CN112480262B - Fusion protein and preparation and application thereof - Google Patents

Abstract

The invention provides a fusion protein, which sequentially comprises the following components from an N end to a C end: 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 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 be specifically targeted and infiltrated into a tumor tissue microenvironment, so that the tumor specificity and vascular permeability of the superantigen are greatly improved, and the killing efficiency on the tumor is improved, so that the survival capability of a patient can be effectively improved when the targeting fusion protein is used for treating the patient, and the targeting fusion protein has a good clinical application value.

Description

A kind of fusion protein and its preparation and application

technical field

The invention relates to the field of genetic engineering, in particular to a fusion protein and its preparation and application.

Background technique

Tumor immunotherapy refers to the application of the principles and methods of immunology to effectively kill tumor cells and inhibit the growth of tumor cells by activating immune cells in the body and enhancing the body's anti-tumor immune response. Because of its small side effects and obvious therapeutic effect, it is gradually becoming the development direction of future tumor treatment.

Targeted fusion protein is a new type of therapeutic strategy proposed on the basis of the development of modern medical biology, which is composed of targeting molecules and cytotoxic molecules. Targeting molecules often use tumor-specific antibodies or short peptides to deliver effector molecules capable of killing target cells to tumor lesions, which is in line with the current development trend of precision tumor treatment.

Internalizing RGD (iRGD, also known as internalizing RGD) is a targeted penetrating peptide with a cyclic structure, with a small molecular weight and high water solubility. The sequence is CRGDKGPDC, and the RGD sequence can specifically Binding to tumor tissue vascular endothelium and integrin αv highly expressed by tumor cells, and then being cleaved by specific proteases, the residue fragment has the structural characteristics of R/KXXR/K, which can interact with NRP-1 (neuropilin-1) , mediates the cell membrane penetration effect. Because of its diverse functions, iRGD is widely used in the research of targeting carriers for antitumor drugs, tumor imaging agents, and some biological products. Among them, integrin αv is a member of the cell adhesion receptor family, which regulates various cell functions, especially in the occurrence, development and metastasis of solid tumors, and its expression is also positively correlated with the malignancy of tumors. As a regulator of the nervous system, NRP-1 plays an important role in tumor local regulation of VEGF-induced angiogenesis. It is closely related to the growth, migration and angiogenesis of tumor cells.

At present, most studies use the combination of iRGD and chemical drugs, but the chemical drugs need to be entrapped in liposomes before they can be coupled with iRGD. This combination of drugs has achieved good tumor inhibitory effects. However, since the chemical drugs entrapped in liposomes need to be released in the tissue to exert their drug effects, the type and mode of liposomes have a great influence on the drug effect. Low liposome encapsulation efficiency has always been the key to liposome drugs, and liposome drugs also have problems such as poor stability and easy to be actively cleared by the body. This has also led to the disadvantages of iRGD-coupled liposomes such as unstable structure and effect, which has added obstacles to its application. In addition, in the application of iRGD combined with chemical drugs, there are still some applications where iRGD and chemical drugs are mixed and administered as two independent parts, resulting in iRGD not being able to actively target the delivery of chemical drugs, and only assisting in part delivery. The chemical drugs accumulated in the tumor vascular tissue penetrate into the tumor tissue. This not only weakens the aggregation efficiency of iRGD-mediated effector molecules in local tumors, but also leads to no improvement in the toxic side effects of chemical drugs on normal tissues due to the weak targeting effect.

It is also disclosed in the prior art that the highly permeable targeting membrane-penetrating peptide iRGD is combined with other protein effector molecules such as Trail and CDD to form a fusion protein, and the effect obtained is slightly better. However, the tumor vasculature and tumor tissue are intricate, so fusion proteins with stronger targeting and stronger tissue penetration remain to be sought.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a fusion protein and its gene in order to overcome the technical defect that the fusion protein in the prior art is not enough to pass through the tumor vasculature and the complex microenvironment of the tumor tissue, resulting in an unsatisfactory anti-tumor effect. , its preparation method and application. In the present invention, for the first time, the targeting membrane-penetrating peptide and superantigen, a tumor immunotherapy drug, are connected through a specific linking short peptide to form a targeting fusion protein superantigen (SAg)-linking short peptide (L)-iRGD, It can specifically target and infiltrate into the microenvironment of tumor tissue with high expression of integrin αv and NRP-1, which greatly improves the tumor specificity and vascular permeability of superantigens, and improves the killing efficiency of tumors, thus using When treating patients, it can effectively improve the survival ability of patients, and has good clinical application value.

The present inventors have conducted a lot of research on targeting molecules, linking short peptides, cytotoxic molecules, etc., and unexpectedly found that when the targeting penetrating peptide iRGD and SEC2 or its modified form WWH in the superantigen of tumor immunotherapy drugs Or WWP or WWT is fused with a linking short peptide to cooperate with a specific rigid linking short peptide (in the construction of a fusion protein, a key issue is the linking short peptide Linker between the two proteins, that is, the length of the linking peptide affects the folding of the protein and Stability is very important. If the linker sequence is too short, it may affect the folding of the high-level structure of the two proteins, thereby interfering with each other; if the linker sequence is too long, it will involve the issue of immunogenicity, because the linker sequence itself is a new antigen), the obtained The tumor specificity and vascular permeability of the fusion protein were significantly enhanced.

In order to solve the above-mentioned technical problems, the first aspect of the present invention provides a fusion protein, the fusion protein includes from N-terminal to C-terminal: SEC2 or its mutants, connecting short peptides and iRGD, and the mutations include 1 to 5 amino acid deletion, insertion and/or substitution, 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 in SEQ ID NO: 29, the amino acid sequence of the iRGD is shown in Shown in SEQ ID NO:30.

Preferably, the mutant is mutated at amino acid residues 102-106 of the sequence shown in SEQ ID NO:31; more preferably, the mutation is 102 of the sequence shown in SEQ ID NO:31 Amino acid residue GKVTG at position 106 is mutated to WWX; further preferably, the WWX is WWH, WWP or WWT, that is, the amino acid sequence of the mutant is such as SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO : The sequence shown in bits 1 to 237 of 6.

In the present invention, X in the above WWX is only used as a reference, and it can refer to any amino acid. In addition, the abbreviations of amino acids in the present invention are conventional meanings in the field unless otherwise specified.

Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12 in the sequence listing; more preferably, the amino acid sequence encoding the fusion protein 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 listing.

To solve the above technical problems, the second aspect of the present invention provides a fusion gene encoding the fusion protein as described in the first aspect of the present invention.

Preferably, its nucleotide sequence is shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:11 in the sequence listing.

In order to solve the above technical problems, the third aspect of the present invention provides a recombinant expression vector containing the fusion gene as described in the second aspect of the present invention.

Preferably, the backbone vector of the recombinant expression vector is pET-28a-TEV.

