CN112480262A - Fusion protein and preparation and application thereof - Google Patents

Fusion protein and preparation and application thereof Download PDF

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CN112480262A
CN112480262A CN201910859687.7A CN201910859687A CN112480262A CN 112480262 A CN112480262 A CN 112480262A CN 201910859687 A CN201910859687 A CN 201910859687A CN 112480262 A CN112480262 A CN 112480262A
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epapkp
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CN112480262B (en
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徐明恺
宋宇博
张惠文
张成刚
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Institute of Applied Ecology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Abstract

The invention provides a fusion protein, which sequentially comprises the following components from an N end to a C end: the peptide-based artificial breeding method comprises SEC2 or a mutant thereof, a connecting short peptide and iRGD, wherein the mutation comprises deletion, insertion and/or substitution of 1-5 amino acids, the amino acid sequence of SEC2 is shown as SEQ ID NO:31, the amino acid sequence of the connecting short peptide is shown as SEQ ID NO:29, and the amino acid sequence of the iRGD is shown as SEQ ID NO: 30. The invention also provides a preparation method and application of the fusion protein. The targeting fusion protein can specifically target and infiltrate into a tumor tissue microenvironment, greatly improves the tumor specificity and vascular permeability of the superantigen, and improves the killing efficiency on tumors, so that the survival capability of patients can be effectively improved when the targeting fusion protein is used for treating the patients, and the targeting fusion protein has good clinical application value.

Description

Fusion protein and preparation and application thereof
Technical Field
The invention relates to the field of genetic engineering, in particular to a fusion protein and preparation and application thereof.
Background
The immunotherapy of tumor is a treatment method which applies the principle and method of immunology and effectively kills tumor cells and inhibits the growth of tumor cells by activating immune cells in vivo and enhancing the anti-tumor immune response of an organism. Due to its small side effect and obvious therapeutic effect, it is becoming the development direction of future tumor therapy.
The targeting fusion protein is a novel treatment strategy proposed on the basis of the development of modern medical biology, and is formed by combining targeting molecules and cytotoxic molecules. The targeting molecule is usually selected from tumor specific antibody or short peptide, and the effector molecule capable of killing target cells is delivered to the affected part of the tumor, so that the development trend of the current tumor precision treatment is met.
The Internalizing RGD (also called internalization RGD) is a targeting cell-penetrating peptide with a ring structure, has smaller molecular weight and higher water solubility, and has a sequence of CRGDKGPDC, wherein the RGD sequence can be specifically combined with tumor tissue vascular endothelium and tumor cell high-expression integrin alphav, and after being cut by specific protease, a residue segment has the structural characteristics of R/KXXR/K and can interact with NRP-1 (neuropilin-1) to mediate cell membrane penetration effect. Due to the function diversity, the iRGD is widely applied to the research of targeting vectors of antitumor drugs, tumor imaging agents, some biological products and the like. Among them, integrin α v is a member of the cell adhesion receptor family, regulates a variety of cellular functions, especially in the occurrence, development and metastasis of solid tumors, and its expression is also positively correlated with the malignancy of tumors. NRP-1 plays an important role as a regulator of the nervous system in the local regulation of VEGF-induced angiogenesis in tumors. It is closely related to the growth, migration and angiogenesis of tumor cells.
Most of the existing researches adopt the combination of iRGD and chemical drugs, but the chemical drugs need to be encapsulated by liposome and then can be coupled with the iRGD, and the combined drugs have good tumor inhibition effect. However, since the chemical drug entrapped in the liposome needs to be released in the tissue to exert the drug effect, the type and mode of the liposome greatly affect the drug effect. The lower liposome encapsulation efficiency is always the key to troubling liposome medicines, and the liposome medicines also have the problems of poor stability, easy active clearing of organisms and the like. This also leads to the disadvantage that the iRGD-coupled liposomes are also unstable in structure and effect, which adds an obstacle to their use. In addition, in the application of combining the iRGD and the chemical drug, some iRGD and chemical drug are mixed and administered as two independent parts, so that the iRGD cannot exert an active targeted delivery effect on the chemical drug, and only can assist part of the chemical drug accumulated in the tumor vascular tissue to penetrate into the tumor tissue. This not only weakens the efficiency of iRGD-mediated effector molecule aggregation locally in tumors, but also does not improve the toxic side effects of chemical drugs on normal tissues due to weak targeting effects.
The prior art also discloses that the targeting cell-penetrating peptide iRGD with high permeability is combined with other protein effector molecules such as Trail and CDD to form fusion protein, and the obtained effect is slightly good. However, the tumor vasculature and tumor tissues are intricate and complex, and therefore fusion proteins with stronger targeting and tissue penetration are further sought.
Disclosure of Invention
The invention aims to solve the technical problem that the fusion protein cannot penetrate through a tumor vasculature and a complex microenvironment of tumor tissues to cause an unsatisfactory anti-tumor effect in the prior art, and provides a fusion protein, a gene thereof, a preparation method thereof and application thereof. The invention connects the targeting cell-penetrating peptide and the tumor immunotherapy medicament of the superantigen through the specific connecting short peptide for the first time to form the targeting fusion protein superantigen (SAg) -connecting short peptide (L) -iRGD, which can specifically target and infiltrate into the tumor tissue microenvironment with high expression integrin alphav and NRP-1, greatly improves the tumor specificity and vascular permeability of the superantigen, improves the killing efficiency of the tumor, thereby effectively improving the survival ability of the patient when being used for treating the patient and having good clinical application value.
The present inventors have conducted extensive studies on targeting molecules, short Linker peptides, cytotoxic molecules, etc., and have unexpectedly found that when targeting cell-penetrating peptide iRGD is fused with SEC2 or its modified WWH or WWP or WWT in tumor immunotherapy drug superantigen to match with a specific rigid short Linker peptide (in constructing fusion proteins, a key issue is Linker of the short Linker peptide between two proteins, i.e., the length of the Linker peptide is very important for the folding and stability of the proteins).
In order to solve the above technical problems, a first aspect of the present invention provides a fusion protein, which comprises, from N-terminus to C-terminus: the peptide-based artificial breeding method comprises SEC2 or a mutant thereof, a connecting short peptide and iRGD, wherein the mutation comprises deletion, insertion and/or substitution of 1-5 amino acids, the amino acid sequence of SEC2 is shown as SEQ ID NO. 31, the amino acid sequence of the connecting short peptide is shown as SEQ ID NO. 29, and the amino acid sequence of the iRGD is shown as SEQ ID NO. 30.
Preferably, the mutant has mutation at amino acid residues 102-106 of the sequence shown as SEQ ID NO. 31; preferably, the mutation is that the 102 th to 106 th amino acid residues GKVTG of the sequence shown as SEQ ID NO. 31 are mutated into WWX; more preferably, WWX is WWH, WWP or WWT, that is, the amino acid sequence of the mutant is shown as the 1 st to 237 th positions of SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 6.
In the present invention, X in the above-mentioned WWX is used merely as a reference, and it may refer to any one amino acid. In addition, the amino acid abbreviations in the present invention have the ordinary meaning in the art unless otherwise specified.
Preferably, the amino acid sequence of the fusion protein is shown as SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6 or SEQ ID NO 12 in the sequence table; more preferably, the nucleotide sequence for coding the fusion protein is shown as SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 11 in the sequence table.
In order to solve the above technical problems, the second aspect of the present invention provides a fusion gene encoding the fusion protein according to the first aspect of the present invention.
Preferably, the nucleotide sequence is shown as SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 11 in the sequence table.
In order to solve the above technical problems, the third aspect of the present invention provides a recombinant expression vector comprising the fusion gene according to the second aspect of the present invention.
Preferably, the skeleton vector of the recombinant expression vector is pET-28 a-TEV.
In order to solve the above-mentioned problems, the fourth aspect of the present invention provides a transformant in which the fusion gene according to the second aspect of the present invention or the recombinant expression vector according to the third aspect of the present invention is introduced into a host.
Preferably, the host is escherichia coli, preferably escherichia coli e.coli BL21(DE3) cells or e.coli TG 1.
In order to solve the above technical problems, a fifth aspect of the present invention provides a method for preparing a fusion protein, comprising the steps of:
(1) obtaining a transformant according to the fourth aspect of the present invention;
(2) screening the transformants, expressing and purifying the fusion protein.
In the step (2), the purification preferably comprises subjecting the expressed thallus to ultrasonication, centrifuging, collecting supernatant, and performing Ni affinity chromatography twice to obtain the soluble fusion protein with biological activity. In general, after two times of Ni affinity chromatography, only the solution after the column penetration of the sample is collected to obtain the fusion protein.
Preferably, the Ni affinity chromatography comprises loading the sample on a pre-equilibrated Ni affinity chromatography column at a loading rate of 0.2-0.8ml/min, washing with 8-12 column volumes of equilibration buffer (to wash away non-specifically bound hetero-proteins) and then eluting with elution buffer, preferably 10 column volumes of equilibration buffer;
more preferably, the equilibration buffer is an equilibration buffer containing 20-80mM imidazole, and the composition of the equilibration buffer is preferably as follows: 20-30mM Tirs-HCl, 800-; and/or the elution buffer is an elution buffer containing 250-300mM imidazole, and the composition of the elution buffer is preferably as follows: 20-30mM Tirs-HCl, 800-; and/or the pH value of the elution buffer solution is 7.2-8.0.
Preferably, the method also comprises a step of ultrafiltration desalination between the two times of Ni affinity chromatography; more preferably, the method also comprises a step of mixing and enzyme digestion with TEV protease after the ultrafiltration desalination; more preferably, the molar ratio of the product after ultrafiltration desalination to the TEV protease is 1: 5; and/or the enzyme digestion time is 24 hours.
In order to solve the above technical problems, a sixth aspect of the present invention provides a fusion protein according to the first aspect of the present invention, a fusion gene according to the second aspect of the present invention, a recombinant expression vector according to the third aspect of the present invention, or a transformant according to the fourth aspect of the present invention for use in the preparation of a medicament; preferably the application in preparing the medicine for treating the tumor, and more preferably the application in preparing the medicine for treating tumor immunity.
