition comprising one or more of the above-mentioned dual signal peptides, polypeptides, polynucleotides, expression vectors, and host cells.

[0042] In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0043] In a seventh aspect, the present invention provides use of the above-mentioned dual signal peptide, polypeptide, polynucleotide, expression vector, host cell or pharmaceutical composition in the preparation of coagulation factor VIII or a drug for treating hemophilia.

[0044] In an eighth aspect of the present invention, a method for secreting coagulation factor VIII comprises using the above-mentioned dual signal peptide, polypeptide, polynucleotide, expression vector, host cell or pharmaceutical composition to secrete coagulation factor VIII.

[0045] In one embodiment of the present invention, the method comprises the following steps: constructing an expression vector containing the polynucleotide encoding the dual signal peptide and the coagulation factor VIII coding sequence, transferring the vector into a host cell, and culturing the host cell under conditions that allow the expression and secretion of the coagulation factor VIII to obtain the coagulation factor VIII.

[0046] In one embodiment of the present invention, the method comprises culturing the host cell under conditions that allow expression and secretion of the coagulation factor VIII, and isolating the coagulation factor VIII from the cell culture.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The present invention unexpectedly discovered through experiments that a single signal peptide cannot achieve good secretory expression of coagulation factor VIII, and its secretion amount cannot be detected or is at a low level. However, when a dual signal peptide is selected for coagulation factor VIII, it shows better secretion effect and coagulation activity. Preferably, the effect is most significant when an exogenous signal peptide and an endogenous signal peptide are combined together to guide the expression and secretion of FVIII. More preferably, when a dual signal peptide combination having an exogenous Gluc signal peptide or an IgG V signal peptide and an endogenous signal peptide is used, the FVIII expression amount is greatly improved compared to a single signal peptide group (only an endogenous signal peptide group or only an exogenous signal peptide group), and the highest has exceeded 200 ng / ml.

[0049] (2) The present invention addresses the problem of FVIII secretion levels in cells and, through experimental optimization, selects a dual signal peptide structure to achieve optimal expression and secretion effects. Compared with a single signal peptide, the secretion effect and coagulation activity are substantially improved. In particular, when a dual signal peptide combination of an exogenous Gluc signal peptide or an IgG V signal peptide and an endogenous signal peptide is selected as a signal peptide, it is significantly superior to other types of signal peptides, and the coagulation activity is also significantly improved. The present invention can significantly increase the production of FVIII, improve its industrial production efficiency, further promote its application in hemophilia treatment drugs, and help reduce treatment costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG1 is a schematic diagram of the exogenous gene expression frame carrying a single signal peptide and a double signal peptide in the present invention.

[0051] FIG2 is a comparison of the effects of different signal peptides or signal peptide combinations on the activity of FVIII expressed and secreted by cells in the HEK-293T cell line in Example 2 of the present invention.

[0052] FIG3 compares the effects of different signal peptides or signal peptide combinations on the expression and secretion of FVIII in the HEK-293T cell line in Example 2 of the present invention. DETAILED DESCRIPTION

[0053] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0054] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. In the specific embodiments of the present invention, the secretion level or expression level of coagulation factor VIII is determined based on the coagulation factor VIII secreted by cells, specifically, the detection result in the supernatant.

[0055] Technical terms

[0056] The term "coagulation factor VIII" is also known as FVIII or F8. B-domain-deleted F8: The intact F8 gene encodes full-length factor VIII (FVIII), which has six structural domains: A1, A2, B, A3, C1, and C2. The B domain is not essential for FVIII's coagulation catalytic activity. The B-domain-deleted F8 gene is relatively small, easily integrated, and encodes FVIII with coagulation activity.