In order to solve the above technical problems, the fourth aspect of the present invention provides a transformant, which introduces the fusion gene as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention into a host.

Preferably, the host is Escherichia coli, preferably E. coli BL21 (DE3) cells or E. coli TG1.

In order to solve the above technical problems, the fifth aspect of the present invention provides a method for preparing a fusion protein, which includes the following steps:

(1) obtaining the transformant as described in the fourth aspect of the present invention;

(2) Screening the transformant, expressing and purifying the fusion protein.

In the above step (2), the purification preferably includes ultrasonically disrupting the expressed bacteria, centrifuging to collect the supernatant, and then obtaining a soluble fusion protein with biological activity after two Ni affinity chromatography. Usually, after two Ni affinity chromatography, it is only necessary to collect the solution after passing through the column to obtain the fusion protein.

Preferably, the Ni affinity chromatography includes loading the sample on a pre-equilibrated Ni affinity chromatography column at a loading rate of 0.2-0.8ml/min, using 8-12 column volumes of equilibration buffer After washing (washing away non-specifically bound foreign proteins), it can be eluted with elution buffer, preferably using 10 column volumes of equilibration buffer for washing;

More preferably, the equilibrium buffer is an equilibrium buffer containing 20-80mM imidazole, and its composition is preferably: 20-30mM Tirs-HCl, 800-1000mM NaCl, 20-80mM imidazole, and/or, the equilibrium buffer The pH value of the solution is 7.2-8.0; and/or, the elution buffer is an elution buffer containing 250-300mM imidazole, and its composition is preferably: 20-30mM Tirs-HCl, 800-1000mM NaCl, 250-300mM imidazole; and/or, the pH value of the elution buffer is 7.2-8.0.

Preferably, the step of ultrafiltration and desalination is also included between the two Ni affinity chromatography; more preferably, the step of mixed enzyme digestion with TEV protease is also included after the ultrafiltration and desalination; further more preferably Preferably, the molar ratio of the ultrafiltration desalted product to the TEV protease is 1:5; and/or, the enzyme cleavage time is 24h.

In order to solve the above technical problems, the sixth aspect of the present invention provides a fusion protein as described in the first aspect of the present invention, a fusion gene as described in the second aspect of the present invention, a recombinant expression gene as described in the third aspect of the present invention The application of the carrier or the transformant according to the fourth aspect of the present invention in the preparation of medicine; preferably the application in the preparation of a medicine for treating tumors, more preferably the application in the preparation of a medicine for treating tumor immunity.

In the present invention, the modified form WWH of SEC2 refers to the 102nd to 106th positions of the superantigen protein Staphylococcal enterotoxin C2 (Staphylococcal enterotoxin C2, SEC2, whose amino acid sequence is shown in SEQ ID NO: 31) The amino acid residues of GKVTG are changed to the SAg WWH isomer of WWH (all referred to as WWH in the present invention, as shown in SEQ ID NO: 31). Similarly, the modified WWP refers to the SAg WWP isomer in which the GKVTG amino acid residues at positions 102-106 of SEC2 are changed to WWP (all referred to as WWP in the present invention). The modified WWT refers to the SAg WWT isomer in which the GKVTG amino acid residues at positions 102-106 of SEC2 are changed to WWT (all referred to as WWT in the present invention). The three modified forms can also refer to the patent application CN201110077088.3, the entire content of which is incorporated herein by reference. Among them, superantigen (superantigen, SAg) is a protein molecule that produces strong immune activation on T lymphocytes at extremely low concentrations, and it can bind to MHC II (histocompatibility) in the antigen-binding region outside the antigen-presenting cell. Complex) molecules combine with the Vβ region of T cells to form a complex, thereby activating the proliferation of a large number of T lymphocytes, and releasing a large number of cytokines and other effector molecules in vitro or in vivo.

In the present invention, "fusion gene" refers to a gene formed by connecting two or more nucleotide sequences from different sources, or two or more nucleotide sequences from the same source but not connected to each other in their natural positions Genes linked by acid sequences. The protein encoded by the fusion gene of the present invention is called fusion protein.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is:

In the present invention, for the first time, the targeting membrane-penetrating peptide is connected with the superantigen to form a targeting fusion protein SAg-L-iRGD (superantigen-linked short peptide-iRGD), which can specifically target and infiltrate highly expressed integrin αv The tumor tissue microenvironment with NRP-1 greatly improves the tumor specificity and vascular permeability of the superantigen, enhances its local enrichment in the tumor, and can significantly inhibit the growth of tumor cells, thereby improving the anti-tumor effect. When applied to clinical treatment, the required dose can be reduced accordingly, that is, a lower dose can obtain better tumor suppression effect, and a lower dose will bring lower toxicity, thereby reducing the side effects during drug use; in addition , The improvement of tumor specificity can make drug molecules more concentrated in the tumor area, reducing the distribution of other non-tumor areas and organs, thereby also reducing toxicity and improving the survival ability of patients. In a certain preferred embodiment of the present invention, the ability of the fusion protein of the present invention to bind tumor cells is increased by more than 3.92 times compared with the protein before fusion, and the tumor inhibition rate is as high as 63.3%. The fusion protein of the present invention is applied to patients with When the mice had melanoma, the average number of days the mice survived was significantly higher than that of the control group. The tumor-suppressing effect of the fusion protein of the present invention is significantly better than the effect of simply mixing the "targeting penetrating peptide" and "superantigen", that is, the effect of 1+1>2 is achieved in the tumor-suppressing effect.

Description of drawings

Figure 1 shows the results of pET-28a-tev-wwh-epapkp-irgd enzyme digestion verification by 1.0% agarose gel electrophoresis analysis, in which: 1 is the λ-EcoT14I/BglII digest DNA marker; 2 is the undigested pET -28a-tev-wwh-epapkp-irgd plasmid; 3 and 4 are the pET-28a-tev-wwh-epapkp-irgd plasmids that have been digested with EcoRI and XhoI, respectively; 5 is the pET that has been digested with EcoRI and XhoI -28a-tev-wwh-epapkp-irgd plasmid; 6 is DL2000 DNA molecular weight standard.

Figure 2 is the 10% SDS-PAGE electrophoresis analysis graph of pET-28a-tev-wwh-epapkp-irgd expression before and after induction, wherein: 1 is the total protein expression of the bacteria before induction; Protein expression level; 3 is the expression level of soluble protein in the supernatant after the cells were broken and centrifuged after induction at 30°C for 4 hours; 4 is the total protein expression level after the cells were broken after induction at 30°C for 4 hours; 5 is the protein marker; 6 7 is the total protein expression of the bacteria before induction; 7 is the total protein expression of the bacteria after induction at 37°C for 4 hours; 8 is the expression of soluble proteins in the supernatant after the bacteria are broken and centrifuged after induction at 37°C for 4 hours; 9 is the expression of soluble proteins at 37°C After 4 hours of induction, the bacterial cells were broken, and the total protein expression was measured.