In the invention, the modified WWH of SEC2 refers to SAg WWH isomer (WWH is shown in SEQ ID NO:31 for short in the invention) of WWH changed from 102 th to 106 th GKVTG amino acid residues of super antigen protein staphylococcus aureus enterotoxin C2 (Staphylococcus enterotoxin C2, SEC2, amino acid sequence is shown in SEQ ID NO: 31). Similarly, the modified WWP refers to SAg WWP isomer (WWP for short) obtained by changing GKVTG amino acid residues at positions 102-106 of SEC2 into WWP. The modified WWT refers to SAg WWT isomer (WWT for short) in which GKVTG amino acid residues at positions 102-106 of SEC2 are changed into WWT. These three modifications are also referred to in patent application CN201110077088.3, which is incorporated herein by reference in its entirety. The superantigen (SAg) is a protein molecule which can generate strong immune activation on T lymphocytes at a very low concentration, and can be combined with MHC II (histocompatibility complex) molecules and T cell V beta regions in an antigen binding region outside an antigen presenting cell to form a complex, so that a large amount of T lymphocytes are activated to proliferate, and a large amount of cytokines and other effector molecules are released in vitro or in vivo.
In the present invention, the "fusion gene" refers to a gene in which two or more nucleotide sequences derived from different sources are linked, or a gene in which two or more nucleotide sequences derived from the same source but not linked to each other in their natural positions are linked. The protein encoded by the fusion gene of the present invention is referred to as a fusion protein.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the invention connects the targeting cell-penetrating peptide and the super antigen for the first time to form targeting fusion protein SAg-L-iRGD (super antigen-connecting short peptide-iRGD), which can specifically target and infiltrate into the microenvironment of tumor tissues with high expression of integrin alphav and NRP-1, greatly improve the tumor specificity and vascular permeability of the super antigen, enhance the local enrichment of the super antigen in the tumor, and obviously inhibit the growth of tumor cells, thereby improving the anti-tumor effect, the required dosage can be reduced when the invention is applied to clinical treatment, namely, a better tumor inhibition effect can be obtained by using a lower dosage, and the lower dosage can bring lower toxicity, thereby reducing the side effect in the using process of the medicine; in addition, the improvement of tumor specificity can enable drug molecules to be more concentrated in a tumor area, and the distribution quantity of other non-tumor areas and organs is reduced, so that the toxicity is also reduced, and the survival capability of a patient is improved. In a preferred embodiment of the present invention, the ability of the fusion protein of the present invention to bind tumor cells is improved by more than 3.92 times compared with the ability of the fusion protein before fusion, and the tumor inhibition rate is as high as 63.3%, and when the fusion protein of the present invention is applied to mice with melanoma, the average survival days of the mice are significantly higher than those of the control group. The tumor inhibition effect of the fusion protein is obviously superior to the effect of simply mixing the targeting cell-penetrating peptide and the superantigen, namely the tumor inhibition effect achieves the effect of 1+1> 2.
Drawings
FIG. 1 shows the results of 1.0% agarose gel electrophoresis analysis of the restriction enzyme validation for the construction of pET-28a-tev-wwh-epapkp-irgd, wherein: 1 is lambda-EcoT 14I/BglII digest DNA marker; 2 is pET-28a-tev-wwh-epapkp-irgd plasmid which is not cut by enzyme; 3 and 4 are respectively pET-28a-tev-wwh-epapkp-irgd plasmid subjected to single enzyme digestion by EcoRI and XhoI; 5 is pET-28a-tev-wwh-epapkp-irgd plasmid subjected to double enzyme digestion by EcoR I and XhoI; 6 is DL2000DNA molecular weight standard.
FIG. 2 is a 10% SDS-PAGE electrophoretic analysis of expression before and after induction of pET-28a-tev-wwh-epapkp-irgd, wherein: 1 is the expression quantity of thallus holoprotein before induction; 2 is the thallus holoprotein expression quantity after being induced for 4 hours at 30 ℃; 3, after the thalli are crushed and centrifuged after being induced for 4 hours at 30 ℃, the expression quantity of soluble protein in supernatant fluid is obtained; 4. after the thalli is broken after being induced for 4 hours at 30 ℃, the total protein expression quantity is obtained; 5 is a protein Marker; 6 is the expression quantity of the whole protein of the thalli before induction; 7 is the thallus holoprotein expression quantity after being induced for 4 hours at 37 ℃; 8 is the soluble protein expression quantity in supernatant after the thalli are crushed and centrifuged after being induced for 4 hours at 37 ℃; 9 is total protein expression amount after the thalli are broken after being induced for 4 hours at 37 ℃.
FIG. 3 is 12% SDS-PAGE electrophoretic analysis chart of soluble expressed cell-penetrating peptide-superantigen fusion protein WWH-EPAPKP-iRGD after two purifications with AKTANI column, wherein: m is 180KD protein Marker; 1 is a secondarily purified fusion protein which is purified by Ni column chromatography, dialyzed and desalted, and then subjected to enzyme digestion by TEV protease to remove a label, wherein the concentration is 300 ng/mu L; 2 is fusion protein which is purified by Ni column chromatography and dialyzed to remove salt, and the concentration is (300 ng/. mu.L).
FIG. 4 shows fusion proteins WWH-EPAPKP-RGD, WWH-EPAPKP-tLyp-1, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, WWH-GGGGS (G)4S) -iRGD, WWH- (GS)5-iRGD, WWH-EPAPK-iRGD, iRGD-EPAPKP-WWH and WWH in vitro binding alpha v+And NRP-1+Experimental results for mouse melanoma cell B16F 10.
FIG. 5 shows fusion proteins WWH-EPAPKP-RGD, WWH-EPAPKP-tLyp-1, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, WWH-GGGGS (G)4S) -iRGD, WWH- (GS)5-iRGD, WWH-EPAPK-iRGD, iRGD-EPAPKP-WWH and WWH in vitro binding alpha v+And NRP-1+Experimental results for mouse breast cancer cell 4T 1.
FIG. 6 shows fusion proteins WWH-EPAPKP-RGD, WWH-EPAPKP-tLyp-1, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, iRGD-EPAPKP-WWH, WWH-GGGGS (G-APKP-RGD), and4s) -iRGD, WWH- (GS)5-iRGD, WWH-EPAPK-iRGD, sTRAIL-EPAPKP-iRGD and WWH + iRGD, BSA, iRGD, WWH, WWP and WWT inhibit alpha v in vitro+And NRP-1+Experimental results of mouse melanoma cell microspheres B16F 10.
FIG. 7 shows fusion proteins WWH-EPAPKP-RGD, WWH-EPAPKP-tLyp-1, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, iRGD-EPAPKP-WWH, WWH-GGGGS (G-APKP-RGD), and4s) -iRGD, WWH- (GS)5-iRGD, WWH-EPAPK-iRGD, sTRAIL-EPAPKP-iRGD and WWH + iRGD, BSA, iRGD, WWH, WWP and WWT inhibit alpha v in vitro+And NRP-1+Experimental results of mouse breast cancer cell microspheres 4T 1.
FIG. 8 shows the in vivo inhibition of α v by fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD and physiological saline, WWH and WWH + iRGD+And NRP-1+Experimental results for mouse melanoma cell B16F 10.
FIG. 9 shows the in vivo inhibition of α v by fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD and physiological saline, WWH and WWH + iRGD+And NRP-1+Experimental results for mouse breast cancer cell 4T 1.
FIG. 10 shows fusionProteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, SEA-EPAPKP-iRGD, SEB-EPAPKP-iRGD, SEC2-EPAPKP-iRGD and PBS and WWH inhibit alpha v in vivo+And NRP-1+Survival curve of mouse melanoma cell B16F 10.
FIG. 11 shows the in vivo inhibition of α v by fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, SEA-EPAPKP-iRGD, SEB-EPAPKP-iRGD, SEC2-EPAPKP-iRGD and PBS and WWH+And NRP-1+Survival curves for mouse breast cancer cells 4T 1.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
DNA encoding the base sequences of all protein molecules in the following examples was synthesized by Beijing Huada Gene Co.
Example 1
1. Fusion protein WWH-EPAPKP-iRGD gene WWH-EPAPKP-iRGD, which has the base sequence shown in SEQ ID NO:1 in Table 1, wherein WWH (WWH refers to the Sag WWH modified body of super antigen protein staphylococcus aureus enterotoxin C2 (Staphyloccal enterotoxin C2, SEC2, amino acid sequence is shown as the sequence shown in SEQ ID NO: 31) with 102 th to 106 th GKVTG amino acid residues mutated into WWH, the following and the description drawings are both referred to as WWH) coding gene WWH has the base sequence shown in 1 st to 711 th of SEQ ID NO:1, iRGD coding gene iRGD has the base sequence shown in 730 th to 756 th of SEQ ID NO:1, and LinkerEPAPKP has the base sequence shown in 712 to 729 th of SEQ ID NO:1 through DNA coding connecting short peptide.
TABLE 1
Figure BDA0002199368460000071
Note: the bold underlined sequence is the superantigen modifier sequence, the bold sequence is the linker sequence, and the underlined sequence is the targeting molecule sequence iRGD.
(1) Information of SEQ ID NO:1 (see sequence Listing)
(a) Sequence characteristics:
length: 756bp
Type (2): nucleic acids
Chain type: double chain
Topological structure: linearity
(b) Molecular type: cDNA
(c) Suppose that: whether or not
(d) Antisense: whether or not
(2) Preparation of fusion Gene wwh-epapkp-irgd:
(a) PCR primer design and reaction conditions: a forward primer (synthesized by Beijing Huada) was designed based on the multiple cloning site of pET28a vector (purchased from Novagen) in combination with the above-mentioned gag isomer WWH, and a reverse primer (synthesized by Beijing Huada) was designed from epapkp-ird gene sequence for PCR:
forward primer (F): 5'-CGGAATTCGAGAGTCAACCAGACCC-3' (SEQ ID NO:27)
Reverse primer (R):
5’-CCCTCGAGTTAACAATCCGGACCTTTATCACCACGACAAGGTTTTGGCGCCGGTTCTCCATTCTTTGTTGTAAGGTGGACTTCTAT-3’(SEQ ID NO:28)
the PCR reaction system was (Pyrobest buffer, dNTP, Pyrobest DNA polymerase were purchased from TAKARA Corp.): 10 XPyrobest buffer 5. mu. L, dNTP 250. mu. mol, forward and reverse primers 25pmol each, 0.1. mu.g of plasmid DNA (shown by the nucleotide sequences from 1 ST to 711 th of SEQ ID NO:1, see also ST-1 in patent application CN 201110077088.3) containing the sag gene as a template, 2U of Pyrobest DNA polymerase, and a sterile ultrapure water-filled volume of 50. mu.L.