[0057] The term "spacer sequence" refers to a nucleotide sequence interval of 1-100bp or longer, or an amino acid sequence interval of 1-100 amino acids or longer. The spacer sequence can be a multiple cloning site, a recombination site, or can contain a binding site for a transcription factor, or other functional fragments. The spacer sequence does not affect the function of the double signal peptide and does not affect the secretory expression of FVIII. In the present invention, the double signal peptides and the signal peptide and the secretory protein (such as FVIII) can be directly connected, or a spacer sequence can be added. Preferably, the double signal peptides and the signal peptide and the secretory protein are directly connected. In the present invention, the promoter and the double signal peptide are connected by a PacI endonuclease recognition sequence (TTAATTAA), and the BDDF8-CO and polyA (pA) are connected by a NotI endonuclease recognition sequence (GCGGCCGC).

[0058] The term "signal peptide" refers to a short peptide chain of 5-30 amino acids in a newly synthesized polypeptide chain that directs the transfer of proteins into the secretory pathway. It is located at the N-terminus of the secretory protein and consists of three parts: a positively charged basic amino terminus at the N-terminus; a central functional region, a hydrophobic sequence formed by neutral amino acids; and a negatively charged processing region at the C-terminus, which serves as the cleavage site for the signal peptide. Newly synthesized proteins are guided into the endoplasmic reticulum lumen by the signal peptide, where the signal peptide sequence is removed by the action of a signal peptidase. The protein then continues to be translated, folded, and modified. Signal peptides can be used with heterologous promoters or with their native promoters. The promoter molecules of the present invention can be operably linked to endogenous signal peptides or to exogenous signal peptides.

[0059] The term "endogenous signal peptide" refers to a signal peptide derived from the corresponding target gene (protein) in an organism. In the present invention, the endogenous signal peptide includes the endogenous signal peptide of coagulation factor VIII, which comprises 19 amino acids, the amino acid sequence of which is shown in SEQ ID NO: 8, or fragments or variants thereof.

[0060] The term "exogenous signal peptide" refers to a signal peptide that is not the native signal peptide of the target gene (protein) in the organism, but is artificially added during gene expression. In the present invention, the amino acid sequence of the exogenous signal peptide includes the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or fragments or variants thereof.

[0061] The term "5' untranslated polynucleotide sequence" refers to a non-translated region at the 5' end of a pre-mRNA or mature mRNA. For example, on mature mRNAs, the 5'-UTR typically has a 7-methylguanosine cap at its 5' end and is involved in many processes, such as splicing, polyadenylation, mRNA export to the cytoplasm, recognition of the 5' end of the mRNA by the translation machinery, and protection of the mRNA from degradation.

[0062] The term "3' non-translated polynucleotide sequence" refers to the untranslated segment at the 3' end of the pre-mRNA or mature mRNA. For example, on mature mRNA, this region has a poly-(A) tail and is known to have multiple functions in mRNA stability, translation initiation, and mRNA export.

[0063] The term "promoter" refers to a gene component that binds to proteins called transcription factors to control the initiation and extent of gene transcription. CMV promoter: A strong promoter discovered in human cytomegalovirus (CMV). In the present invention, promoters include, but are not limited to, the EF1a promoter, CMV promoter, CAG promoter, PKG promoter, CBA promoter, and tissue-specific promoters.

[0064] The term "PolyA (pA)": polyadenylic acid, which plays an important role in maintaining the stability of mRNA and preventing its degradation during the nuclear transport and translation of mRNA. Mammalian expression plasmids usually add a transcription terminator and PolyA after the target gene to promote polyadenylation and transcription termination. Poly (A) can be AATAAA. The AATAAA sequence can be replaced by other hexanucleotide sequences that are homologous to AATAAA and can signal polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATTAAC, AATTAGA, AATTAAA or AATAAG (see, for example, WO 06 / 12414). In some embodiments, the poly (A) signal sequence can be a synthetic polyadenylation site (see, for example, Genes Dev. 3 (7): 1019-1025, 1989). Other examples of poly(A) signal sequences are known in the art.

