Dual-signal peptide for improving secretion level of blood coagulation factor VIII and application of dual-signal peptide

Abstract

The invention provides a dual-signal peptide, the dual-signal peptide comprises a first signal peptide and a second signal peptide, the first signal peptide comprises an exogenous signal peptide, the second signal peptide comprises an endogenous signal peptide of a blood coagulation factor VIII, and the first signal peptide and the second signal peptide are connected in series. According to the dual-signal peptide, the secretion effect and the blood coagulation activity of the blood coagulation factor VIII are substantially improved, the production of FVIII can be greatly improved, the industrial production efficiency of FVIII is improved, the application of FVIII in hemophilia treatment drugs is further promoted, and the treatment cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of blood coagulation factor biosynthesis, and particularly relates to a dual signal peptide for improving the secretion level of blood coagulation factor VIII and its application. Background Art

[0002] Hemophilia A (HA) is an X-linked recessive genetic disease, and its pathogenesis is due to abnormal genes encoding blood coagulation factor VIII, resulting in the absence or functional defect of blood coagulation factor VIII. The structure of blood coagulation factor VIII is composed of several different regions, including 3 A regions, 1 B region, and 2 C regions. The B region is highly glycosylated, and the absence of the entire B region structure or most of the B region does not affect the activity of blood coagulation factor VIII. Research shows that whether it is wild-type blood coagulation factor VIII or recombinantly improved blood coagulation factor VIII, their molecular structures are generally large. After introducing the exogenous gene expressing blood coagulation factor VIII into cells, its expression level in cells is relatively low, far lower than the actual demand. Therefore, promoting the expression and secretion of blood coagulation factor VIII in cells is one of the technical problems to be solved by those skilled in the art.

[0003] A signal peptide, also known as a leader peptide, signal sequence, transit peptide, etc., abbreviated as SP, is located at the N-terminus, i.e., the amino terminus, of a nascent protein, and is generally composed of 5 - 30 amino acids. It is a short peptide chain that guides the transport and secretion of newly synthesized proteins. A signal peptide usually includes three parts: 1. a positively charged N-terminus; 2. a middle hydrophobic sequence, which is often mainly composed of neutral amino acids and can form a helical structure, and is the main functional region of the signal peptide; 3. a negatively charged C-terminus, including small amino acids, which is the sequence cleavage site of the signal peptide and belongs to the processing region of the signal peptide. A signal peptide is a signal recognition element attached to the endoplasmic reticulum membrane. Through its guidance, the nascent protein is imported into the lumen of the endoplasmic reticulum, and then the signal peptide can be excised under the action of signal peptidase. For secreted proteins, signal peptides play an important role in the transport and secretion of secreted proteins. By promoting transport in cells, secreted proteins are secreted outside the cells. For secreted proteins expressed by exogenous genes, without a signal peptide, their secretion effect is poor or they do not secrete. After connecting a signal peptide, good secretion can be obtained. However, both the types of secreted proteins and signal peptides are diverse. For the same secreted protein, different signal peptides have different secretion effects; for the same signal peptide, when used for different secreted proteins, it also often shows different secretion effects.

[0004] US5880327A discloses transgenic mammals expressing human blood coagulation factor VIII. CN109929029A discloses a method for improving the high-efficiency expression of recombinant human blood coagulation factor VIII. However, the secretion amount and secretion effect of human blood coagulation factor VIII in these patents are not satisfactory. Summary of the Invention

[0005] In view of how to promote the expression and secretion of coagulation factor VIII in cells, the inventors of the present application unexpectedly found through experiments that when coagulation factor VIII adopts a dual signal peptide, its secretion level in cells can be greatly improved.

[0006] In a first aspect of the present invention, there is provided a dual signal peptide, which comprises a first signal peptide and a second signal peptide. The first signal peptide comprises an exogenous signal peptide, and the second signal peptide comprises an endogenous signal peptide of coagulation factor VIII. The first signal peptide and the second signal peptide are connected in series.