The PCR reaction conditions are as follows: the first stage is as follows: 95 ℃ for 5 min; and a second stage: 94 ℃, 55 s; 60 ℃ for 2 min; 72 ℃ for 2 min; a total of 30 cycles; and a third stage: 72 ℃ for 10 min.
(b) And (3) recovering a PCR product: the PCR amplification product is analyzed by 1.0% agarose gel electrophoresis, and a 753bp target band is recovered by cutting gel, and the operation method is carried out according to the instructions of the kit for recovering and purifying the gel of Jiangsukang century GmbH. Obtaining the product targeting cell-penetrating peptide-superantigen fusion gene wwh-epapkp-irgd.
The fusion protein WWH-EPAPKP-iRGD coded by the nucleotide shown in SEQ ID NO. 1 has an amino acid sequence shown in SEQ ID NO. 2 (see Table 1 specifically).
Wherein, the information of SEQ ID NO 2 is as follows
(a) Sequence characterization
Length: 252 residue
Type: amino acids
Chain type: single strand
Topology structure: linearity
(b) Molecular type: protein
(c) Suppose that: whether or not
(d) Antisense: whether or not
(e) The initial sources were: artificial sequences.
2. A fusion protein WWP-EPAPKP-iRGD gene WWP-EPAPKP-iRGD has a base sequence shown in SEQ ID NO:3 in Table 2, wherein a gene WWP encoding WWP (WWP refers to a Sag WWP mutant in which GKVTG amino acid residues at positions 102-106 of SEC2 are mutated into WWP, and WWP is abbreviated as WWP in the following and the attached drawings) has a base sequence from position 1 to position 711 of SEQ ID NO:3, and an iRGD encoding gene iRGD has a base sequence from position 730 to position 756 of SEQ ID NO:3, and has a base sequence from position 712 to position 729 of SEQ ID NO:3 through DNA Linker EPAPKP encoding a connecting short peptide.
TABLE 2
Figure BDA0002199368460000091
Note: the bold underlined sequence is the superantigen modifier sequence, the bold sequence is the linker sequence, and the underlined sequence is the targeting molecule sequence iRGD.
(1) Information of SEQ ID NO:3 (see Table 2)
(a) Sequence characteristics:
length: 756bp
Type (2): nucleic acids
Chain type: double chain
Topological structure: linearity
(b) Molecular type: cDNA
(c) Suppose that: whether or not
(d) Antisense: whether or not
(2) The fusion gene wwp-epapkp-irgd was prepared as described in section 1 above (only the template was different), and the template was also referred to the sequence shown in ST-3 of patent application CN 201110077088.3. The fusion protein WWP-EPAPKP-iRGD coded by the nucleotide shown in SEQ ID NO. 3 has the amino acid sequence shown in SEQ ID NO. 4 (see Table 2 specifically). Wherein, the information of SEQ ID NO. 4 is as follows:
(a) sequence characterization
Length: 252 residue
Type: amino acids
Chain type: single strand
Topology structure: linearity
(b) Molecular type: protein
(c) Suppose that: whether or not
(d) Antisense: whether or not
(e) The initial sources were: artificial sequences.
3. A fusion protein WWT-EPAPKP-iRGD gene WWT-EPAPKP-iRGD has a base sequence shown in SEQ ID NO:5 in Table 3, wherein a gene WWT encoding WWT (WWT refers to a Sag WWT mutant in which GKVTG amino acid residues at positions 102-106 of SEC2 are mutated into WWT, and WWT is abbreviated as WWT in the following and the attached drawings) has a base sequence from position 1 to position 711 of SEQ ID NO:5, and an iRGD encoding gene iRGD has a base sequence from position 730 to position 756 of SEQ ID NO:5, and has a base sequence from position 712 to position 729 of SEQ ID NO:5 through DNA Linker EPAPKP encoding a connecting short peptide.
TABLE 3
Figure BDA0002199368460000101
Note: the bold underlined sequence is the superantigen modifier sequence, the bold sequence is the linker sequence, and the underlined sequence is the targeting molecule sequence iRGD.
(1) Information of SEQ ID NO:5 (see sequence Listing)
(a) Sequence characteristics:
length: 756bp
Type (2): nucleic acids
Chain type: double chain
Topological structure: linearity
(b) Molecular type: cDNA
(c) Suppose that: whether or not
(d) Antisense: whether or not
(2) Preparation of fusion Gene wwt-epapkp-irgd in section 1 above (template only), the template can also be referred to the sequence shown in ST-2 in patent application CN 201110077088.3. The fusion protein WWT-EPAPKP-iRGD coded by the nucleotide shown in SEQ ID NO. 5 has the amino acid sequence shown in SEQ ID NO. 6 (see Table 3 specifically). Wherein, the information of SEQ ID NO 6 is as follows:
(a) sequence characterization
Length: 252 residue
Type: amino acids
Chain type: single strand
Topology structure: linearity
(b) Molecular type: protein
(c) Suppose that: whether or not
(d) Antisense: whether or not
(e) The initial sources were: artificial sequences.
Example 2
The three targeting cell-penetrating peptide-superantigen fusion genes wwh-L-iRGD (i.e. wwh-epapkp-iRGD), wwp-L-iRGD (i.e. wwp-epapkp-iRGD) and wwt-L-iRGD (i.e. wwt-epapkp-iRGD) prepared in example 1 are connected to a prokaryotic expression vector pET-28a-TEV, so as to realize the expression of the targeting cell-penetrating peptide-superantigen fusion protein SAg-L-iRGD in Escherichia coli, specifically:
the fusion genes sag-l-irgd (i.e., wwh-l-irgd, wwp-l-irgd, wwwt-l-irgd described above) were ligated into expression vector pET-28a-TEV (available from Novagen Co.): plasmid DNA of an expression vector pET-28a-TEV and a gene DNA fragment of sag-l-irgd are subjected to double enzyme digestion by EcoRI (purchased from Dalian Bio Inc.) and XhoI (purchased from Dalian Bio Inc.), respectively, the plasmid DNA and the gene DNA fragment of sag-l-irgd are subjected to electrophoresis by 1.0% agarose gel, the sag-l-irgd fragment and the DNA large fragment of the plasmid pET-28a-TEV are recovered by gel electrophoresis, and the fragments are connected overnight at 16 ℃ by T4DNA ligase (purchased from Dalian Bio Inc) to construct a targeting transmembrane peptide-superantigen fusion protein expression vector pET28 a-TEV-sag-l-irgd. The ligation product was transformed into E.coli DH 5. alpha. competent cells (purchased from Dalibao Biopsis). Transformants were selected for kanamycin resistance (purchased from Sigma), recombinant monoclonals were selected for expansion, plasmid DNA was extracted, and correct recombinant clones were identified by double restriction with EcoR I and XhoI (fig. 1). And the recombinant clone plasmid which is verified to be correct by double enzyme digestion is sent to Shanghai bio-chemical company for sequencing. The correctly sequenced plasmids were transformed into competent cells of E.coli BL21(DE3) (from Beijing Tiangen Biochemical technology).
(1) Expression of fusion proteins SAg-L-iRGD (i.e., WWH-EPAPKP-iRGD), WWP-L-iRGD (i.e., WWP-EPAPKP-iRGD) and WWT-L-iRGD (i.e., WWT-EPAPKP-iRGD) A single colony of BL21(DE3) inoculated with the above-mentioned transformed recombinant plasmid pET28 a-TEV-SAg-L-irdd was inoculated in liquid LB of kanamycin 60 μ g/ml overnight at 37 ℃ and transferred to the next generation on the following day at a volume ratio of 1:100 (volume ratio), cultured at 37 ℃ until OD600 is 0.8, and introduced at final concentration of 10mM IPTG (purchased from Sigma) at 30 ℃ and 37 ℃ for 4 h.
(2) Collecting the supernatant containing the fusion protein: the cells after induction expression were collected by centrifugation, the cells per 100ml of the original culture were resuspended in 10ml of equilibration buffer (20mM Tris-HCl, 500mM NaCl, 50mM imidazole, pH 7.9), sonicated at 0 ℃ until the cells became clear, and centrifuged at 100000rpm for 10min at an ultra high speed (10 ten thousand rpm was used to remove impurities such as nucleic acids and cell debris, which is advantageous for subsequent purification treatment, and the 10 ten thousand rpm was found not to decrease the yield of protein). The supernatant was collected.
(3) The supernatants before and after centrifugation were taken as samples of whole cell protein after induction and whole cell soluble protein after induction, and the soluble expression level was analyzed by 12% SDS-PAGE, and the results are shown in FIG. 2. As can be seen in the figure, the location of target protein expression has been marked with a white box, and significant target protein expression can be seen; in addition, the target protein expressed under the condition of 37 degrees has more soluble components (namely, the target protein in the supernatant after the disruption and centrifugation is more). Here, WWH-L-iRGD is taken as an example, and the results are similar for the other two fusion proteins.
(4) The fusion protein was purified using AKTA purificator (product of GE, USA): the centrifuged supernatant was applied (application rate 0.2-0.8ml/min) to an AKTANI affinity chromatography column (GE, USA), and the column was rinsed with ten column volumes of equilibration buffer (20mM drugs-HCl, 500mM NaCl, 50mM imidazole, pH 7.9) until the UV detection value was stable. Finally, the target protein was eluted using elution buffer (20mM peptides-HCl, 500mM NaCl, 250mM imidazole, pH 7.9) and collected after the UV detection value started to rise until UV plateaus. The collected TEV-SAg-L-iRGD protein eluate was dialyzed to remove salts and analyzed for purity by SDS-PAGE, as shown in lane 2 of FIG. 3.
(5) Mixing the dialyzed fusion protein TEV-SAg-L-iRGD with TEV protease (the used pET28a vector is self-provided with a His-tag purification tag, so that the fusion protein TEV-SAg-L-iRGD is subjected to affinity tag cleavage) in a molar ratio of 1:5, carrying out enzyme digestion for 24h, loading the mixed system on a Ni column of an AKTA (alkyl ketene dimer) purifier, collecting a first UV peak during loading, namely the fusion protein SAg-L-iRGD, dialyzing to remove salt, and analyzing the purity by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), wherein the result is shown in a No. 1 lane and is higher than that of a No. 2 lane shown in figure 3.
Example 3 tumor targeting study of Targeted cell-penetrating peptide-superantigen fusion protein SAg-L-iRGD
Western blot was selected to verify that murine melanoma cells B16F10 and murine breast cancer cells 4T1, which highly express integrin alphav and NRP-1, were used as target cells (the cells used were purchased from "cell bank of the culture Collection of type culture of Chinese academy of sciences").