[0065] The terms "sequence identity" or "identity" can be used interchangeably and refer to the identical nucleic acid residues or amino acid residues in two sequences when the maximum correspondence is achieved over a specific comparison window by alignment. The percentage of sequence identity is a numerical value determined by comparing sequences (e.g., nucleic acid sequences or amino acid sequences) that are optimally aligned over the comparison window, wherein the portion of a sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) to achieve optimal alignment of the two sequences. Percentages are calculated by determining the number of positions at which the identical nucleic acid or amino acid residues appear in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce percent sequence identity. Methods for aligning sequences for comparison are well known. Various programs and alignment algorithms are described, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24: 307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174: 247-50.

[0066] The term "tandem connection" refers to the connection of two or more polypeptides in the order of amino to carboxyl terminus. In the present invention, the tandem connection includes the connection of the C-terminus of the first signal peptide to the N-terminus of the second signal peptide.

[0067] The term "operably linked" refers to elements or structures in a nucleic acid sequence that are connected by operational capability rather than physical location. These elements or structures are capable of or characterized by completing a desired operation. Those of ordinary skill in the art recognize that elements or structures in a nucleic acid sequence do not necessarily have to be operably linked in a series or adjacent order. "Operably linked" can include a situation in which a selected nucleotide sequence and a regulatory nucleotide sequence are covalently linked in a manner that places the expression of the nucleotide coding sequence under the influence or control of the regulatory sequence. Therefore, if the regulatory sequence is capable of influencing the transcription of part or all of the nucleotide coding sequence that forms the selected nucleotide sequence, the regulatory sequence is operably linked to the selected nucleotide sequence. Where appropriate, the resulting transcript can then be translated into the desired protein or polypeptide. In the present invention, a promoter, a polynucleotide sequence encoding a first signal peptide, a polynucleotide sequence encoding a second signal peptide, a sequence encoding Factor VIII, and a 3' untranslated polynucleotide sequence are operably linked.

[0068] Example 1 F8 gene expression vectors carrying different signal peptides

[0069] Three exogenous signal peptides (SPs) were selected and linked to the N-terminus of the codon-optimized B-domain deleted F8 (hereafter referred to as BDDF8-CO, encoding FVIII protein) gene to improve the expression and secretion of FVIII. At the same time, the endogenous signal peptide of the F8 gene was carried or not carried. The nucleotide sequences encoding the above structures were inserted into the multiple cloning site of the Psmart-hCkan expression vector by Shanghai Sangon Biotechnology Co., Ltd. to construct BDDF8-CO expression plasmids containing only the endogenous signal peptide, BDDF8-CO expression plasmids without the endogenous signal peptide (exogenous signal peptide only) guided by three different exogenous signal peptides, and BDDF8-CO expression plasmids containing both the endogenous signal peptide and the endogenous signal peptide (double signal peptide) guided by three different exogenous signal peptides.

[0070] The two signal peptides are directly connected to each other, the signal peptide is directly connected to BDDF8-CO, the CMV promoter and the signal peptide are connected by the PacI endonuclease recognition sequence (TTAATTAA), and the BDDF8-CO and polyA (pA) are connected by the NotI endonuclease recognition sequence (GCGGCCGC). The above plasmids were transfected into human embryonic kidney cells (HEK-293T cells) to detect the expression and secretion of the target protein FVIII. The schematic diagram of the expression frame of BDDF8-CO with only the endogenous signal peptide, BDDF8-CO with only the exogenous signal peptide, and BDDF8-CO guided by the exogenous signal peptide and containing the endogenous signal peptide (double signal peptide) is shown in Figure 1.