Figure 3 is the 12% SDS-PAGE electrophoresis analysis diagram of the soluble expressed membrane-penetrating peptide-superantigen fusion protein WWH-EPAPKP-iRGD purified twice by AKTANi column, wherein: M is 180KD protein marker; The concentration is (300ng/μL); 2 is the fusion protein purified by Ni column chromatography and then dialyzed to desalt, the concentration is (300ng/μL).

Fig. 4 is fusion protein WWH-EPAPKP-RGD, WWH-EPAPKP-tLyp-1, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, WWH-GGGGS(G ₄ S)-iRGD, WWH- (GS) Experimental results of 5-iRGD, WWH-EPAPK-iRGD, iRGD-EPAPKP-WWH and WWH binding to αv ⁺ and NRP-1 ⁺ mouse melanoma cells B16F10 in vitro.

Fig. 5 is fusion protein WWH-EPAPKP-RGD, WWH-EPAPKP-tLyp-1, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, WWH-GGGGS(G ₄ S)-iRGD, WWH- (GS) Experimental results of 5-iRGD, WWH-EPAPK-iRGD, iRGD-EPAPKP-WWH and WWH binding to αv ⁺ and NRP-1 ⁺ mouse breast cancer cells 4T1 in vitro.

Fig. 6 is fusion protein WWH-EPAPKP-RGD, WWH-EPAPKP-tLyp-1, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, iRGD-EPAPKP-WWH, WWH-GGGGS (G ₄ S )-iRGD, WWH-(GS)5-iRGD, WWH-EPAPK-iRGD, sTRAIL-EPAPKP-iRGD and WWH+iRGD, BSA, iRGD, WWH, WWP and WWT inhibited αv ⁺ and NRP-1 ⁺ mouse melanin in vitro Experimental results of tumor cell microspheres B16F10.

Fig. 7 is fusion protein WWH-EPAPKP-RGD, WWH-EPAPKP-tLyp-1, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, iRGD-EPAPKP-WWH, WWH-GGGGS (G ₄ S )-iRGD, WWH-(GS)5-iRGD, WWH-EPAPK-iRGD, sTRAIL-EPAPKP-iRGD and WWH+iRGD, BSA, iRGD, WWH, WWP and WWT inhibited αv ⁺ and NRP-1 ⁺ mouse mammary glands in vitro Experimental results of cancer cell microspheres 4T1.

Figure 8 shows the experimental results of fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, and saline, WWH and WWH+iRGD inhibiting αv ⁺ and NRP-1 ⁺ mouse melanoma cells B16F10 in vivo.

Figure 9 shows the experimental results of fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, and saline, WWH and WWH+iRGD inhibiting αv ⁺ and NRP-1 ⁺ mouse breast cancer cells 4T1 in vivo.

Fig. 10 is fusion protein WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, SEA-EPAPKP-iRGD, SEB-EPAPKP-iRGD, SEC2-EPAPKP-iRGD and PBS and WWH inhibit αv ⁺ and NRP in vivo Survival curves of ^-1+ mouse melanoma cells B16F10.

Fig. 11 is fusion protein WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT-EPAPKP-iRGD, SEA-EPAPKP-iRGD, SEB-EPAPKP-iRGD, SEC2-EPAPKP-iRGD and PBS and WWH inhibit αv ⁺ and NRP in vivo Survival curves of ^-1+ mouse breast cancer cells 4T1.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

The coding base sequence DNAs of all protein molecules in the following examples were synthesized by Beijing Huada Gene Company.

Example 1

1. Fusion protein WWH-EPAPKP-iRGD gene wwh-epapkp-irgd, which has a base sequence as in SEQ ID NO: 1 in Table 1, wherein WWH (WWH refers to the superantigen protein Staphylococcus aureus enterotoxin C2 (Staphylococcal enterotoxin C2, SEC2, the amino acid sequence is the sequence shown in SEQ ID NO: 31), the 102nd to 106th GKVTG amino acid residues are mutated into the Sag WWH variant of WWH, hereinafter and in the drawings of the description are referred to as WWH) encoding gene wwh has the base sequence shown in the 1st to 711 positions of SEQ ID NO:1, and the iRGD encoding gene irgd has the base sequence shown in the 730th to the 756th positions of SEQ ID NO:1 , the DNALinkerEPAPKP connecting the short peptide by coding has the base sequence as shown in the 712th to 729th positions of SEQ ID NO:1.

Table 1

Note: The bold and underlined sequence is the superantigen modified 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 features:

Length: 756bp

Type: nucleic acid

Chain type: double chain

Topology: Linear

(b) Molecular type: cDNA

(c) Assumption: No

(d) Antisense: No

(2) Preparation of fusion gene wwh-epapkp-irgd:

(a) PCR primer design and reaction conditions: According to the multiple cloning site of the pET28a vector (purchased from Novagen) combined with the above-mentioned sag isomer WWH, the forward primer (synthesized by Beijing Huada Gene Company) was designed, and the epapkp-irgd gene sequence Design reverse primers (synthesized by Beijing Huada Gene Company) for PCR:

Forward primer (F): 5'-CGGAATTCGAGAGTCAACCAGACCC-3' (SEQ ID NO:27)

Reverse primer (R):

5'-CCCTCGAGTTAACAATCCGGACCTTTATCACCACGACAAGGTTTTGGCGCCGGTTTCCATTCTTTGTTGTAAGGTGGACTTCTAT-3' (SEQ ID NO: 28)

The PCR reaction system was (Pyrobest buffer, dNTP, and pyrobest DNA polymerase were all purchased from TAKARA Company): 5 μL of 10×Pyrobest buffer, 250 μmol of dNTP, 25 pmol of forward and reverse primers, and the template was plasmid DNA containing the sag gene (sequence shown in SEQ ID The base sequence from the 1st position to the 711th position of NO: 1 is shown, and the sequence shown in ST-1 in the patent application CN201110077088.3 can also be referred to) 0.1 μg, pyrobest DNA polymerase 2U, supplemented with sterile ultrapure water Bring the volume up to 50 μL.

The PCR reaction conditions are: first stage: 95°C, 5min; second stage: 94°C, 55s; 60°C, 2min; 72°C, 2min; a total of 30 cycles; third stage: 72°C, 10min.

(b) PCR product recovery: The PCR amplification product was analyzed by 1.0% agarose gel electrophoresis and the 753bp target band was recovered by cutting the gel. The operation method was carried out according to the instructions of the gel recovery and purification kit of Jiangsu Kangwei Century Co., Ltd. The product-targeting membrane-penetrating peptide-superantigen fusion gene wwh-epapkp-irgd was obtained.

The fusion protein WWH-EPAPKP-iRGD encoded by the nucleotide shown in SEQ ID NO:1 has the amino acid sequence shown in SEQ ID NO:2 (see Table 1 for details).