The targeting verification of fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD: fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD and superantigen WWH were labeled with the fluorescent dye AlexaFlour 647 (available from thermo Co.), respectively, target cells B16F10 and 4T1 were fixed, mixed with the above-mentioned fluorescent labeled proteins, incubated for 20min, centrifuged at 1000g for 5min, and the supernatant was removed. Resuspend with PBS and centrifuge. After resuspending again with 200. mu.l of PBS solution, detection was performed by flow cytometry. The experimental results are shown in fig. 4 and 5, which show that fluorescence labeled WWH-epakp-iRGD, WWP-epakp-iRGD and WWT-epakp-iRGD can generate strong target binding effect on target cells, the binding ability is obviously stronger than that of the control group, and the binding strength between the three is almost the same.
Wherein, the capacity of WWH-EPAPKP-iRGD for combining B16F10 cells is 4.26 times of that of WWH, the capacity of WWP-EPAPKP-iRGD for combining B16F10 cells is 4.49 times of that of WWH, and the capacity of WWT-EPAPKP-iRGD for combining B16F10 cells is 4.41 times of that of WWH.
The ability of WWH-EPAPKP-iRGD to bind to 4T1 cells is 4.02 times that of WWH, the ability of WWP-EPAPKP-iRGD to bind to 4T1 cells is 3.92 times that of WWH, and the ability of WWT-EPAPKP-iRGD to bind to 4T1 cells is 3.97 times that of WWH.
Example 4 in vitro anti-tumor Activity study of Targeted cell-penetrating peptide-superantigen fusion protein SAg-L-iRGD
In vitro anti-tumor activity verification of fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD: B16F10 and 4T1 cells at 1X 104cells/well were added to a U-shaped cell culture plate (model 4515 Corning USA), and after 48h of culture to form stable tumor microspheres, the isolated individual mouse splenocytes were added at a 1:10 effective target ratio. Then, the experimental group fusion proteins a, WWH-EPAPKP-iRGD, b, WWP-EPAPKP-iRGD, C, WWT-EPAPKP-iRGD, d, WWH, WWT were used individually, e, iRGD-EPAPKP-WWH (the difference from a is that the direction of the fusion proteins is opposite, i RGD, EPAPKP and mutant WWH are from N end to C end, f, iRGD, g, iRGD + WWH are mixed and combined (i.e. WWH + iRGD group), the same substance amount of 350pmol/μ l is added into each hole, and blank control holes (only adding culture medium RPMI-1640, product of Gibco company, USA), tumor cell control holes (only adding tumor cells) are arranged, 3 multiple holes are arranged. Wells were also prepared using bovine serum albumin BSA (purchased from Sigma) as a negative control. According to the conventional conditions (37 ℃, 5% CO)2Concentration) was added to each well after 48 hours of culture, 100. mu.l of Cell-tilterGlo 3D Cell viability assay reagent solution (purchased from Promega). After standing at room temperature for 25min, bioluminescence in each well was detected using a microplate reader.
Tumor inhibition rate (Tumor growth inhibition,%) 100- [ (experimental well-blank control well)/(Tumor cell control well-blank control well) ] × 100.
The experimental results show (fig. 6 and 7):
at the concentration of 350 pmol/mul, the tumor inhibition effect of WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD on B16F10 is the most obvious, and the tumor inhibition effect respectively reaches 65.2%, 67.5% and 66.3%. Wherein WWH-EPAPKP-iRGD is increased by 29.9% compared with WWH, WWP-EPAPKP-iRGD is increased by 30.12% compared with WWP, WWT-EPAPKP-iRGD is increased by 28.7% compared with WWT, and the tumor inhibition rate is obviously enhanced.
At the concentration of 350 pmol/mul, the tumor inhibition effect of WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD on 4T1 is the most obvious, and the tumor inhibition effect respectively reaches 64.6%, 63.3% and 67.5%. Wherein WWH-EPAPKP-iRGD is improved by 29.3% compared with WWH, WWP-EPAPKP-iRGD is improved by 27.1% compared with WWP, WWT-EPAPKP-iRGD is improved by 32.8% compared with WWT, and the tumor inhibition rate is obviously enhanced.
In addition, more importantly, the experimental results aiming at the two cell strains show that the tumor inhibition rate of WWH-EPAPKP-iRGD is not only obviously higher than that of a BSA control group, an iRGD single-use group and a WWH single-use group, but also obviously higher than that of an iRGD + WWH combined treatment group, which indicates that the tumor inhibition effect of the fusion protein is obviously higher than that of the combined treatment effect of two molecules which are not fused together, and indicates that the fusion of the protein is very critical.
Example 5 in vivo anti-tumor solid tumor Activity study of Targeted cell-penetrating peptide-superantigen fusion protein SAg-L-iRGD
The activity of fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD in the body of a mouse against the tumor solid tumor is verified: subcutaneous inoculation 10 respectively6A single melanoma cell (B16F 10) was inoculated into the left and back of C57 mice (five weeks old, N.K.)6One breast cancer cell/mouse 4T1 was inoculated at the breast pad site of BALB/c mice (Experimental animals technology, Inc., Wei Tong Li, Beijing, five weeks old). When the tumor grows to about 100mm3Starting to administer drug via tail vein, setting treatment groups of a, WWH-EPAPKP-iRGD, b, WWP-EPAPKP-iRGD,wwt-EPAPKP-iRGD, d.wwh, e.irgd and WWH were combined in a mixed manner (i.e., WWH + iRGD group), and normal saline was used as a control treatment group at a dosing concentration of 70 pmol/one, once every three days, for six dosing. Tumor volume changes during dosing were recorded (fig. 8 and 9), and death endpoints of mice were recorded to plot survival curves (fig. 10 and 11). The results show that the fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD can generate specific target killing effect on solid tumors with high integrin alphav and NRP-1 expression, and the effect is obviously stronger than that of other experimental groups (d.WWH, e.iRGD and WWH are mixed and used) and control groups. The tumor suppression rates for each group at the dosing endpoint day16 are shown in tables 4 and 5:
TABLE 4 tumor suppression Rate of the respective fusion proteins in C57 mice modeled as B16F10
Figure BDA0002199368460000141
Figure BDA0002199368460000151
*Tumor inhibition rate (control tumor volume-experimental tumor volume)/control tumor volume 100%
TABLE 5 tumor suppression Rate of each fusion protein in BALB/c mice modeled with 4T1
Figure BDA0002199368460000152
*Tumor inhibition rate (control tumor volume-experimental tumor volume)/control tumor volume 100%
As can be seen from the table:
the tumor inhibition rate of WWH-EPAPKP-iRGD on B16F10 modeled C57 mice reaches 71.24%, the tumor inhibition rate of WWP-EPAPKP-iRGD on B16F10 modeled C57 mice reaches 70.61%, the tumor inhibition rate of WWT-EPAPKP-iRGD on B16F10 modeled C57 mice reaches 72.06%, the significance is higher than 19.65% of that of WWH group, and the significance is also higher than 33.68% of that of WWH + iRGD combined group.
The tumor inhibition rate of WWH-EPAPKP-iRGD on the BALB/c mouse modeled by 4T1 reaches 67.45%, the tumor inhibition rate of WWP-EPAPKP-iRGD on the BALB/c mouse modeled by 4T1 reaches 64.78%, the tumor inhibition rate of WWT-EPAPKP-iRGD on the BALB/c mouse modeled by 4T1 reaches 69.55%, the tumor inhibition rate is obviously higher than 22.73% of the WWH group, and is also obviously higher than 27.45% of the WWH + iRGD combined group.
The results of survival experiments in mice dosed with the drug are shown in fig. 10 and 11:
the average survival days of the mice molded by the fusion protein WWH-EPAPKP-iRGD administration group B16F10 are 26.83 days, the average survival days of the mice molded by the WWP-EPAPKP-iRGD administration group B16F10 are 26.83 days, and the average survival days of the mice molded by the WWT-EPAPKP-iRGD administration group B16F10 are 26.50 days, which are obviously higher than that of the single SAg protein (22.33 days) and the control group (18.17 days).
The average survival days of the fusion protein WWH-EPAPKP-iRGD administration group 4T1 model-making mice are 57.50 days, the average survival days of the WWP-EPAPKP-iRGD administration group 4T1 model-making mice are 58.00 days, and the average survival days of the WWT-EPAPKP-iRGD administration group 4T1 model-making mice are 59.17 days, which are obviously higher than that of the single SAg protein (41.83 days) and the control group (35.67 days).
Comparative example:
(1) the invention synthesizes and expresses the following fusion proteins:
the coding base sequence DNAs of all protein molecules in the comparative example are synthesized by Beijing Huada Gene company, the construction of expression vectors, protein expression and purification, and protein quantification methods of all protein molecules are the same as those in example 2, and the activity detection of all protein molecules is the same as those in examples 3, 4, and 5.
The fusion protein SEA-EPAPKP-iRGD has the amino acid sequence shown in SEQ ID NO. 8 in the following table 6, and the coding gene SEA-EPAPKP-iRGD has the base sequence shown in SEQ ID NO. 7 in the following table 6.
TABLE 6
Figure BDA0002199368460000161
Note: the bold sequence is the linker sequence and the underlined sequence is the targeting molecule sequence iRGD.
The fusion protein SEB-EPAPKP-iRGD has an amino acid sequence shown in SEQ ID NO:10 in the following table 7, and a coding gene SEB-EPAPKP-iRGD thereof has a base sequence shown in SEQ ID NO:9 in the following table 7.
TABLE 7
Figure BDA0002199368460000171
Note: the bold sequence is the linker sequence and the underlined sequence is the targeting molecule sequence iRGD.
The fusion protein SEC2-EPAPKP-iRGD has the amino acid sequence shown in SEQ ID NO:12 in the following table 8, and the coding gene SEC2-EPAPKP-iRGD has the base sequence shown in SEQ ID NO:11 in the following table 8.
TABLE 8
Figure BDA0002199368460000172
Note: the bold sequence is the linker sequence and the underlined sequence is the targeting molecule sequence iRGD.
Fusion protein WWH-GGGGS (G)4S) -iRGD has the amino acid sequence in SEQ ID NO:14 in Table 9 below, and encodes gene wwh-ggggs-iRGD, having the base sequence in SEQ ID NO:13 in Table 9 below.
TABLE 9
Figure BDA0002199368460000181
Note: the bold underlined sequence is the superantigen modifier sequence, the bold sequence is the linker sequence, and the underlined sequence is the targeting molecule sequence iRGD.