[0007] In one embodiment of the present invention, the dual signal peptide is used to promote the secretion of coagulation factor VIII and / or improve the activity of coagulation factor VIII.

[0008] In a preferred embodiment of the present invention, the exogenous signal peptide is one or both of Gaussia luciferase and IgG V.

[0009] 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 has at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with any of the sequences of SEQ ID NO:4 and SEQ ID NO:6, and has the same activity.

[0010] 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 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.

[0011] In one embodiment of the present invention, the connection in series of the first signal peptide and the second signal peptide comprises the connection of the C-terminus of the first signal peptide and the N-terminus of the second signal peptide.

[0012] In one embodiment of the present invention, there is or is not a spacer sequence between the first signal peptide and the second signal peptide. In one embodiment of the present invention, there is no spacer sequence 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 through a single peptide bond. In another embodiment, there is a spacer sequence of one or more amino acids between the first signal peptide and the second signal peptide. 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.

[0013] 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% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO: 11.

[0014] The second aspect of the present invention provides a polypeptide, which comprises the above-mentioned dual signal peptide and coagulation factor VIII.

[0015] In one embodiment of the present invention, the coagulation factor VIII includes modified or unmodified coagulation factor VIII.

[0016] 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 lacking the B domain or its variants, including but not limited to BDDF8-CO variant, BDDF8-SQ variant, BDDF8-N222 variant, BDDF8-N6 variant, etc.

[0017] 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 having at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO: 10 and having the same activity.

[0018] In one embodiment of the present invention, the C-terminus of the dual signal peptide is connected to the N-terminus of the coagulation factor VIII.

[0019] In one embodiment of the present invention, there is or is not a spacer sequence between the dual signal peptide and the coagulation factor VIII. In one embodiment of the present invention, there is no spacer sequence between the dual signal peptide and the coagulation factor VIII. In other words, the dual signal peptide and the coagulation factor are covalently bonded through a single peptide bond. In another embodiment, there is a spacer sequence of one or more amino acids between the dual signal peptide and the 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.

[0020] 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% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO: 12.

[0021] The third aspect of the present invention provides a polynucleotide, which encodes the above-mentioned dual signal peptide or polypeptide.

[0022] 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 shown in any one of SEQ ID NO:1 and SEQ ID NO:3.

[0023] 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% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO:7.

[0024] 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.

[0025] 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.

[0026] In one embodiment of the present invention, the polynucleotide includes, 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.

[0027] 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% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO:13.

[0028] In one embodiment of the present invention, the polynucleotide further includes a promoter and a 3'-untranslated polynucleotide sequence. In a preferred embodiment of the present invention, the 3'-untranslated polynucleotide sequence includes a PolyA tail.

[0029] In one embodiment of the present invention, the promoter includes a homologous promoter or a heterologous promoter.

[0030] In one embodiment of the present invention, the promoter has tissue-preferred expression.

[0031] In one embodiment of the present invention, the promoter, the polynucleotide, and the 3'-untranslated polynucleotide sequence are operably linked.

[0032] 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.

[0033] 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 sequence encoding factor VIII - a 3' untranslated polynucleotide sequence.

[0034] In one embodiment of the present invention, the polynucleotide comprises, from the 5'-end to the 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.

[0035] The fourth aspect of the present invention provides an expression vector comprising the above-mentioned polynucleotide.

[0036] In one embodiment of the present invention, the expression vector can 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.

[0037] The fifth aspect of the present invention provides a host cell comprising one or more of the above-mentioned dual signal peptides, polypeptides, polynucleotides, or expression vectors.

[0038] In one embodiment of the present invention, the host cell includes bacteria, fungi, viruses, animal cells, and / or plant cells.

[0039] The sixth aspect of 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.

[0040] In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0041] The seventh aspect of the present invention provides the use of the above-mentioned dual signal peptides, polypeptides, polynucleotides, expression vectors, host cells, or pharmaceutical compositions in the preparation of factor VIII or in the preparation of drugs for treating hemophilia.