[0071] The nucleotide sequence of the exogenous signal peptide is as follows:

[0072] Gaussia luciferase (Gluc) (SEQ ID NO: 1):

[0073] Human IgK VIII (SEQ ID NO: 2):

[0074] Human IgG V (SEQ ID NO: 3):

[0075] The amino acid sequence of the exogenous signal peptide is as follows:

[0076] Gaussia luciferase (Gluc) (SEQ ID NO: 4): MGVKVLFALICIAVAEA;

[0077] Human IgK VIII (SEQ ID NO: 5): MDMRVPAQLLGLLLLWLRGARC;

[0078] Human IgG V (SEQ ID NO: 6): MDWTWRFLFVVAAATGVQS.

[0079] The nucleotide sequence of the BDDF8-CO endogenous signal peptide is as follows (SEQ ID NO: 7):

[0080] The amino acid sequence of the BDDF8-CO endogenous signal peptide is as follows (SEQ ID NO: 8):

[0081] The nucleotide sequence of BDDF8-CO (excluding the endogenous signal peptide) is as follows (SEQ ID NO: 9):

[0082] The amino acid sequence of BDDF8-CO (excluding the endogenous signal peptide) is as follows (SEQ ID NO: 10):

[0083] The amino acid sequence of the Gluc double signal peptide is as follows (SEQ ID NO: 11):

[0084] The amino acid sequence of the combination of the Gluc double signal peptide and the F8 expression cassette is as follows (SEQ ID NO: 12):

[0085] The nucleotide sequence of the combination of the Gluc double signal peptide and the F8 expression cassette is as follows (SEQ ID NO: 13):

[0086] Example 2 Signal Peptide Efficiency Verification Plasmid Transfection HEK-293T Cell Line and Detection of FVIII Expression and Secretion Levels

[0087] 1. Transfection of signal peptide efficiency verification plasmid into HEK-293T cell line

[0088] Human embryonic kidney cell line HEK-293T cell line (Wuhan Punosai Life Science Technology Co., Ltd., #CL-0005, hereinafter referred to as 293T cells) was cultured and grown to a confluence of about 80%. The complete medium (high-glucose DMEM medium (Gibco, #C11995500BT) containing 10% FBS (Gibco, #12664025)) was discarded, and the cells were gently washed twice with DPBS and then lysed with 0.05% trypsin (Gibco, #15050065) for 2-3 hr. ) for 2 minutes, discard the digestion solution, and resuspend the cells in complete culture medium. 4 × 105 cells were seeded per well of a 12-well plate. When the 293T cells reached 40% confluency, 1 μg of the efficiency verification plasmid (obtained in Example 1) was transfected into each well using Lipo2000 transfection reagent (Thermo, #11668019) (the control group served as a negative control). Transfection procedures were performed according to the manufacturer's instructions. After 12 hours, 1 mL of fresh complete culture medium was replaced and cultured. The transfection experiment was repeated three times for each group.

[0089] 2. Detection of FVIII coagulation activity in cell supernatant using a fully automatic coagulation analyzer

[0090] To examine the effects of different signal peptides or their combinations on FVIII secretion and corresponding coagulation activity, cell culture supernatants were collected 72 hours after efficiency verification plasmid transfection. The percentage of FVIII activity in the culture supernatants of each group was measured using an automated coagulation analyzer (Sysmex, #CS-2400) using an intrinsic coagulation factor activity assay. The procedure was performed according to the instrument's instructions and using the reagents supplied with the instrument. The FVIII activity assay results are shown in Figure 2.

[0091] As shown in Figure 2, the FVIII activity was almost undetectable in the endogenous signal peptide group (Endogenous SP only), and the FVIII activity in the three groups with only exogenous signal peptides (Without endogenous SP) was also low, with the detection values ​​of each group not exceeding 10%. In the group with both exogenous Gluc signal peptide and endogenous signal peptide, the FVIII activity exceeded 400%, approximately 4 IU / ml; the FVIII activity in the group with both exogenous Human IgG V signal peptide and endogenous signal peptide was also high, approximately 300%. This shows that compared with a single signal peptide or other types of dual signal peptide combinations, the dual signal peptide combination composed of an exogenous Gluc signal peptide and an endogenous signal peptide achieved the best coagulation activity effect, far exceeding the coagulation effect that can be achieved by other single signal peptides.