Fusion protein WWH- (GS)5-iRGD has the amino acid sequence in SEQ ID NO 16 in Table 10 below, which codes for the gene wwh-gssgsgs-iRGD, having the sequence in Table 10 below15 in SEQ ID NO.
Watch 10
Figure BDA0002199368460000182
Note: the bold underlined sequence is the superantigen modifier sequence, the bold sequence is the linker sequence, and the underlined sequence is the targeting molecule sequence iRGD.
The fusion protein WWH-EPAPK-iRGD has an amino acid sequence shown in SEQ ID NO:18 in the following table 11, encodes a gene WWH-EPAPK-iRGD, and has a base sequence shown in SEQ ID NO:17 in the following table 11.
TABLE 11
Figure BDA0002199368460000191
Note: the bold underlined sequence is the superantigen modifier sequence, the bold sequence is the linker sequence, and the underlined sequence is the targeting molecule sequence iRGD.
The fusion protein iRGD-EPAPKP-WWH has an amino acid sequence shown in SEQ ID NO:20 in the following table 12, a coding gene iRGD-EPAPKP-WWH and a base sequence shown in SEQ ID NO:19 in the following table 12.
TABLE 12
Figure BDA0002199368460000192
Figure BDA0002199368460000201
Note: the bold sequence is the linker sequence and the underlined sequence is the targeting molecule sequence iRGD.
The fusion protein WWH-EPAPKP-RGD has an amino acid sequence shown in SEQ ID NO:22 in the following table 13, a coding gene WWH-EPAPKP-RGD, and a base sequence shown in SEQ ID NO:21 in the following table 13:
watch 13
Figure BDA0002199368460000202
Note: the bold sequence is the linker sequence and the underlined sequence is the targeting molecule sequence RGD.
The fusion protein WWH-EPAPKP-tLyp-1 has an amino acid sequence shown in SEQ ID NO:24 in the following table 14, and a coding gene WWH-EPAPKP-tLyp-1 thereof has a base sequence shown in SEQ ID NO:23 in the following table 14:
TABLE 14
Figure BDA0002199368460000203
Figure BDA0002199368460000211
Note: the bold sequence is the linker sequence and the underlined sequence is the targeting molecule sequence iRGD.
The fusion protein sTRAIL-EPAPKP-iRGD has an amino acid sequence shown in SEQ ID NO. 26 in the following table 15, and a coding gene strain-EPAPKP-iRGD thereof has a base sequence shown in SEQ ID NO. 25 in the following table 15:
watch 15
Figure BDA0002199368460000212
Note: the bold sequence is the linker sequence and the underlined sequence is the targeting molecule sequence iRGD.
(2) In order to compare the effects of using different short peptides targeting the same receptor, the present inventors constructed fusion proteins a.wwh-epappk-RGD, b.wwh-epappk-tllp-1, consisting of superantigens linked to target targeting molecules RGD (for target integrin α v) and tllp-1 (for target neuropilin NRP-1), wherein RGD had the ability to target integrin α v, and tllp-1 had the ability to target NRP-1 and enhance drug tumor tissue penetration by CendR. After three proteins are constructed and purified, the target verification experiment and the anti-tumor activity of the three proteins are respectively tested.
The results of the targeting verification experiment are shown in FIG. 4 and FIG. 5, which show that at the same concentration, the strength of WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD binding to target cells B16F10 and 4T1 is substantially the same, and the capacity of WWH-EPAPKP-iRGD binding to B16F10 cells is 2.84 times that of WWH-EPAPKP-RGD and 1.71 times that of WWH-EPAPKP-tLyp-1. The ability of WWH-EPAPKP-iRGD to bind to 4T1 cells is 2.24 times that of WWH-EPAPKP-RGD and 1.64 times that of WWH-EPAPKP-tLyp-1. It is demonstrated that three fusion proteins, namely WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD, have stronger binding force on target cells.
The experimental method for verifying the in vitro antitumor activity is the same as that in example 4, and the results are shown in FIG. 6 and FIG. 7, wherein at a concentration of 350 pmol/. mu.l, the growth inhibition abilities of WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD on target cell microspheres B16F10 and 4T1 are basically the same, and the tumor inhibition rate of WWH-EPAPKP-iRGD on B16F10 is increased by 25.1% compared with WWH-EPAPKP-RGD and by 23.1% compared with WWH-EPAPKP-tLyp-1. The tumor inhibition rate of WWH-EPAPKP-iRGD on 4T1 is increased by 24.5% compared with WWH-EPAPKP-RGD, and is increased by 22.5% compared with WWH-EPAPKP-tLyp-1. The comparison of the in-vitro tumor inhibition results shows that compared with other two targeting short peptides, the soluble fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD can better carry effector molecules to permeate into a tumor microenvironment to play a role in inhibition.
(3) The invention compares the targeting ability of fusion protein formed by different connection modes with the in vitro anti-tumor microsphere activity. In order to compare different connection modes of the iRGD connection short peptide and the SAg, a comparative example fusion protein iRGD-EPAPKP-WWH which connects the iRGD to the N end of the superantigen Sag is constructed based on WWH-EPAPKP-iRGD (the iRGD is at the C end of the Sag). To compare the difference between the linker of the present invention and other commonly used linker, G4S (a flexible linker, GGGGS), (GS) was constructed5(a flexible connecting short peptide) and EPAPK (rigid connecting short peptide with one less amino acid than EPAPKP) which are connected to form a fusion protein WWH-GGGGS (G)4S)-iRGD、WWH-(GS)5-iRGD, WWH-EPAPK-iRGD three fusion proteins. And the combination of targeting molecule iRGD and effector molecule WWH as two independent individuals (WWH + iRGD) was also used as a comparative example.
The results of the targeting verification performed in the same manner as in example 3 are shown in FIGS. 4 and 5, and the ability of WWH-EPAPKP-iRGD to bind to B16F10 cells at the same concentration is WWH-GGGGS (G)41.37 times of S) -iRGD, 1.42 times of WWH- (GS)5-iRGD, 1.42 times of WWH-EPAPK-iRGD and 3.00 times of iRGD-EPAPKP-WWH. The ability of WWH-EPAPKP-iRGD to bind to 4T1 cells is WWH-GGGGS (G)41.50 times of S) -iRGD, 1.80 times of WWH- (GS)5-iRGD, 1.52 times of WWH-EPAPK-iRGD and 2.69 times of iRGD-EPAPKP-WWH. These comparative data demonstrate that: compared with other linkers, the fusion protein formed by using EPAPKP as the Linker can better play the binding role of target cells; furthermore, linking iRGD to the C-terminus of the superantigen enables the fusion protein to better exert target cell binding effects than linking iRGD to the N-terminus of the superantigen.
The results of the in vitro antitumor microsphere experiments conducted in the same manner as in example 4 are shown in FIGS. 6 and 7, and at a concentration of 350 pmol/. mu.l, the inhibition rate of WWH-EPAPKP-iRGD on B16F10 was improved by 20.9% as compared with iRGD-EPAPKP-WWH, by 24.6% as compared with WWH + iRGD, and by WWH-GGGGS (G-GGGGS)4The S) -iRGD tumor inhibition rate is improved by 19.9 percent, is improved by 21 percent compared with the WWH- (GS)5-iRGD tumor inhibition rate, and is improved by 16 percent compared with the WWH-EPAPK-iRGD tumor inhibition rate; the tumor inhibition rate of WWH-EPAPKP-iRGD on 4T1 is increased by 19.4% compared with iRGD-EPAPKP-WWH, by 25.4% compared with WWH + iRGD, and compared with WWH-GGGGS (G)4The S) -iRGD tumor inhibition rate is improved by 18.3 percent, is improved by 19 percent compared with the WWH- (GS)5-iRGD tumor inhibition rate, and is improved by 17.4 percent compared with the WWH-EPAPK-iRGD tumor inhibition rate. These comparative data demonstrate that: compared with other linkers, the fusion protein formed by using EPAPKP as the Linker can better play a role in inhibiting tumors; in addition, the attachment of iRGD to the C-terminus of superantigen enables the fusion protein to exert a better tumor-inhibiting effect than the attachment of iRGD to the N-terminus of superantigen.
(4) In order to compare the difference of the anti-tumor effect of the same connecting peptide and iRGD after connecting WWH, WWP and WWT with other anti-tumor effector molecules (including other super antigens), four fusion proteins SEA-EPAPKP-iRGD, SEB-EPAPKP-iRGD, SEC2-EPAPKP-iRGD and sTRAIL-EPAPKP-iRGD which are formed by connecting different effector molecules in the same connecting mode are constructed, and the biological activities are compared. The experimental procedure was as in example 5.
The results show that the fusion proteins SEA-EPAPKP-iRGD and SEB-EPAPKP-iRGD constructed by SEA and SEB have strong toxicity, and cause animal death in animal experiments (figure 10 and figure 11); the fusion protein SEC2-EPAPKP-iRGD constructed by SEC2 has no similar tumor inhibiting effect to the modified bodies WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD under the same dosage (figure 10 and figure 11).
The tumor inhibition effects of the fusion protein sTRAIL-EPAPKP-iRGD with similar molecular weight were selected for comparison, and the experimental method is the same as that of example 4, and the results are shown in FIG. 6 and FIG. 7, which show that the tumor inhibition rate of WWH-EPAPKP-iRGD to B16F10 is 52% higher than that of sTRAIL-EPAPKP-iRGD and that of 4T1 is 51.4% higher than that of sTRAIL-EPAPKP-iRGD under the same concentration (350pmol/μ l). It is demonstrated that the combined mode of fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD is changed into other types of effect molecules such as sTRAIL-EPAPKP-iRGD, and the same ideal effect cannot be shown.