[0042] The eighth aspect of the present invention provides a method for secreting factor VIII, comprising secreting factor VIII by using the above-mentioned dual signal peptides, polypeptides, polynucleotides, expression vectors, host cells, or pharmaceutical compositions.

[0043] In one embodiment of the present invention, the method comprises the following steps: constructing an expression vector containing the polynucleotide encoding the above double signal peptides and the coding sequence of coagulation factor VIII, transferring it into a host cell, and culturing the host cell under conditions allowing the expression and secretion of the coagulation factor VIII to obtain the coagulation factor VIII.

[0044] In one embodiment of the present invention, the method comprises culturing the above host cell under conditions allowing the expression and secretion of the coagulation factor VIII, and isolating the coagulation factor VIII from the cell culture.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) In the present invention, it was unexpectedly found through experiments that a single signal peptide could not achieve good secretion and expression of coagulation factor VIII, and its secretion amount could not be detected or was at a low level. However, when double signal peptides were selected for coagulation factor VIII, it showed better secretion effect and coagulation activity. Preferably, the simultaneous combination of an exogenous signal peptide and an endogenous signal peptide for guiding the expression and secretion of FVIII had the most significant effect. More preferably, when the double signal peptide combination of an exogenous Gluc signal peptide or IgG V signal peptide and an endogenous signal peptide was used, the FVIII expression level was significantly increased compared with the single signal peptide group (the group with only the endogenous signal peptide or the group with only the exogenous signal peptide), and the highest exceeded 200 ng / ml.

[0047] (2) Aiming at the secretion level problem of FVIII in cells, the present invention optimized and selected the double signal peptide structure through experiments, achieving the best expression and secretion effects. Compared with a single signal peptide, both the secretion effect and coagulation activity were substantially improved. Especially when the double signal peptide combination of an exogenous Gluc signal peptide or IgG V signal peptide and an endogenous signal peptide was used as the signal peptide, it was also significantly better than other types of signal peptides, and the coagulation activity was also greatly improved. The present invention can greatly increase the production amount of FVIII, improve its industrial production efficiency, further promote its application in hemophilia treatment drugs, and is beneficial to reducing the treatment cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a schematic diagram of the exogenous gene expression frames carrying single signal peptides and double signal peptides in the present invention.

[0049] Figure 2 It shows the influence of different signal peptides or signal peptide combinations on the activity of FVIII expressed and secreted by cells in HEK-293T cell line in Example 2 of the present invention.

[0050] Figure 3This is to compare the effects of different signal peptides or signal peptide combinations on the expression and secretion levels of FVIII in the HEK-293T cell line in Example 2 of the present invention. Detailed implementation manners

[0051] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate and explain the present invention by way of example, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0052] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods. In the specific implementation manners of the present invention, the secretion level or expression level of coagulation factor VIII is detected with the coagulation factor VIII secreted by cells. Specifically, the detection results in the supernatant are used as the standard.

[0053] Technical terms

[0054] The term "coagulation factor VIII", also known as FVIII or F8. F8 lacking the B domain: The complete F8 gene encodes full-length factor VIII (FVIII). Full-length FVIII has six domains: A1, A2, B, A3, C1, and C2. Among them, the B domain is not essential for the coagulation catalytic activity of FVIII. The F8 gene lacking the B domain is relatively small and easy to integrate, and also encodes FVIII with coagulation activity.

[0055] The term "spacer sequence" refers to a nucleotide sequence spacer of 1-100 bp or longer, or an amino acid sequence spacer of 1-100 amino acids or longer. The spacer sequence can be a multiple cloning site, a recombination site, can also contain a binding site for a transcription factor, or other functional fragments, etc. The spacer sequence does not affect the function of the dual signal peptide and does not affect the secretion and expression of FVIII. In the present invention, the dual signal peptides can be directly connected to each other, and a spacer sequence can also be added between the signal peptide and the secreted protein (such as FVIII). Preferably, the dual signal peptides are directly connected to each other, and the signal peptide and the secreted protein are also directly connected. In the present invention, the promoter and the dual signal peptide are connected by the PacI endonuclease recognition sequence (TTAATTAA), and BDDF8-CO and polyA (pA) are connected by the NotI endonuclease recognition sequence (GCGGCCGC).