Wherein, the information of SEQ ID NO:2 is as follows

(a) Sequential features

* Length: 252 residues

*Type: amino acid

* Chain type: single chain

*Topology: Linear

(b) Molecule type: protein

(c) Assumption: No

(d) Antisense: No

(e) Original source: artificial sequence.

2. The fusion protein WWP-EPAPKP-iRGD gene wwp-epapkp-irgd has the base sequence in SEQ ID NO: 3 in Table 2, wherein WWP (WWP refers to the 102nd to 106th GKVTG amino acids of SEC2 The Sag WWP transformant whose residue is mutated into WWP, hereinafter referred to as WWP in the accompanying drawings) The coding gene wwp has the base sequence from the first to the 711th position of SEQ ID NO:3, and the iRGD coding gene irgd has the SEQ ID The 730th to 756th base sequence of NO: 3 has the 712th to 729th base sequence of SEQ ID NO: 3 through the DNA Linker EPAPKP that encodes the linking short peptide.

Table 2

Note: The bold and underlined sequence is the superantigen modified 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 features:

Length: 756bp

Type: nucleic acid

Chain type: double chain

Topology: Linear

(b) Molecular type: cDNA

(c) Assumption: No

(d) Antisense: No

(2) The preparation process of the fusion gene wwp-epapkp-irgd is the same as the above-mentioned part 1 (only the template is different), and the template can also refer to the sequence shown in ST-3 in the patent application CN201110077088.3. The fusion protein WWP-EPAPKP-iRGD encoded by the nucleotide shown in SEQ ID NO:3 has the amino acid sequence shown in SEQ ID NO:4 (see Table 2 for details). Wherein, the information of SEQ ID NO:4 is as follows:

(a) Sequential features

* Length: 252 residues

*Type: amino acid

* Chain type: single chain

*Topology: Linear

(b) Molecule type: protein

(c) Assumption: No

(d) Antisense: No

(e) Original source: artificial sequence.

3. The fusion protein WWT-EPAPKP-iRGD gene wwt-epapkp-irgd has the base sequence in SEQ ID NO: 5 in Table 3, wherein WWT (WWT refers to the 102nd to 106th GKVTG amino acids of SEC2 The Sag WWT transformant whose residue is mutated into WWT, hereinafter referred to as WWT in the accompanying drawings) The coding gene wwt has the base sequence from the 1st to the 711th position of SEQ ID NO:5, and the iRGD coding gene irgd has the SEQ ID The 730th to 756th base sequence of NO: 5 has the 712th to 729th base sequence of SEQ ID NO: 5 through the DNA Linker EPAPKP encoding the linking short peptide.

table 3

Note: The bold and underlined sequence is the superantigen modified 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 features:

Length: 756bp

Type: nucleic acid

Chain type: double chain

Topology: Linear

(b) Molecular type: cDNA

(c) Assumption: No

(d) Antisense: no

(2) The preparation process of the fusion gene wwt-epapkp-irgd The above-mentioned part 1 (only the template is different), the template can also refer to the sequence shown in ST-2 in the patent application CN201110077088.3. The fusion protein WWT-EPAPKP-iRGD encoded by the nucleotide shown in SEQ ID NO:5 has the amino acid sequence shown in SEQ ID NO:6 (see Table 3 for details). Wherein, the information of SEQ ID NO:6 is as follows:

(a) Sequential features

* Length: 252 residues

*Type: amino acid

* Chain type: single chain

*Topology: Linear

(b) Molecule type: protein

(c) Assumption: No

(d) Antisense: No

(e) Original source: artificial sequence.

Example 2

The three targeting membrane-penetrating peptide-superantigen fusion genes wwh-l-irgd (ie wwh-epapkp-irgd), wwp-l-irgd (ie wwp-epapkp-irgd), and wwt-l-irgd (i.e. wwt-epapkp-irgd) was connected to the prokaryotic expression vector pET-28a-TEV to realize the expression of targeted penetrating peptide-superantigen fusion protein SAg-L-iRGD in Escherichia coli, specifically :

The fusion gene sag-l-irgd (i.e. the above-mentioned wwh-l-irgd, wwp-l-irgd, wwt-l-irgd) was connected into the expression vector pET-28a-TEV (purchased from Novagen): the expression vector pET The plasmid DNA of -28a-TEV and the gene DNA fragment of sag-l-irgd were digested with EcoRI (purchased from Dalian Bao Biological Company) and XhoI (purchased from Dalian Bao Biological Company) respectively, and subjected to 1.0% agarose gel electrophoresis , the sag-l-irgd fragment and the large DNA fragment of the plasmid pET-28a-TEV were recovered from the gel, and T4 DNA ligase (purchased from Dalian Bao Biological Co., Ltd.) was used to connect overnight at 16°C to construct a targeting membrane-penetrating peptide-superantigen fusion protein Expression vector pET28a-TEV-sag-l-irgd. The ligation product was transformed into Escherichia coli DH5α competent cells (purchased from Dalian Bao Biological Company). Transformants were screened by kanapenicillin (purchased from Sigma) resistance, recombinant single clones were selected for expansion, plasmid DNA was extracted, and correct recombinant clones were identified by double digestion with EcoR I and XhoI (Figure 1). And the correct recombinant cloning plasmid verified by double enzyme digestion was sent to Shanghai Sangong Company for sequencing. The plasmids with correct sequencing were transformed into Escherichia coli BL21 (DE3) competent cells (purchased from Beijing Tiangen Biochemical Technology Company).

(1) Fusion protein SAg-L-iRGD (ie WWH-L-iRGD (ie WWH-EPAPKP-iRGD), WWP-L-iRGD (ie WWP-EPAPKP-iRGD), WWT-L-iRGD (ie WWT-EPAPKP-iRGD) -iRGD) expression: inoculate the BL21 (DE3) single colony of above-mentioned transformation recombinant plasmid pET28a-TEV-sag-l-irgd in the liquid LB of 60 μ g/ml kanamycin overnight at 37 ℃, the next day by 1:100 (volume Ratio) was transferred to the next generation, cultured at 37°C until OD600 was 0.8, and induced at 30°C and 37°C for 4 hours by adding a final concentration of 10 mMIPTG (purchased from Sigma).

(2) Collect the supernatant containing the fusion protein: centrifuge to collect the cells after induced expression, and resuspend the cells of every 100 ml of the original culture in 10 ml of equilibration buffer (20 mM Tirs-HCl, 500 mM NaCl, 50 mM imidazole, pH=7.9). Ultrasonic crushing at 0°C until the bacterial liquid becomes clear, and ultra-high-speed centrifugation at 100,000 rpm for 10 minutes (100,000 rpm can better remove impurities such as nucleic acid and cell debris, which is beneficial to subsequent purification, and experiments have found that 100,000 rpm does not reduce protein yield). Collect the supernatant.