The fusion proteins SEA-EPAPKP-iRGD, SEB-EPAPKP-iRGD, SEC2-EPAPKP-iRGD composed of the effector molecules replaced with the superantigens SEA, SEB, SEC2 and linked in the same manner, and the mouse survival curves obtained according to the experimental method consistent with example 5 are shown in FIG. 10 (P0 for wild SEC2 and P0.001 for wild SEC2 and SEB) and FIG. 11 (P0.001 for wild SEC2 and P0.001 for wild SEC2 and SEB) showing that the mice modelled by the fusion protein SEA-EPAPKP-iRGD group B16F 7 had an average survival time of 11.17 days, the mice modelled by the fusion protein SEA-EPAPKP-iRGD group B16F10 had an average survival time of 11.17 days, the fusion protein SEA-EPAPKP-iRGD group B16F10 had an average survival time of less than that of the mice dosed by the fusion protein PBS, and the fusion protein was found by the control group B16F 3517.18-APRGD (average survival time of the fusion protein of 3 days), the average survival time of the fusion protein WWH-EPAPKP-iRGD is 26.83 days, the average survival time of the WWP-EPAPKP-iRGD is 26.83 days, and the average survival time of the WWT-EPAPKP-iRGD is 26.50 days. The average survival days of the fusion protein SEA-EPAPKP-iRGD administration group 4T1 model-made mice were 25 days, the average survival days of the fusion protein SEB-EPAPKP-iRGD administration group 4T1 model-made mice were 25.83 days, which were lower than the average survival days of the control group (PBS) (35.67 days), while the average survival days of the fusion protein SEC2-EPAPKP-iRGD administration group 4T1 model-made mice were 47.5 days, the average survival days of the fusion protein WWH-EPAPKP-iRGD was 57.5 days, the average survival days of the WWP-EPAPKP-iRGD was 58 days, and the average survival days of the WWT-EPAPKP-iRGD was 59.17 days. The result shows that the fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD show ideal tumor inhibition effect, and have no universality on other superantigen or non-superantigen effector molecules.
SEQUENCE LISTING
<110> Shenyang application ecological research institute of Chinese academy of sciences
<120> fusion protein, preparation and application thereof
<130> P19012196C
<160> 31
<170> PatentIn version 3.5
<210> 1
<211> 756
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene wwh-epapkp-irgd
<400> 1
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtatggtggc atggtaaaac ttgtatgtat ggaggaataa caaaacatga aggaaaccac 360
tttgataatg ggaacttaca aaatgtactt ataagagttt atgaaaataa aagaaacaca 420
atttcttttg aagtgcaaac tgataagaaa agtgtaacag ctcaagaact agacataaaa 480
gctaggaatt ttttaattaa taaaaaaaat ttgtatgagt ttaacagttc accatatgaa 540
acaggatata taaaatttat tgaaaataac ggcaatactt tttggtatga tatgatgcct 600
gcaccaggcg ataagtttga ccaatctaaa tatttaatga tgtacaacga caataaaacg 660
gttgattcta aaagtgtgaa gatagaagtc caccttacaa caaagaatgg agaaccggcg 720
ccaaaacctt gtcgtggtga taaaggtccg gattgt 756
<210> 2
<211> 252
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein WWH-EPAPKP-iRGD
<400> 2
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Trp Trp His Gly Lys Thr Cys Met Tyr Gly Gly
100 105 110
Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln Asn
115 120 125
Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe Glu
130 135 140
Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile Lys
145 150 155 160
Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn Ser
165 170 175
Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly Asn
180 185 190
Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln
195 200 205
Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser Lys
210 215 220
Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu Pro Ala
225 230 235 240
Pro Lys Pro Cys Arg Gly Asp Lys Gly Pro Asp Cys
245 250
<210> 3
<211> 756
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene wwp-epapkp-irgd
<400> 3
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtatggtggc caggtaaaac ttgtatgtat ggaggaataa caaaacatga aggaaaccac 360
tttgataatg ggaacttaca aaatgtactt ataagagttt atgaaaataa aagaaacaca 420
atttcttttg aagtgcaaac tgataagaaa agtgtaacag ctcaagaact agacataaaa 480
gctaggaatt ttttaattaa taaaaaaaat ttgtatgagt ttaacagttc accatatgaa 540
acaggatata taaaatttat tgaaaataac ggcaatactt tttggtatga tatgatgcct 600
gcaccaggcg ataagtttga ccaatctaaa tatttaatga tgtacaacga caataaaacg 660
gttgattcta aaagtgtgaa gatagaagtc caccttacaa caaagaatgg agaaccggcg 720
ccaaaacctt gtcgtggtga taaaggtccg gattgt 756
<210> 4
<211> 252
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein WWP-EPAPKP-iRGD
<400> 4
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Trp Trp Pro Gly Lys Thr Cys Met Tyr Gly Gly
100 105 110
Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln Asn
115 120 125
Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe Glu
130 135 140
Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile Lys
145 150 155 160
Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn Ser
165 170 175
Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly Asn
180 185 190
Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln
195 200 205
Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser Lys
210 215 220
Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu Pro Ala
225 230 235 240
Pro Lys Pro Cys Arg Gly Asp Lys Gly Pro Asp Cys
245 250
<210> 5
<211> 756
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene wwt-epapkp-irgd
<400> 5
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtatggtgga caggtaaaac ttgtatgtat ggaggaataa caaaacatga aggaaaccac 360
tttgataatg ggaacttaca aaatgtactt ataagagttt atgaaaataa aagaaacaca 420
atttcttttg aagtgcaaac tgataagaaa agtgtaacag ctcaagaact agacataaaa 480
gctaggaatt ttttaattaa taaaaaaaat ttgtatgagt ttaacagttc accatatgaa 540
acaggatata taaaatttat tgaaaataac ggcaatactt tttggtatga tatgatgcct 600
gcaccaggcg ataagtttga ccaatctaaa tatttaatga tgtacaacga caataaaacg 660
gttgattcta aaagtgtgaa gatagaagtc caccttacaa caaagaatgg agaaccggcg 720
ccaaaacctt gtcgtggtga taaaggtccg gattgt 756
<210> 6
<211> 252
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein WWT-EPAPKP-iRGD
<400> 6
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Trp Trp Thr Gly Lys Thr Cys Met Tyr Gly Gly
100 105 110
Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln Asn
115 120 125
Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe Glu
130 135 140
Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile Lys
145 150 155 160
Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn Ser
165 170 175
Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly Asn
180 185 190
Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln
195 200 205
Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser Lys
210 215 220
Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu Pro Ala
225 230 235 240
Pro Lys Pro Cys Arg Gly Asp Lys Gly Pro Asp Cys
245 250
<210> 7
<211> 813
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene sea-epapkp-irgd
<400> 7
aaaaaaacag catttacatt acttttattc attgccctaa cgttgacaac aagtccactt 60
gtaaatggta gcgagaaaag cgaagaaata aatgaaaaag atttgcgaaa aaagtctgaa 120
ttgcagggaa cagctttagg caatcttaaa caaatctatt attacaatga aaaagctaaa 180
actgaaaata aagagagtca cgatcaattt ttacagcata ctatattgtt taaaggcttt 240
tttacagatc attcgtggta taacgattta ttagtagatt ttgattcaaa ggatattgtt 300
gataaatata aagggaaaaa agtagacttg tatggtgctt attatggtta tcaatgtgcg 360
ggtggtacac caaacaaaac agcttgtatg tatggtggtg taacgttaca tgataataat 420
cgattgaccg aagagaaaaa agtgccgatc aatttatggc tagacggtaa acaaaataca 480
gtacctttgg aaacggttaa aacgaataag aaaaatgtaa ctgttcagga gttggatctt 540
caagcaagac gttatttaca ggaaaaatat aatttatata actctgatgt ttttgatggg 600
aaggttcaga ggggattaat cgtgtttcat acttctacag aaccttcggt taattacgat 660
ttatttggtg ctcaaggaca gtattcaaat acactattaa gaatatatag agataataaa 720
acgattaact ctgaaaacat gcatattgat atatatttat atacaagtga accggcgcca 780
aaaccttgtc gtggtgataa aggtccggat tgt 813
<210> 8
<211> 271
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein SEA-EPAPKP-iRGD
<400> 8
Lys Lys Thr Ala Phe Thr Leu Leu Leu Phe Ile Ala Leu Thr Leu Thr
1 5 10 15
Thr Ser Pro Leu Val Asn Gly Ser Glu Lys Ser Glu Glu Ile Asn Glu
20 25 30
Lys Asp Leu Arg Lys Lys Ser Glu Leu Gln Gly Thr Ala Leu Gly Asn
35 40 45
Leu Lys Gln Ile Tyr Tyr Tyr Asn Glu Lys Ala Lys Thr Glu Asn Lys
50 55 60
Glu Ser His Asp Gln Phe Leu Gln His Thr Ile Leu Phe Lys Gly Phe
65 70 75 80
Phe Thr Asp His Ser Trp Tyr Asn Asp Leu Leu Val Asp Phe Asp Ser
85 90 95
Lys Asp Ile Val Asp Lys Tyr Lys Gly Lys Lys Val Asp Leu Tyr Gly
100 105 110
Ala Tyr Tyr Gly Tyr Gln Cys Ala Gly Gly Thr Pro Asn Lys Thr Ala
115 120 125
Cys Met Tyr Gly Gly Val Thr Leu His Asp Asn Asn Arg Leu Thr Glu
130 135 140
Glu Lys Lys Val Pro Ile Asn Leu Trp Leu Asp Gly Lys Gln Asn Thr
145 150 155 160
Val Pro Leu Glu Thr Val Lys Thr Asn Lys Lys Asn Val Thr Val Gln
165 170 175
Glu Leu Asp Leu Gln Ala Arg Arg Tyr Leu Gln Glu Lys Tyr Asn Leu
180 185 190
Tyr Asn Ser Asp Val Phe Asp Gly Lys Val Gln Arg Gly Leu Ile Val
195 200 205
Phe His Thr Ser Thr Glu Pro Ser Val Asn Tyr Asp Leu Phe Gly Ala
210 215 220
Gln Gly Gln Tyr Ser Asn Thr Leu Leu Arg Ile Tyr Arg Asp Asn Lys
225 230 235 240
Thr Ile Asn Ser Glu Asn Met His Ile Asp Ile Tyr Leu Tyr Thr Ser
245 250 255
Glu Pro Ala Pro Lys Pro Cys Arg Gly Asp Lys Gly Pro Asp Cys
260 265 270
<210> 9
<211> 840
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene seb-epapkp-irgd
<400> 9
tataagagat tatttatttc acatgtaatt ttgatattcg cactgatatt agttatttct 60
acacccaacg ttttagcaga gagtcaacca gatcctaaac cagatgagtt gcacaaatcg 120
agtaaattca ctggtttgat ggaaaatatg aaagttttgt atgatgataa tcatgtatca 180
gcaataaacg ttaaatctat agatcaattt ctatactttg acttaatata ttctattaag 240
gacactaagt tagggaatta tgataatgtt cgagtcgaat ttaaaaacaa agatttagct 300
gataaataca aagataaata cgtagatgtg tttggagcta attattatta tcaatgttat 360
ttttctaaaa aaacgaatga tattaattcg catcaaactg acaaacgaaa aacttgtatg 420
tatggtggtg taactgagca taatggaaac caattagata aatatagaag tattactgtt 480
cgggtatttg aagatggtaa aaatttatta tcttttgacg tacaaactaa taagaaaaag 540
gtgactgctc aagaattaga ttacctaact cgtcactatt tggtgaaaaa taaaaaactc 600