[0056] The term "signal peptide" refers to a short peptide chain consisting of 5 - 30 amino acids in a newly synthesized polypeptide chain that guides the protein to the secretory pathway; it is located at the N - terminus of the secreted protein and consists of three parts: the N - terminus is a positively charged basic amino terminus; the middle is the main functional region, a hydrophobic sequence formed by neutral amino acids; the C - terminus is a negatively charged processing region, which is the cleavage site of the signal peptide. The newly synthesized protein is guided by the signal peptide into the lumen of the endoplasmic reticulum, and the signal peptide sequence is excised under the action of signal peptidase. Subsequently, the protein continues translation while undergoing folding and modification. The signal peptide can be used with a heterologous promoter or with their native promoter. The promoter molecule of the present invention can be operably linked to an endogenous signal peptide or can be operably linked to an exogenous signal peptide.

[0057] The term "endogenous signal peptide": a signal peptide derived from the corresponding target gene (protein) within an organism itself. In the present invention, the endogenous signal peptide includes the endogenous signal peptide of coagulation factor VIII, which consists of 19 amino acids, and its amino acid sequence is as shown in SEQ ID NO:8, or its fragments or variants.

[0058] The term "exogenous signal peptide": not the signal peptide of the target gene (protein) within an organism itself, but other signal peptides artificially added during gene expression. In the present invention, the amino acid sequences of the exogenous signal peptide include the amino acid sequences in SEQ ID NO:1 and SEQ ID NO:3, or their fragments or variants.

[0059] The term "5' untranslated polynucleotide sequence": the untranslated region at the 5' end of pre - mRNA or mature mRNA. For example, in mature mRNA, the 5’ - UTR usually 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 mRNA by the translation machinery, and protection of mRNA from degradation.

[0060] The term "3' untranslated polynucleotide sequence": the untranslated region at the 3' end of pre - mRNA or mature mRNA. For example, in mature mRNA, this region has a poly - (A) tail and is known to have multiple functions in mRNA stability, translation initiation, and mRNA export.

[0061] The term "promoter": a gene component that binds to a protein such as a transcription factor to control the start time and expression level of gene transcription and expression. CMV promoter: a strong promoter found in human cytomegalovirus (CMV). In the present invention, the promoter includes but is not limited to EF1a promoter, CMV promoter, CAG promoter, PKG promoter, CBA promoter, tissue - specific promoter.

[0062] The term "PolyA (pA)": polyadenylic acid, which plays an important role in maintaining the stability of mRNA and preventing its degradation during the nuclear export 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 capable of signaling polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA 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.

[0063] The terms "sequence identity" or "identity" are used interchangeably and refer to the identical nucleic acid or amino acid residues in two sequences when there is a maximum correspondence over a specified comparison window upon alignment. The percentage of sequence identity is a value determined by comparing the sequences that are optimally aligned over a comparison window (e.g., a nucleic acid sequence or an amino acid sequence), where a portion of one sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to a reference sequence (which does not contain additions or deletions) so that the two sequences achieve optimal alignment. The percentage is calculated by determining the number of positions at which the identical nucleic acid or amino acid residue occurs in both sequences to yield 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 yield the percent sequence identity. Methods for aligning sequences for comparison are well known. Various programs and alignment algorithms are described in the following, for example: 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. U.S.A. 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.

[0064] The term "tandem connection" refers to the connection of more than two polypeptides in the order from the amino terminus to the carboxyl terminus. In the present invention, the tandem connection includes the connection of the C-terminus of the first signal peptide and the N-terminus of the second signal peptide.