[0092] 3. ELISA detection of F8 content in cell supernatant

[0093] To further verify the coagulation factor secretion achieved by different signal peptides, cell culture supernatants were collected 72 hours after transfection and the Factor VIII content in the culture supernatants of each group was measured using a human FVIII ELISA kit (CEDARLANE, #CL20035K) according to the kit instructions. The results of the ELISA assay for FVIII secretion are shown in Figure 3.

[0094] As shown in Figure 3, the FVIII content in the endogenous signal peptide only group (Endogenous SP only) was almost undetectable, and the FVIII content in the three groups with only exogenous signal peptides (Without endogenous SP) increased slightly, but was still low, and did not exceed 10ng / ml. The most significant effect was achieved when the exogenous signal peptide and the endogenous signal peptide were combined to guide FVIII expression. Among them, the dual signal peptide combination with both the exogenous Gluc signal peptide and the endogenous signal peptide significantly increased the FVIII expression level compared to the single signal peptide group (endogenous signal peptide only group or exogenous signal peptide only group), exceeding 200ng / ml.

[0095] From the above experimental results, it can be seen that for FVIII, due to its large protein and the large number of amino acids that make up the protein, a single signal peptide is often difficult to achieve a good secretion effect in the cell, and the secretion amount and coagulation activity are extremely low. In the specific embodiment of the present invention, a dual signal peptide mode is particularly adopted to achieve an increase in secretion amount and coagulation activity. Preferably, the exogenous signal peptide and the endogenous signal peptide of the protein itself are combined together to form a dual signal peptide structure to enhance the expression and secretion of the target protein. In the specific embodiment of the present invention, it is confirmed from both the coagulation activity and the expression amount that the dual signal peptide combination can significantly and substantially improve the expression and secretion of the target protein compared with a single exogenous signal peptide or a single endogenous signal peptide, among which the dual signal peptide combination of the Gluc exogenous signal peptide and the endogenous signal peptide has the best effect.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the 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 purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A double signal peptide, characterized in that: The double signal peptide includes a first signal peptide and a second signal peptide. The first signal peptide includes an exogenous signal peptide, and the second signal peptide includes the endogenous signal peptide of factor VIII. The first signal peptide and the second signal peptide are connected in series.

2. The double signal peptide according to claim 1, characterized in that: The exogenous signal peptide includes any available signal peptide that can promote the secretion of factor VIII from cells. Preferably, the exogenous signal peptide is one or both of Gaussia luciferase and IgG V.

3. A dual signal peptide according to claim 2, characterized in that: The exogenous signal peptide includes the following amino acid sequence: the amino acid sequence of the exogenous signal peptide is shown as any one of SEQ ID NO: 4 and SEQ ID NO: 6, or has at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with any one of the sequences of SEQ ID NO: 4 and SEQ ID NO: 6, and has the same activity.

4. A double signal peptide as claimed in claim 1, characterized in that: The amino acid sequence of the endogenous signal peptide is shown as SEQ ID NO: 8, or has at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO: 8, and has the same activity.

5. A dual signal peptide as claimed in claim 1, wherein: The connection in series of the first signal peptide and the second signal peptide includes the connection of the C-terminus of the first signal peptide and the N-terminus of the second signal peptide. Preferably, there is or is not a spacer sequence between the first signal peptide and the second signal peptide. Preferably, the amino acid sequence of the double signal peptide is shown as SEQ ID NO: 11, or has at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO:

11.