(3) The supernatants before and after centrifugation were taken respectively as samples of induced whole-cell protein and induced whole-cell soluble protein, and the soluble expression was analyzed by 12% SDS-PAGE, and the results were shown in FIG. 2 . It can be seen from the figure that the expression position of the target protein has been marked with a white frame, and the expression of the target protein can be seen clearly; in addition, the target protein expressed under the condition of 37 degrees has more soluble components (that is, the soluble component after crushing and centrifugation). more target protein in serum). Taking WWH-L-iRGD as an example here, the results of the other two fusion proteins are similar.

(4) Purify the fusion protein using AKTA purification instrument (product of GE company in the United States): load the supernatant after centrifugation (the loading speed is 0.2-0.8ml/min) on the AKTANi affinity chromatography column (product of GE company in the United States) , rinse with ten column volumes of equilibration buffer (20 mM Tirs-HCl, 500 mM NaCl, 50 mM imidazole, pH=7.9) until the UV detection value is stable. Finally, use the elution buffer (20mM Tirs-HCl, 500mM NaCl, 250mM imidazole, pH=7.9) to elute the target protein, and collect after the UV detection value starts to rise until the UV becomes stable. The collected TEV-SAg-L-iRGD protein eluate was dialyzed to desalt, and the purity was analyzed by SDS-PAGE. The result is shown in lane 2 in FIG. 3 .

(5) The dialyzed fusion protein TEV-SAg-L-iRGD and TEV protease (the used pET28a vector has its own His-tag purification tag, so the fusion protein TEV-SAg-L-iRGD is affinity Tag cutting) was mixed at a molar ratio of 1:5, and after 24 hours of enzyme digestion, the mixed system was loaded on the Ni column of the AKTA purification instrument, and the first UV peak was collected when loading the sample, which was the fusion protein SAg-L-iRGD. Dialyzed to remove salt and analyzed the purity by SDS-PAGE, the result is shown in the No. 1 swimming lane shown in Figure 3, which is higher than the No. 2 swimming lane.

Example 3 Tumor Targeting Study of Targeted Penetrating Peptide-Superantigen Fusion Protein SAg-L-iRGD

Mouse-derived melanoma cells B16F10 and mouse-derived breast cancer cells 4T1, which were verified by western blot to highly express integrin αv and NRP-1, were selected as target cells (the cells used were purchased from the "Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences").

Targeting verification of fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD: use fluorescent dye AlexaFlour 647 (purchased from thermo company) to target fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP- iRGD, WWT-EPAPKP-iRGD, and superantigen WWH were labeled, and the target cells B16F10 and 4T1 were fixed and mixed with the above-mentioned fluorescently-labeled proteins for incubation. After incubation for 20 minutes, centrifuge at 1000g for 5 minutes, and remove the supernatant. Resuspend in PBS and centrifuge. After resuspending with 200 μl PBS solution again, use a flow cytometer to detect. The experimental results are shown in Figure 4 and Figure 5, which show that fluorescently labeled WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD can have a strong targeting binding effect on target cells, and the binding ability is obviously strong Compared with the control group, there was almost no difference in the binding strength among the three.

Among them, the ability of WWH-EPAPKP-iRGD to bind B16F10 cells was 4.26 times that of WWH, the ability of WWP-EPAPKP-iRGD to bind B16F10 cells was 4.49 times that of WWH, and the ability of WWT-EPAPKP-iRGD to bind B16F10 cells was 4.41 times that of WWH.

The ability of WWH-EPAPKP-iRGD to bind 4T1 cells was 4.02 times that of WWH, the ability of WWP-EPAPKP-iRGD to bind 4T1 cells was 3.92 times that of WWH, and the ability of WWT-EPAPKP-iRGD to bind 4T1 cells was 3.97 times that of WWH.

Example 4 In vitro anti-tumor activity of targeting membrane-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 were added to U-shaped cell culture plates (Corning 4515, USA) at 1×10 ⁴ cells/well type), after 48 hours of culture to form stable tumor microspheres, the isolated single mouse splenocytes were added according to the effect-to-target ratio of 1:10. Then the experimental group fusion protein a.WWH-EPAPKP-iRGD, b.WWP-EPAPKP-iRGD, c.WWT-EPAPKP-iRGD, d.WWH, WWP, WWT were used alone, e.iRGD-EPAPKP-WWH (with The difference in a is that the direction of the two is opposite, from the N-terminal to the C-terminal are iRGD, EPAPKP and mutant WWH), f. 350 pmol/μl of the substance was added to each well, and blank control wells (only medium RPMI-1640, product of Gibco, USA) and tumor cell control wells (only tumor cells were added) were set up, with 3 replicate wells for each sample. In the same way, each well was set up with bovine serum albumin BSA (purchased from Sigma) as a negative control. After culturing for 48 hours under conventional conditions (37° C., 5% CO ₂ concentration), 100 μl of Cell-tilterGlo 3D cell viability detection reagent (purchased from Promega) was added to each well. After standing at room temperature for 25 min, a microplate reader was used to detect the bioluminescence in each well.

Tumor growth inhibition rate (Tumor growth inhibition, %)=100-[(experimental well-blank control well)/(tumor cell control well-blank control well)]×100.

Experimental results show (Figure 6 and Figure 7):

At the concentration of 350pmol/μl, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD had the most obvious inhibitory effects on B16F10, reaching 65.2%, 67.5%, and 66.3%, respectively. Among them, WWH-EPAPKP-iRGD increased by 29.9% compared with WWH, WWP-EPAPKP-iRGD increased by 30.12% compared with WWP, WWT-EPAPKP-iRGD increased by 28.7% compared with WWT, and the tumor inhibition rate was significantly enhanced.

At the concentration of 350pmol/μl, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD had the most obvious inhibitory effects on 4T1, reaching 64.6%, 63.3%, and 67.5%, respectively. Among them, WWH-EPAPKP-iRGD increased by 29.3% compared with WWH, WWP-EPAPKP-iRGD increased by 27.1% compared with WWP, WWT-EPAPKP-iRGD increased by 32.8% compared with WWT, and the tumor inhibition rate was significantly enhanced.

In addition, more importantly, the experimental results for the two cell lines showed that the tumor inhibition rate of WWH-EPAPKP-iRGD was not only significantly higher than that of the BSA control group, iRGD alone group, and WWH alone group, but also significantly higher than that of iRGD+ In the WWH combined treatment group, it shows that the tumor inhibitory effect of the fusion protein is significantly higher than the combined treatment effect of the two molecules that have not been fused together, indicating that the fusion of the protein is very critical.