[0065] The term "operably linked" refers to elements or structures in a nucleic acid sequence that are linked by the ability to operate rather than by physical position. These elements or structures are capable of or characterized by performing a desired operation. Those of ordinary skill in the art recognize that the elements or structures in a nucleic acid sequence do not have to be operably linked in a tandem or adjacent order. "Operably linked" can include a situation in which a selected nucleotide sequence and a regulatory nucleotide sequence are covalently linked in such a way as to place the expression of the nucleotide coding sequence under the influence or control of the regulatory sequence. Thus, a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence can affect the transcription of the nucleotide coding sequence that forms part or all of the selected nucleotide sequence. In an appropriate case, 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 coagulation factor VIII, and a 3' untranslated polynucleotide sequence are operably linked.

[0066] Example 1 F8 gene expression vectors carrying different signal peptides

[0067] Three exogenous signal peptides (Signal peptide, SP) were selected and these exogenous signal peptides were respectively linked to the N-terminus of the codon-optimized F8 lacking the B domain (hereinafter referred to as BDDF8-CO, encoding the FVIII protein) gene to improve the expression and secretion of FVIII, and at the same time, with or without the endogenous signal peptide of the F8 gene itself. The Shanghai Sangon Biotech Co., Ltd. inserted the nucleotide sequences encoding the above structures into the multiple cloning sites of the Psmart-hCkan expression vector respectively to construct the BDDF8-CO expression plasmid with only the endogenous signal peptide, the BDDF8-CO expression plasmids without the endogenous signal peptide (only exogenous signal peptides) respectively guided by 3 different exogenous signal peptides, and the BDDF8-CO expression plasmids with both endogenous and exogenous signal peptides (dual signal peptides) respectively guided by 3 different exogenous signal peptides.

[0068] Among them, the two signal peptides are directly connected, and the signal peptide and BDDF8-CO are directly connected. The CMV promoter and the signal peptide are connected by the PacI endonuclease recognition sequence (TTAATTAA), and 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 diagrams of the expression cassettes of BDDF8-CO with only the endogenous signal peptide, BDDF8-CO with only the exogenous signal peptide, and BDDF8-CO with both endogenous and exogenous signal peptides (dual signal peptides) are as Figure 1 shown.

[0069] The nucleotide sequences of the exogenous signal peptides are as follows:

[0070] Gaussia luciferase (Gluc) (SEQ ID NO:1):

[0071] ATGGGCGTGAAGGTGCTGTTTGCCCTGATTTGCATCGCCGTGGCCGAGGCC;

[0072] Human IgK VIII (SEQ ID NO:2):

[0073] ATGGACATGAGAGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGCTGAGGGGAGCTAGATGT;

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

[0075] ATGGACTGGACCTGGCGGTTCCTGTTTGTGGTGGCCGCCGCCACCGGCGTGCAGAGC。

[0076] The amino acid sequences of the exogenous signal peptides are as follows:

[0077] Gaussia luciferase (Gluc) (SEQ ID NO:4): MGVKVLFALICIAVAEA;

[0078] Human IgK VIII (SEQ ID NO:5): MDMRVPAQLLGLLLLWLRGARC;

[0079] Human IgG V (SEQ ID NO:6): MDWTWRFLFVVAAATGVQS。

[0080] The nucleotide sequence of the BDDF8-CO endogenous signal peptide is as follows (SEQ ID NO:7):

[0081] ATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCT。

[0082] The amino acid sequence of the BDDF8-CO endogenous signal peptide is as follows (SEQ ID NO:8):

[0083] MQIELSTCFFLCLLRFCFS。

[0084] The nucleotide sequence of BDDF8-CO (without the endogenous signal peptide) is as follows (SEQ ID NO: 9):

[0085]

[0086]

[0087]

[0088] The amino acid sequence of BDDF8-CO (without the endogenous signal peptide) is as follows (SEQ ID NO: 10):

[0089]

[0090] The amino acid sequence of the Gluc dual signal peptide is as follows (SEQ ID NO: 11):

[0091] MGVKVLFALICIAVAEAMQIELSTCFFLCLLRFCFS.