tatgaattta acaactcgcc ttatgaaacg ggatatatta aatttataga aaatgagaat 660
agcttttggt atgacatgat gcctgcacca ggagataaat ttgaccaatc taaatattta 720
atgatgtaca atgacaataa aatggttgat tctaaagatg tgaagattga agtttatctt 780
acgacaaaga aaaaggaacc ggcgccaaaa ccttgtcgtg gtgataaagg tccggattgt 840
<210> 10
<211> 280
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein SEB-EPAPKP-iRGD
<400> 10
Tyr Lys Arg Leu Phe Ile Ser His Val Ile Leu Ile Phe Ala Leu Ile
1 5 10 15
Leu Val Ile Ser Thr Pro Asn Val Leu Ala Glu Ser Gln Pro Asp Pro
20 25 30
Lys Pro Asp Glu Leu His Lys Ser Ser Lys Phe Thr Gly Leu Met Glu
35 40 45
Asn Met Lys Val Leu Tyr Asp Asp Asn His Val Ser Ala Ile Asn Val
50 55 60
Lys Ser Ile Asp Gln Phe Leu Tyr Phe Asp Leu Ile Tyr Ser Ile Lys
65 70 75 80
Asp Thr Lys Leu Gly Asn Tyr Asp Asn Val Arg Val Glu Phe Lys Asn
85 90 95
Lys Asp Leu Ala Asp Lys Tyr Lys Asp Lys Tyr Val Asp Val Phe Gly
100 105 110
Ala Asn Tyr Tyr Tyr Gln Cys Tyr Phe Ser Lys Lys Thr Asn Asp Ile
115 120 125
Asn Ser His Gln Thr Asp Lys Arg Lys Thr Cys Met Tyr Gly Gly Val
130 135 140
Thr Glu His Asn Gly Asn Gln Leu Asp Lys Tyr Arg Ser Ile Thr Val
145 150 155 160
Arg Val Phe Glu Asp Gly Lys Asn Leu Leu Ser Phe Asp Val Gln Thr
165 170 175
Asn Lys Lys Lys Val Thr Ala Gln Glu Leu Asp Tyr Leu Thr Arg His
180 185 190
Tyr Leu Val Lys Asn Lys Lys Leu Tyr Glu Phe Asn Asn Ser Pro Tyr
195 200 205
Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Glu Asn Ser Phe Trp Tyr
210 215 220
Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln Ser Lys Tyr Leu
225 230 235 240
Met Met Tyr Asn Asp Asn Lys Met Val Asp Ser Lys Asp Val Lys Ile
245 250 255
Glu Val Tyr Leu Thr Thr Lys Lys Lys Glu Pro Ala Pro Lys Pro Cys
260 265 270
Arg Gly Asp Lys Gly Pro Asp Cys
275 280
<210> 11
<211> 762
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene sec2-epapkp-irgd
<400> 11
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa acatgaagga 360
aaccactttg ataatgggaa cttacaaaat gtacttataa gagtttatga aaataaaaga 420
aacacaattt cttttgaagt gcaaactgat aagaaaagtg taacagctca agaactagac 480
ataaaagcta ggaatttttt aattaataaa aaaaatttgt atgagtttaa cagttcacca 540
tatgaaacag gatatataaa atttattgaa aataacggca atactttttg gtatgatatg 600
atgcctgcac caggcgataa gtttgaccaa tctaaatatt taatgatgta caacgacaat 660
aaaacggttg attctaaaag tgtgaagata gaagtccacc ttacaacaaa gaatggagaa 720
ccggcgccaa aaccttgtcg tggtgataaa ggtccggatt gt 762
<210> 12
<211> 254
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein SEC2-EPAPKP-iRGD
<400> 12
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu
225 230 235 240
Pro Ala Pro Lys Pro Cys Arg Gly Asp Lys Gly Pro Asp Cys
245 250
<210> 13
<211> 753
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene wwh-gggggs-irgd
<400> 13
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtatggtggc atggtaaaac ttgtatgtat ggaggaataa caaaacatga aggaaaccac 360
tttgataatg ggaacttaca aaatgtactt ataagagttt atgaaaataa aagaaacaca 420
atttcttttg aagtgcaaac tgataagaaa agtgtaacag ctcaagaact agacataaaa 480
gctaggaatt ttttaattaa taaaaaaaat ttgtatgagt ttaacagttc accatatgaa 540
acaggatata taaaatttat tgaaaataac ggcaatactt tttggtatga tatgatgcct 600
gcaccaggcg ataagtttga ccaatctaaa tatttaatga tgtacaacga caataaaacg 660
gttgattcta aaagtgtgaa gatagaagtc caccttacaa caaagaatgg aggtggcgga 720
ggttcatgtc gtggtgataa aggtccggat tgt 753
<210> 14
<211> 251
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein WWH-G4S-iRGD
<400> 14
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Trp Trp His Gly Lys Thr Cys Met Tyr Gly Gly
100 105 110
Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln Asn
115 120 125
Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe Glu
130 135 140
Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile Lys
145 150 155 160
Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn Ser
165 170 175
Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly Asn
180 185 190
Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln
195 200 205
Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser Lys
210 215 220
Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Gly Gly Gly
225 230 235 240
Gly Ser Cys Arg Gly Asp Lys Gly Pro Asp Cys
245 250
<210> 15
<211> 768
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene wwh-gsgssgs-irgd
<400> 15
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtatggtggc atggtaaaac ttgtatgtat ggaggaataa caaaacatga aggaaaccac 360
tttgataatg ggaacttaca aaatgtactt ataagagttt atgaaaataa aagaaacaca 420
atttcttttg aagtgcaaac tgataagaaa agtgtaacag ctcaagaact agacataaaa 480
gctaggaatt ttttaattaa taaaaaaaat ttgtatgagt ttaacagttc accatatgaa 540
acaggatata taaaatttat tgaaaataac ggcaatactt tttggtatga tatgatgcct 600
gcaccaggcg ataagtttga ccaatctaaa tatttaatga tgtacaacga caataaaacg 660
gttgattcta aaagtgtgaa gatagaagtc caccttacaa caaagaatgg aggttcaggc 720
tccggaagcg gttcaggttc ctgtcgtggt gataaaggtc cggattgt 768
<210> 16
<211> 256
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein WWH- (GS)5-iRGD
<400> 16
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Trp Trp His Gly Lys Thr Cys Met Tyr Gly Gly
100 105 110
Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln Asn
115 120 125
Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe Glu
130 135 140
Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile Lys
145 150 155 160
Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn Ser
165 170 175
Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly Asn
180 185 190
Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln
195 200 205
Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser Lys
210 215 220
Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Gly Ser Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Ser Cys Arg Gly Asp Lys Gly Pro Asp Cys
245 250 255
<210> 17
<211> 753
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene wwh-epapk-irgd
<400> 17
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtatggtggc atggtaaaac ttgtatgtat ggaggaataa caaaacatga aggaaaccac 360
tttgataatg ggaacttaca aaatgtactt ataagagttt atgaaaataa aagaaacaca 420
atttcttttg aagtgcaaac tgataagaaa agtgtaacag ctcaagaact agacataaaa 480
gctaggaatt ttttaattaa taaaaaaaat ttgtatgagt ttaacagttc accatatgaa 540
acaggatata taaaatttat tgaaaataac ggcaatactt tttggtatga tatgatgcct 600
gcaccaggcg ataagtttga ccaatctaaa tatttaatga tgtacaacga caataaaacg 660
gttgattcta aaagtgtgaa gatagaagtc caccttacaa caaagaatgg agaaccggcg 720
ccaaaatgtc gtggtgataa aggtccggat tgt 753
<210> 18
<211> 251
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein WWH-EPAPK-iRGD
<400> 18
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Trp Trp His Gly Lys Thr Cys Met Tyr Gly Gly
100 105 110
Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln Asn
115 120 125
Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe Glu
130 135 140
Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile Lys
145 150 155 160
Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn Ser
165 170 175
Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly Asn
180 185 190
Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln
195 200 205
Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser Lys
210 215 220
Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu Pro Ala
225 230 235 240
Pro Lys Cys Arg Gly Asp Lys Gly Pro Asp Cys
245 250
<210> 19
<211> 756
<212> DNA
<213> Artificial Sequence
<220>
<223> gene irgd-epapkp-wwh
<400> 19
tgtcgtggtg ataaaggtcc ggattgtgaa ccggcgccaa aacctgagag tcaaccagac 60
cctacgccag atgagttgca caaatcaagt gagtttactg gtacgatggg taatatgaaa 120
tatttatatg atgatcatta tgtatcagca actaaagtta tgtctgtaga taaatttttg 180
gcacatgatt taatttataa cattagtgat aaaaaactaa aaaattatga caaagtgaaa 240
acagagttat taaatgaaga tttagcaaag aagtacaaag atgaagtagt tgatgtgtat 300
ggatcaaatt actatgtaaa ctgctatttt tcatccaaag ataatgtatg gtggcatggt 360
aaaacttgta tgtatggagg aataacaaaa catgaaggaa accactttga taatgggaac 420
ttacaaaatg tacttataag agtttatgaa aataaaagaa acacaatttc ttttgaagtg 480
caaactgata agaaaagtgt aacagctcaa gaactagaca taaaagctag gaatttttta 540
attaataaaa aaaatttgta tgagtttaac agttcaccat atgaaacagg atatataaaa 600
tttattgaaa ataacggcaa tactttttgg tatgatatga tgcctgcacc aggcgataag 660
tttgaccaat ctaaatattt aatgatgtac aacgacaata aaacggttga ttctaaaagt 720
gtgaagatag aagtccacct tacaacaaag aatgga 756
<210> 20
<211> 252
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein iRGD-EPAPKP-WWH
<400> 20
Cys Arg Gly Asp Lys Gly Pro Asp Cys Glu Pro Ala Pro Lys Pro Glu
1 5 10 15
Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu Phe
20 25 30
Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr Val
35 40 45
Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp Leu
50 55 60
Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val Lys
65 70 75 80
Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu Val
85 90 95
Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser Ser
100 105 110
Lys Asp Asn Val Trp Trp His Gly Lys Thr Cys Met Tyr Gly Gly Ile
115 120 125
Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln Asn Val
130 135 140
Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe Glu Val
145 150 155 160
Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile Lys Ala
165 170 175
Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn Ser Ser
180 185 190
Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly Asn Thr
195 200 205
Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln Ser
210 215 220
Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser Lys Ser
225 230 235 240
Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly
245 250
<210> 21
<211> 738
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene wwh-epapkp-rgd
<400> 21
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtatggtggc atggtaaaac ttgtatgtat ggaggaataa caaaacatga aggaaaccac 360
tttgataatg ggaacttaca aaatgtactt ataagagttt atgaaaataa aagaaacaca 420
atttcttttg aagtgcaaac tgataagaaa agtgtaacag ctcaagaact agacataaaa 480
gctaggaatt ttttaattaa taaaaaaaat ttgtatgagt ttaacagttc accatatgaa 540
acaggatata taaaatttat tgaaaataac ggcaatactt tttggtatga tatgatgcct 600
gcaccaggcg ataagtttga ccaatctaaa tatttaatga tgtacaacga caataaaacg 660