Example 5 In vivo anti-tumor solid tumor activity of targeting membrane-penetrating peptide-superantigen fusion protein SAg-L-iRGD

Fusion protein WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, WWT- ^EPAPKP -iRGD in vivo anti-tumor solid tumor activity verification in mice: subcutaneously inoculate 106 melanoma cells B16F10 in C57 mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., inoculated at the age of five weeks) on the left side of the back, inoculated 10 ⁶ breast cancer cells 4T1 in BALB/c mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., five weeks old Vaccination at the time) the breast pad area. When the tumor grows to about 100mm ³ start to administer through the tail vein, set up the treatment groups a. Mixed combination (that is, WWH+iRGD group), physiological saline was used as the control treatment group, and the administration concentration was 70 pmol/rat, administered once every three days, and administered six times in total. The changes in tumor volume during the administration period were recorded (Figure 8 and Figure 9), and the death endpoint of the mice was recorded to draw the survival curve (Figure 10 and Figure 11). The results showed that the fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD can produce specific targeting and killing effects on solid tumors that highly express integrin αv and NRP-1, and their effects are significantly stronger. In other experimental groups (d.WWH, e.iRGD mixed with WWH) and the control group. At the end of administration day 16, the tumor inhibition rates of each group are shown in Table 4 and Table 5:

Table 4. Tumor inhibition rate of each fusion protein on C57 mice modeled with B16F10

^* Tumor inhibition rate=(tumor volume of control group-tumor volume of experimental group)/tumor volume of control group*100%

Table 5. Tumor inhibition rate of each fusion protein on 4T1 modeled BALB/c mice

^* Tumor inhibition rate=(tumor volume of control group-tumor volume of experimental group)/tumor volume of control group*100%

It can be seen from the table:

The tumor inhibition rate of WWH-EPAPKP-iRGD on B16F10-created C57 mice reached 71.24%, the tumor inhibition rate of WWP-EPAPKP-iRGD on B16F10-created C57 mice reached 70.61%, and the tumor inhibition rate of WWT-EPAPKP-iRGD on B16F10 The tumor inhibition rate of the model C57 mice reached 72.06%, significantly higher than the 19.65% of the WWH group, and also significantly higher than the 33.68% of the WWH+iRGD combined group.

The tumor inhibition rate of WWH-EPAPKP-iRGD on 4T1-modeled BALB/c mice reached 67.45%, and the tumor inhibition rate of WWP-EPAPKP-iRGD on 4T1-modeled BALB/c mice reached 64.78%. WWT-EPAPKP- The tumor inhibition rate of iRGD on 4T1-modeled BALB/c mice reached 69.55%, which was significantly higher than the 22.73% of the WWH group, and also significantly higher than the 27.45% of the WWH+iRGD combined group.

The results of the survival experiment of the administered mice are shown in Figure 10 and Figure 11:

The average survival days of B16F10 model mice in the fusion protein WWH-EPAPKP-iRGD administration group was 26.83 days, and the average survival days of B16F10 model mice in the WWP-EPAPKP-iRGD administration group was 26.83 days. The average survival days of the B16F10 model mice in the drug group was 26.50 days, which was significantly higher than that of the SAg protein alone (22.33 days) and the control group (18.17 days).

The average survival days of 4T1 model mice in the fusion protein WWH-EPAPKP-iRGD administration group was 57.50 days, and the average survival days of 4T1 model mice in the WWP-EPAPKP-iRGD administration group was 58.00 days. The average survival days of the 4T1 model mice in the drug group was 59.17 days, which was significantly higher than that of the SAg protein alone (41.83 days) and the control group (35.67 days).

Comparative example:

(1) The present invention synthesized and expressed the following fusion proteins:

The coding base sequence DNA of all protein molecules in this comparative example was synthesized by Beijing Huada Gene Company. The construction of expression vectors, protein expression and purification, and protein quantification methods of all protein molecules were the same as in Example 2. All protein molecules The activity detection of all is identical with embodiment 3,4,5.

The fusion protein SEA-EPAPKP-iRGD has the amino acid sequence of SEQ ID NO:8 in Table 6 below, and its encoding gene sea-epapkp-irgd has the base sequence of SEQ ID NO:7 in Table 6 below.

Table 6

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 the amino acid sequence of SEQ ID NO:10 in Table 7 below, and its encoding gene seb-epapkp-irgd has the base sequence of SEQ ID NO:9 in Table 7 below.

Table 7

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 of SEQ ID NO:12 in Table 8 below, and its coding gene sec2-epapkp-irgd has the base sequence of SEQ ID NO:11 in Table 8 below.

Table 8

Note: The bold sequence is the linker sequence, and the underlined sequence is the targeting molecule sequence iRGD.

The fusion protein WWH-GGGGS (G ₄ S)-iRGD has the amino acid sequence in SEQ ID NO: 14 in the following table 9, and its coding gene wwh-ggggs-irgd has the amino acid sequence in the SEQ ID NO: 13 in the following table 9 base sequence.

Table 9

Note: The bold and underlined sequence is the superantigen modified sequence, the bold sequence is the linker sequence, and the underlined sequence is the targeting molecule sequence iRGD.

The fusion protein WWH-(GS) ₅ -iRGD has the amino acid sequence in SEQ ID NO:16 in the following table 10, and its coding gene wwh-gsgsgsgsgs-irgd has the base in the SEQ ID NO:15 in the following table 10 sequence.

Table 10

Note: The bold and underlined sequence is the superantigen modified 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 the amino acid sequence of SEQ ID NO:18 in Table 11 below, and its encoding gene wwh-epapk-irgd has the base sequence of SEQ ID NO:17 in Table 11 below.

Table 11

Note: The bold and underlined sequence is the superantigen modified 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 the amino acid sequence of SEQ ID NO:20 in Table 12 below, and its coding gene irgd-epapkp-wwh has the base sequence of SEQ ID NO:19 in Table 12 below.

Table 12

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 the amino acid sequence in SEQ ID NO:22 in the following table 13, and its coding gene wwh-epapkp-rgd has the base sequence in the SEQ ID NO:21 in the following table 13:

Table 13

Note: The sequence in bold is the linker sequence, and the underlined sequence is the targeting molecule sequence RGD.

The fusion protein WWH-EPAPKP-tLyp-1 has the amino acid sequence in SEQ ID NO:24 in Table 14 below, and its encoding gene wwh-epapkp-tlyp-1 has the base in SEQ ID NO:23 in Table 14 below base sequence:

Table 14

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 the amino acid sequence in SEQ ID NO:26 in the following table 15, and its coding gene strail-epapkp-irgd has the base sequence in the SEQ ID NO:25 in the following table 15:

Table 15

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 linking short peptides targeting the same receptor, the inventors constructed the same target targeting molecules RGD (for the target integrin αv) and tLyp-1 (for the target neuropil Protein NRP-1) is a fusion protein a.WWH-EPAPKP-RGD, b.WWH-EPAPKP-tLyp-1, wherein RGD has the ability to target integrin αv, and tLyp-1 has the ability to target NRP- 1 and through CendR to enhance the ability of drugs to penetrate tumor tissues. After the three proteins were constructed and purified, their targeting verification experiments and anti-tumor activities were tested respectively.