[0092] The combined amino acid sequence of the Gluc dual signal peptide and the F8 expression cassette is as follows (SEQ ID NO: 12):

[0093]

[0094] The combined nucleotide sequence of the Gluc dual signal peptide and the F8 expression cassette is as follows (SEQ ID NO: 13):

[0095]

[0096]

[0097]

[0098]

[0099] Example 2 Verification of signal peptide efficiency: The plasmid for verifying signal peptide efficiency was transfected into the HEK-293T cell line, and the expression and secretion levels of FVIII were detected

[0100] 1. Transfect the plasmid for verifying signal peptide efficiency into the HEK-293T cell line

[0101] Culture the human embryonic kidney cell line HEK-293T cell line (Wuhan Punosai Life Science Co., Ltd., #CL-0005, hereinafter referred to as 293T cells). When its confluence reaches about 80%, discard the complete medium (high-glucose DMEM medium (Gibco, #C11995500BT) containing 10% FBS (Gibco, #12664025)), gently wash it twice with DPBS, then digest it with 0.05% trypsin (Gibco, #15050065) for 2 minutes, discard the digestion solution and resuspend the cells with the complete medium. Seed 4×105 cells into each well of a 12-well plate; when the confluence of 293T cells reaches 40%, use lipo2000 transfection reagent (Thermo, #11668019) to transfect 1 μg of the efficiency verification plasmid (the plasmid obtained in Example 1) into each well (the Control group is the negative control), and perform the transfection operation according to the instruction manual; after 12 h, replace it with 1 mL of fresh complete medium and continue the culture. Each group independently repeats the transfection experiment 3 times.

[0102] 2. Detection of FVIII coagulation activity in cell supernatant by automatic coagulation analyzer

[0103] To detect the effects of different signal peptides or their combinations on FVIII secretion and corresponding coagulation activity, 72 h after the transfection of the efficiency verification plasmid, collect the cell culture supernatant, and use an automatic coagulation analyzer (Sysmex, #CS-2400) to detect the percentage of FVIII activity in the culture supernatant of each group through the endogenous coagulation factor activity detection test. The operation is carried out according to the instrument instruction manual and the reagents supporting the use of the instrument. The FVIII activity detection results are as Figure 2 shown.

[0104] As Figure 2 can be seen, almost no FVIII activity can be detected in the group with only the endogenous signal peptide (Endogenous SP only), and the FVIII activity is also low in the three groups with only exogenous signal peptides (Without endogenous SP), and the detection values of each group do not exceed 10%. However, the FVIII activity in the group with both exogenous Gluc signal peptide and endogenous signal peptide exceeds 400%, about 4 IU / ml; the FVIII activity in the group with both exogenous Human IgG V signal peptide and endogenous signal peptide is also relatively high, about 300%. It can be seen from this that, compared with a single signal peptide or other types of double signal peptide combinations, the double signal peptide combination composed of exogenous Gluc signal peptide and endogenous signal peptide has achieved the best coagulation activity effect, far exceeding the coagulation effect that can be achieved by other single signal peptides.

[0105] 3. Detection of F8 content in cell supernatant by ELISA

[0106] To further verify the secretion levels of coagulation factors achieved by different signal peptides, 72 h after transfection, the cell culture supernatants were collected, and the content of factor VIII in the culture supernatants of each group was detected using a human FVIII ELISA detection kit (CEDARLANE, #CL20035K). The operation was carried out according to the kit instructions. The results of the ELISA detection of FVIII secretion levels are specifically as Figure 3 shown.

[0107] As Figure 3 can be seen, the content of FVIII was hardly detectable in the group with only the endogenous signal peptide (Endogenous SP only). In the three groups with only exogenous signal peptides (Without endogenous SP), the content of FVIII increased slightly but was still low, all not exceeding 10 ng / ml. When the exogenous signal peptide and the endogenous signal peptide were combined simultaneously to direct the expression of FVIII, the effect was the most significant. Among them, for the dual-signal peptide combination with both the exogenous Gluc signal peptide and the endogenous signal peptide, the expression level of FVIII increased significantly compared to the single-signal peptide groups (the group with only the endogenous signal peptide or the group with only the exogenous signal peptide), exceeding 200 ng / ml.