6. A polypeptide, characterized in that: The polypeptide includes the double signal peptide described in any one of claims 1-5 and factor VIII. Preferably, the factor VIII includes modified or unmodified factor VIII. Preferably, the factor VIII is selected from full-length factor VIII or its variants, factor VIII with B-domain deletion or its variants; more preferably, the factor VIII includes BDDF8-CO variant, BDDF8-SQ variant, BDDF8-N222 variant, BDDF8-N6 variant. Preferably, the amino acid sequence of the factor VIII is shown as SEQ ID NO: 10, or has at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO: 10, and has the same activity. Preferably, the C-terminus of the double signal peptide is connected to the N-terminus of factor VIII. Preferably, there is or is not a spacer sequence between the double signal peptide and factor VIII. Preferably, the amino acid sequence of the polypeptide is shown as SEQ ID NO: 12, or has at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO:

12.

7. A polynucleotide, characterized in that: The polynucleotide encodes the double signal peptide described in any one of claims 1-5 or the polypeptide described in claim 6. Preferably, the polynucleotide sequence encoding the first signal peptide is as shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3, or a nucleotide sequence having at least 95%, or 96%, or 97%, or 98%, or at least 99% sequence identity with the polynucleotide sequence encoding the first signal peptide shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3; Preferably, the polynucleotide sequence encoding the second signal peptide is as shown in SEQ ID NO: 7, or a nucleotide sequence having at least 95%, or 96%, or 97%, or 98%, or at least 99% sequence identity with SEQ ID NO: 7; Preferably, the sequence encoding coagulation factor VIII is as shown in SEQ ID NO: 9, or a nucleotide sequence having at least 95%, or 96%, or 97%, or 98%, or at least 99% sequence identity with SEQ ID NO: 9; Preferably, the polynucleotide sequence encoding the first signal peptide, the polynucleotide sequence encoding the second signal peptide, and the sequence encoding coagulation factor VIII are operably linked; Preferably, the polynucleotide comprises, from the 5'-end to the 3'-end, a polynucleotide sequence encoding the first signal peptide - a polynucleotide sequence encoding the second signal peptide - a sequence encoding coagulation factor VIII; Preferably, the sequence of the polynucleotide is as shown in SEQ ID NO: 13, or a nucleotide sequence having at least 95%, or 96%, or 97%, or 98%, or at least 99% sequence identity with SEQ ID NO: 13; Preferably, the polynucleotide further comprises a promoter and a 3'-untranslated polynucleotide sequence; more preferably, the 3'-untranslated polynucleotide sequence comprises a PolyA tail; Preferably, the promoter, the polynucleotide, and the 3'-untranslated polynucleotide sequence are operably linked.

8. An expression vector, characterized in that: Comprising the polynucleotide according to claim 7.

9. A host cell, characterized in that: Comprising one or more of the dual signal peptides according to any one of claims 1-5, the polypeptide according to claim 6, the polynucleotide according to claim 7, or the expression vector according to claim 8.

10. A pharmaceutical composition, characterized in that: Comprising one or more of the dual signal peptides according to any one of claims 1-5, the polypeptide according to claim 6, the polynucleotide according to claim 7, the expression vector according to claim 8, or the host cell according to claim 9.

11. Use of one or more of the dual signal peptides according to any one of claims 1-5, the polypeptide according to claim 6, the polynucleotide according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, or the pharmaceutical composition according to claim 10 in the preparation of coagulation factor VIII or in the preparation of a medicament for treating hemophilia.

12. A method for secreting coagulation factor VIII, comprising secreting coagulation factor VIII by using one or more of the dual signal peptides according to any one of claims 1-5, the polypeptide according to claim 6, the polynucleotide according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, or the pharmaceutical composition according to claim 10; Preferably, the method includes the following steps: Construct an expression vector containing the polynucleotide encoding the double signal peptide and the coding sequence of coagulation factor VIII, transfer it into a host cell, and culture the host cell under conditions allowing the expression and secretion of the coagulation factor VIII to obtain the coagulation factor VIII; Preferably, the method includes culturing the host cell under conditions allowing the expression and secretion of the coagulation factor VIII, and isolating the coagulation factor VIII from the cell culture.

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