The target verification experiment method is the same as that in Example 3, and the results are shown in Figure 4 and Figure 5. The figure shows that at the same concentration, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD bind to the target cell B16F10 The strength of 4T1 was basically the same, while the ability of WWH-EPAPKP-iRGD to bind B16F10 cells was 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 4T1 cells was 2.24 times that of WWH-EPAPKP-RGD and 1.64 times that of WWH-EPAPKP-tLyp-1. It shows that the three fusion proteins of WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD and WWT-EPAPKP-iRGD have stronger binding force to target cells.

The in vitro anti-tumor activity verification test method is the same as that in Example 4, and the results are shown in Figure 6 and Figure 7. At a concentration of 350 pmol/μl, WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD inhibited the target cell microspheres The growth abilities of B16F10 and 4T1 were basically the same, and the tumor inhibition rate of WWH-EPAPKP-iRGD on B16F10 was 25.1% higher than that of WWH-EPAPKP-RGD, and 23.1% higher than that of WWH-EPAPKP-tLyp-1. The tumor inhibition rate of WWH-EPAPKP-iRGD on 4T1 was 24.5% higher than that of WWH-EPAPKP-RGD and 22.5% higher than that of WWH-EPAPKP-tLyp-1. This comparison of tumor inhibition results in vitro shows that the soluble fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD can carry effector molecules better than the other two targeting short peptides. function in the tumor microenvironment.

(3) The present invention compares the targeting ability and in vitro anti-tumor microsphere activity of fusion proteins formed by linking in different ways. In order to compare the different ways of linking iRGD-linked short peptides to SAg, based on WWH-EPAPKP-iRGD (iRGD is at the C-terminus of Sag), a comparative fusion protein iRGD-EPAPKP-WWH, which links iRGD to the N-terminus of superantigen Sag, was constructed. In order to compare the difference between the short linker peptide (linker) in the present invention and other commonly used linker short peptides, G4S (a kind of flexible linker short peptide, GGGGS), (GS) ₅ (a kind of flexible linker short peptide), EPAPK (Rigidly linked short peptide with one amino acid less than EPAPKP) Three fusion proteins WWH-GGGGS(G ₄ S)-iRGD, WWH-(GS) ₅ -iRGD, and WWH-EPAPK-iRGD formed by three different Linker connections protein. And the combination of targeting molecule iRGD and effector molecule WWH as two independent individuals (WWH+iRGD) is also used as a comparative example.

The experimental method for targeting verification is the same as in Example 3, and the results are shown in Figure 4 and Figure 5. At the same concentration, the ability of WWH-EPAPKP-iRGD to bind to B16F10 cells is 1.37 times that of WWH-GGGGS(G ₄ S)-iRGD , 1.42 times that of WWH-(GS)5-iRGD, 1.42 times that of WWH-EPAPK-iRGD, and 3.00 times that of iRGD-EPAPKP-WWH. The ability of WWH-EPAPKP-iRGD to bind 4T1 cells was 1.50 times that of WWH-GGGGS(G ₄ S)-iRGD, 1.80 times that of WWH-(GS)5-iRGD, 1.52 times that of WWH-EPAPK-iRGD, and iRGD - 2.69 times that of EPAPKP-WWH. These comparative data prove that: compared with other linkers, the fusion protein formed when using EPAPKP as a Linker can better play a role in target cell binding; in addition, compared with connecting iRGD to the N-terminal of the superantigen, the Linking iRGD to the C-terminus of the superantigen can make the fusion protein play a better role in binding to target cells.

The experimental method of anti-tumor microspheres in vitro is the same as that in Example 4. The results are shown in Figure 6 and Figure 7. At a concentration of 350 pmol/μl, the tumor inhibition rate of WWH-EPAPKP-iRGD on B16F10 is 20.9% higher than that of iRGD-EPAPKP-WWH Compared with WWH+iRGD, the tumor inhibition rate increased by 24.6%, compared with WWH-GGGGS(G ₄ S)-iRGD, the tumor inhibition rate increased by 19.9%, and compared with WWH-(GS)5-iRGD, the tumor inhibition rate increased by 21%. The tumor inhibition rate of WWH-EPAPK-iRGD increased by 16%; the tumor inhibition rate of WWH-EPAPKP-iRGD on 4T1 increased by 19.4% compared with iRGD-EPAPKP-WWH, and the tumor inhibition rate increased by 25.4% compared with WWH+iRGD, compared with WWH- The tumor inhibition rate of GGGGS(G ₄ S)-iRGD increased by 18.3%, which was 19% higher than that of WWH-(GS)5-iRGD, and 17.4% higher than that of WWH-EPAPK-iRGD. These comparative data prove that: compared with other linkers, the fusion protein composed of EPAPKP as the Linker can exert a better anti-tumor effect; in addition, compared with the N-terminus of the superantigen, the iRGD Linking to the C-terminus of the superantigen can make the fusion protein play a better role in suppressing tumors.

(4) In order to compare the difference in the anti-tumor effect of WWH, WWP, WWT in the present invention and other anti-tumor effector molecules (including other superantigens) after linking the same linking peptide and iRGD, a method of linking different effector molecules in the same linking manner was constructed. Four fusion proteins SEA-EPAPKP-iRGD, SEB-EPAPKP-iRGD, SEC2-EPAPKP-iRGD, sTRAIL-EPAPKP-iRGD composed of molecules were compared, and their biological activities were compared. The experimental procedure is the same as in Example 5.

As a result, it was found that the fusion proteins SEA-EPAPKP-iRGD and SEB-EPAPKP-iRGD constructed with SEA and SEB were very toxic, and the animal experiments quickly led to animal death (Fig. 10, Fig. 11); the fusion protein SEC2-iRGD constructed with SEC2- The antitumor effect of EPAPKP-iRGD at the same dose was not as good as that of the modified WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD (Figure 10, Figure 11).

The tumor inhibitory effect of the fusion protein sTRAIL-EPAPKP-iRGD with similar molecular weight was selected for comparison. The experimental method was the same as in Example 4. The results are shown in Figure 6 and Figure 7. The tumor inhibition rate of sTRAIL-EPAPKP-iRGD was 52% higher than that of sTRAIL-EPAPKP-iRGD, and the tumor inhibition rate of 4T1 was 51.4% higher than that of sTRAIL-EPAPKP-iRGD. It shows that the combination of fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD cannot show the same ideal effect when replaced with other types of effector molecules such as sTRAIL-EPAPKP-iRGD.