[0108] From the above experimental results, for FVIII, due to its large protein size and the large number of amino acids that make up this protein, a single signal peptide often has poor secretion effects in cells, and both the secretion level and the coagulation activity are extremely low. In the specific implementation manner of the present invention, a dual-signal peptide mode is particularly adopted, which realizes the improvement of the secretion level and the coagulation activity. Preferably, the combination of the exogenous signal peptide and the endogenous signal peptide of the protein itself constitutes a dual-signal peptide structure to enhance the expression and secretion of the target protein. In the specific embodiments of the present invention, it has been confirmed from both aspects of the coagulation activity and the expression level that the dual-signal peptide combination can significantly and greatly improve the expression and secretion of the target protein compared with a single exogenous signal peptide or a single endogenous signal peptide. Among them, the dual-signal peptide combination of the Gluc exogenous signal peptide and the endogenous signal peptide has the best effect.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A double signal peptide, characterized in that: The dual 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 an endogenous signal peptide of coagulation 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 coagulation factor VIII from cells. Preferably, the exogenous signal peptide is one or both of Gaussia luciferase and IgG V.

3. A double signal peptide as claimed in claim 2, wherein: The exogenous signal peptide includes the following amino acid sequences: the amino acid sequence of the exogenous signal peptide is any one of SEQ ID NO:4 and SEQ ID NO:6, or an amino acid sequence having 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 having the same activity.

4. A dual signal peptide as claimed in claim 1, characterized in that: The amino acid sequence of the endogenous signal peptide is as shown in SEQ ID NO:8, or an amino acid sequence having at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO:8 and having 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 dual signal peptide is as shown in SEQ ID NO:11, or an amino acid sequence having 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 dual signal peptide according to any one of claims 1-5 and coagulation factor VIII. Preferably, the coagulation factor VIII includes a modified or unmodified coagulation factor VIII. Preferably, the coagulation factor VIII is selected from full-length coagulation factor VIII or its variants, coagulation factor VIII lacking the B domain or its variants; more preferably, the coagulation factor VIII includes BDDF8-CO variant, BDDF8-SQ variant, BDDF8-N222 variant, BDDF8-N6 variant. Preferably, the amino acid sequence of the coagulation factor VIII is as shown in SEQ ID NO:10, or an amino acid sequence having at least 95% or 96% or 97% or 98%, or at least 99% sequence identity with SEQ ID NO:10 and having the same activity. Preferably, the C-terminus of the dual signal peptide is connected to the N-terminus of coagulation factor VIII. Preferably, there is or is not a spacer sequence between the dual signal peptide and coagulation factor VIII. Preferably, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:12, or an amino acid sequence having 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 dual signal peptide according to any one of claims 1-5 or the polypeptide according to 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 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 factor VIII are operably linked; Preferably, the polynucleotide from the 5' end to the 3' end includes a polynucleotide sequence encoding the first signal peptide - a polynucleotide sequence encoding the second signal peptide - a sequence encoding 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 includes a promoter and a 3' untranslated polynucleotide sequence; more preferably, the 3' untranslated polynucleotide sequence includes 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, and 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, and the pharmaceutical composition according to claim 10 in the preparation of factor VIII or in the preparation of a medicament for treating hemophilia.

12. A method for secreting factor VIII, comprising 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, and the pharmaceutical composition according to claim 10 to secrete factor VIII; Preferably, the method includes the following steps: Construct an expression vector containing the polynucleotide encoding the dual signal peptide and the coding sequence of coagulation factor VIII, transfer it into a host cell, and culture the host cell under conditions that permit 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 that permit the expression and secretion of the coagulation factor VIII and isolating the coagulation factor VIII from the cell culture.

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