gttgattcta aaagtgtgaa gatagaagtc caccttacaa caaagaatgg agaaccggcg 720
ccaaaacctc gcggcgat 738
<210> 22
<211> 246
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein WWH-EPAPKP-RGD
<400> 22
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Trp Trp His Gly Lys Thr Cys Met Tyr Gly Gly
100 105 110
Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln Asn
115 120 125
Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe Glu
130 135 140
Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile Lys
145 150 155 160
Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn Ser
165 170 175
Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly Asn
180 185 190
Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln
195 200 205
Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser Lys
210 215 220
Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu Pro Ala
225 230 235 240
Pro Lys Pro Arg Gly Asp
245
<210> 23
<211> 750
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene wwh-epapkp-tlyp-1
<400> 23
gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60
atgggtaata tgaaatattt atatgatgat cattatgtat cagcaactaa agttatgtct 120
gtagataaat ttttggcaca tgatttaatt tataacatta gtgataaaaa actaaaaaat 180
tatgacaaag tgaaaacaga gttattaaat gaagatttag caaagaagta caaagatgaa 240
gtagttgatg tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat 300
gtatggtggc atggtaaaac ttgtatgtat ggaggaataa caaaacatga aggaaaccac 360
tttgataatg ggaacttaca aaatgtactt ataagagttt atgaaaataa aagaaacaca 420
atttcttttg aagtgcaaac tgataagaaa agtgtaacag ctcaagaact agacataaaa 480
gctaggaatt ttttaattaa taaaaaaaat ttgtatgagt ttaacagttc accatatgaa 540
acaggatata taaaatttat tgaaaataac ggcaatactt tttggtatga tatgatgcct 600
gcaccaggcg ataagtttga ccaatctaaa tatttaatga tgtacaacga caataaaacg 660
gttgattcta aaagtgtgaa gatagaagtc caccttacaa caaagaatgg agaaccggcg 720
ccaaaacctt gcggcaacaa acgcacccgt 750
<210> 24
<211> 250
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein WWH-EPAPKP-tLyp-1
<400> 24
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Trp Trp His Gly Lys Thr Cys Met Tyr Gly Gly
100 105 110
Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu Gln Asn
115 120 125
Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser Phe Glu
130 135 140
Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp Ile Lys
145 150 155 160
Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe Asn Ser
165 170 175
Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly Asn
180 185 190
Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln
195 200 205
Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp Ser Lys
210 215 220
Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly Glu Pro Ala
225 230 235 240
Pro Lys Pro Cys Gly Asn Lys Arg Thr Arg
245 250
<210> 25
<211> 549
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene strain-epapkp-irgd
<400> 25
gtgagagaaa gaggtcctca gagagtagca gctcacataa ctgggaccag aggaagaagc 60
aacacattgt cttctccaaa ctccaagaat gaaaaggctc tgggccgcaa aataaactcc 120
tgggaatcat caaggagtgg gcattcattc ctgagcaact tgcacttgag gaatggtgaa 180
ctggtcatcc atgaaaaagg gttttactac atctattccc aaacatactt tcgatttcag 240
gaggaaataa aagaaaacac aaagaacgac aaacaaatgg tccaatatat ttacaaatac 300
acaagttatc ctgaccctat attgttgatg aaaagtgcta gaaatagttg ttggtctaaa 360
gatgcagaat atggactcta ttccatctat caagggggaa tatttgagct taaggaaaat 420
gacagaattt ttgtttctgt aacaaatgag cacttgatag acatggacca tgaagccagt 480
tttttcgggg cctttttagt tggcgaaccg gcgccaaaac cttgtcgtgg tgataaaggt 540
ccggattgt 549
<210> 26
<211> 183
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein sTRAIL-EPAPKP-iRGD
<400> 26
Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly Thr
1 5 10 15
Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys
20 25 30
Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His
35 40 45
Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His
50 55 60
Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln
65 70 75 80
Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr
85 90 95
Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser
100 105 110
Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser
115 120 125
Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe
130 135 140
Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser
145 150 155 160
Phe Phe Gly Ala Phe Leu Val Gly Glu Pro Ala Pro Lys Pro Cys Arg
165 170 175
Gly Asp Lys Gly Pro Asp Cys
180
<210> 27
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Forward primer (F)
<400> 27
cggaattcga gagtcaacca gaccc 25
<210> 28
<211> 86
<212> DNA
<213> Artificial Sequence
<220>
<223> reverse primer (R)
<400> 28
ccctcgagtt aacaatccgg acctttatca ccacgacaag gttttggcgc cggttctcca 60
ttctttgttg taaggtggac ttctat 86
<210> 29
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> connecting short peptide
<400> 29
Glu Pro Ala Pro Lys Pro
1 5
<210> 30
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> iRGD
<400> 30
Cys Arg Gly Asp Lys Gly Pro Asp Cys
1 5
<210> 31
<211> 239
<212> PRT
<213> Staphylococcus aureus
<400> 31
Glu Ser Gln Pro Asp Pro Thr Pro Asp Glu Leu His Lys Ser Ser Glu
1 5 10 15
Phe Thr Gly Thr Met Gly Asn Met Lys Tyr Leu Tyr Asp Asp His Tyr
20 25 30
Val Ser Ala Thr Lys Val Met Ser Val Asp Lys Phe Leu Ala His Asp
35 40 45
Leu Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys Val
50 55 60
Lys Thr Glu Leu Leu Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp Glu
65 70 75 80
Val Val Asp Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys Tyr Phe Ser
85 90 95
Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys Met Tyr
100 105 110
Gly Gly Ile Thr Lys His Glu Gly Asn His Phe Asp Asn Gly Asn Leu
115 120 125
Gln Asn Val Leu Ile Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile Ser
130 135 140
Phe Glu Val Gln Thr Asp Lys Lys Ser Val Thr Ala Gln Glu Leu Asp
145 150 155 160
Ile Lys Ala Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu Tyr Glu Phe
165 170 175
Asn Ser Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Asn
180 185 190
Gly Asn Thr Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe
195 200 205
Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Thr Val Asp
210 215 220
Ser Lys Ser Val Lys Ile Glu Val His Leu Thr Thr Lys Asn Gly
225 230 235

Claims (10)

1. A fusion protein comprising, in order from N-terminus to C-terminus: SEC2 or its mutant, connecting short peptide and iRGD,
the mutation comprises deletion, insertion and/or substitution of 1-5 amino acids,
the amino acid sequence of the SEC2 is shown in SEQ ID NO. 31,
the amino acid sequence of the connecting short peptide is shown as SEQ ID NO. 29,
the amino acid sequence of the iRGD is shown in SEQ ID NO: 30.
2. The fusion protein of claim 1, wherein the mutant has a mutation at amino acid residues 102-106 of the sequence shown in SEQ ID NO. 31; preferably, the mutation is that the 102 th to 106 th amino acid residues GKVTG of the sequence shown as SEQ ID NO. 31 are mutated into WWX; more preferably, said WWX is WWH, WWP or WWT; namely, the amino acid sequence of the mutant is shown as the 1 st to 237 th positions of SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 6.
3. The fusion protein of claim 1 or 2, wherein the nucleotide sequence encoding the fusion protein is as set forth in SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 11 of the sequence Listing.
4. A fusion gene encoding the fusion protein of claim 1 or 2; preferably, the nucleotide sequence is shown as SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 11 in the sequence table.
5. A recombinant expression vector comprising the fusion gene of claim 4; preferably, the skeleton vector of the recombinant expression vector is pET-28 a-TEV.
6. A transformant obtained by introducing the fusion gene according to claim 4 or the recombinant expression vector according to claim 5 into a host; preferably, the host is escherichia coli, preferably escherichia coli e.coli bl21(DE3) cells or e.coli TG 1.
7. A method of preparing a fusion protein comprising the steps of:
(1) obtaining the transformant of claim 6;
(2) screening the transformants, expressing and purifying the fusion protein.
8. The method according to claim 7, wherein in the step (2), the purification comprises subjecting the cells obtained by the expression to ultrasonication, centrifuging the cells, collecting the supernatant, and subjecting the supernatant to two times of Ni affinity chromatography;
preferably, the Ni affinity chromatography comprises loading a sample onto a pre-equilibrated Ni affinity chromatography column at a loading rate of 0.2-0.8 ml/min; washing with 8-12 column volumes of equilibration buffer solution, and eluting with elution buffer solution, preferably washing with 10 column volumes of equilibration buffer solution;
more preferably, the equilibration buffer is an equilibration buffer containing 20-80mM imidazole, and the composition of the equilibration buffer is preferably as follows: 20-30mM Tirs-HCl, 800-; and/or the elution buffer is an elution buffer containing 250-300mM imidazole, and the composition of the elution buffer is preferably as follows: 20-30mM Tirs-HCl, 800-; and/or the pH value of the elution buffer solution is 7.2-8.0.
9. The method according to claim 8, wherein the method further comprises a step of desalting by ultrafiltration between the two times of Ni affinity chromatography, and preferably further comprises a step of mixed enzyme digestion with TEV protease after the desalting by ultrafiltration; more preferably, the molar ratio of the product after ultrafiltration desalination to the TEV protease is 1: 5; and/or the enzyme digestion time is 24 hours.
10. Use of the fusion protein according to any one of claims 1 to 3, the fusion gene according to claim 4, the recombinant expression vector according to claim 5, or the transformant according to claim 6 for the preparation of a medicament; preferably the application in preparing the medicine for treating the tumor, and more preferably the application in preparing the medicine for treating tumor immunity.
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