Replace the effector molecules with superantigens SEA, SEB, and SEC2, and connect the fusion proteins SEA-EPAPKP-iRGD, SEB-EPAPKP-iRGD, and SEC2-EPAPKP-iRGD composed of iRGD in the same way, according to the same experiment as in Example 5 The mouse survival curve obtained by the method is shown in Figure 10 (wild SEC2 to P=0 of SEA, wild SEC2 to SEB P=0.001) and Figure 11 (wild SEC2 to P=0.001 of SEA, wild SEC2 to SEB P=0.001 ), the figure shows that the average survival days of the B16F10 model mice in the fusion protein SEA-EPAPKP-iRGD administration group was 11.17 days, and the average survival days of the B16F10 model mice in the fusion protein SEB-EPAPKP-iRGD administration group was 12 days, which was lower than the average survival days (18.17 days) of the control group (PBS), while the average survival days of the B16F10 model mice in the fusion protein SEC2-EPAPKP-iRGD administration group was 20.50 days, and the fusion protein WWH-EPAPKP- The average survival days of iRGD was 26.83 days, the average survival days of WWP-EPAPKP-iRGD was 26.83 days, and the average survival days of WWT-EPAPKP-iRGD was 26.50 days. The average survival days of 4T1 model mice in the fusion protein SEA-EPAPKP-iRGD administration group was 25 days, and the average survival days of 4T1 model mice in the fusion protein SEB-EPAPKP-iRGD administration group was 25.83 days, which was lower than that of the control group (PBS) average survival days (35.67 days), while the average survival days of fusion protein SEC2-EPAPKP-iRGD administration group 4T1 model mice was 47.5 days, and the average survival days of fusion protein WWH-EPAPKP-iRGD was 57.5 days , the average survival days of WWP-EPAPKP-iRGD was 58 days, and the average survival days of WWT-EPAPKP-iRGD was 59.17 days. This result shows that the ideal tumor inhibitory effect of fusion proteins WWH-EPAPKP-iRGD, WWP-EPAPKP-iRGD, and WWT-EPAPKP-iRGD is not universal on other superantigen or non-superantigen effector molecules.

SEQUENCE LISTING

<110> Shenyang Institute of Applied Ecology, Chinese Academy of Sciences

<120> A fusion protein and its preparation and application

<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 catttatgtat 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 aaagagttt 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 catttatgtat 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 aaagagttt 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 catttatgtat 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 aaagagttt 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 taacgatta 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 atttattatta tcaatgttat 360

ttttctaaaaaaacgaatga tattaattcg catcaaactg acaaacgaaaaacttgtatg 420

tatggtggtg taactgagca taatggaaac caattagata aatatagaag tattactgtt 480

cgggtatttg aagatggtaa aaattttatta tcttttgacg tacaaactaa taagaaaaag 540

gtgactgctc aagaattaga ttacctaact cgtcactatt tggtgaaaaa taaaaaactc 600

tatgaattta acaactcgcc ttatgaaacg ggatatatta aatttataga aaatgagaat 660

agcttttggt atgacatgat gcctgcacca ggagataaat ttgaccaatc taaatatta 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 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 catttatgtat 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 catttatgtat 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 aaagagttt 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-gsgsgsgsgs-irgd

<400> 15

gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt tactggtacg 60

atgggtaata tgaaatattt atatgatgat catttatgtat 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 aaagagttt 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 catttatgtat 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 aaagagttt 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 catttatgtat 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 aaagagttt 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 catttatgtat 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 aaagagttt 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 strail-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> linked 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 (21)

1. A fusion protein, characterized in that the fusion protein comprises sequentially from the N-terminus to the C-terminus: SEC2 or its mutant, connecting short peptides and iRGD, 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 in SEQ ID NO: 29, The amino acid sequence of the iRGD is shown in SEQ ID NO:30; The amino acid sequence of the mutant is as shown in the 1st to 237th positions of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. 2. fusion protein as claimed in claim 1, is characterized in that, the nucleotide sequence of coding described fusion protein is as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID in sequence listing NO:11 shown. 3. A fusion gene, characterized in that it encodes the fusion protein according to claim 1. 4. fusion gene as claimed in claim 3 is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:11 in the sequence listing . 5. A recombinant expression vector, characterized in that the recombinant expression vector contains the fusion gene according to claim 3 or 4. 6. The recombinant expression vector according to claim 5, wherein the backbone vector of the recombinant expression vector is pET-28a-TEV. 7. A transformant, characterized in that the fusion gene according to claim 3 or 4 or the recombinant expression vector according to claim 5 or 6 is introduced into the host. 8. The transformant according to claim 7, wherein the host is Escherichia coli. 9. The transformant according to claim 8, wherein the host is E. coli E. coli BL21 (DE3) cells or E. coli TG1. 10. A method for preparing a fusion protein, comprising the following steps: (1) obtaining the transformant as described in any one of claims 7-9; (2) Screening the transformant, expressing and purifying the fusion protein. 11. The preparation method according to claim 10, characterized in that, in step (2), said purifying comprises centrifuging and collecting supernatant after said expressed thallus is subjected to ultrasonic crushing, and undergoes two Ni-affinity Chromatography is enough. 12. preparation method as claimed in claim 11, is characterized in that, described Ni affinity chromatography comprises with the loading speed of 0.2-0.8ml/min sample is loaded on the Ni affinity chromatography column that balances in advance ;Elute with elution buffer after washing with 8-12 column volumes of equilibration buffer. 13. The preparation method according to claim 12, wherein 10 column volumes of equilibration buffer are used for washing. 14. The preparation method according to claim 13, wherein the equilibrium buffer is an equilibrium buffer containing 20-80 mM imidazole; and/or, the pH of the equilibrium buffer is 7.2 to 8.0; and /or, the elution buffer is an elution buffer containing 250-300 mM imidazole; and/or, the pH of the elution buffer is 7.2-8.0. 15. The preparation method according to claim 14, wherein the equilibrium buffer consists of: 20-30mM Tirs-HCl, 800-1000mM NaCl, 20-80mM imidazole; and/or, the elution buffer The composition of the solution is: 20-30mM Tirs-HCl, 800-1000mM NaCl, 250-300mM imidazole. 16. The preparation method according to claim 11, characterized in that, a step of ultrafiltration and desalting is also included between the two Ni affinity chromatography. 17. The preparation method according to claim 16, characterized in that, after the ultrafiltration and desalination, it further comprises the step of mixing with TEV protease to digest. 18. The preparation method according to claim 17, characterized in that, the molar ratio of the ultrafiltration desalted product to the TEV protease is 1:5; and/or, the enzyme cleavage time is 24h. 19. A fusion protein as claimed in claim 1 or 2, a fusion gene as claimed in claim 3 or 4, a recombinant expression vector as claimed in claim 5 or 6, or any one of claims 7-9 The use of the transformant described in the item in the preparation of medicines. 20. The use according to claim 19, characterized in that the use is in the preparation of drugs for treating tumors. 21. The use according to claim 20, characterized in that the use is in the preparation of drugs for the treatment of tumor immunity.

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