CN113354738B

CN113354738B - Fusion toxin VEGF 165b mGEL and its coding gene and application

Info

Publication number: CN113354738B
Application number: CN202010145502.9A
Authority: CN
Inventors: 孙红琰; 邵帅; 刘海昌; 胡栋
Original assignee: Shaoxing Defanghua Biotechnology Co ltd
Current assignee: Shao Shuai; Sun Hongyan; Zhejiang Yuchang Biotechnology Co ltd
Priority date: 2020-03-05
Filing date: 2020-03-05
Publication date: 2022-09-09
Anticipated expiration: 2040-03-05
Also published as: CN113354738A

Abstract

The invention discloses a fusion toxin VEGF _165b The mGEL and its coding gene and application belong to the field of biological pharmacy. Fusion toxin VEGF provided _165b /mGEL comprises VEGF ₁₆₅ Inhibitory splice isoforms VEGF _165b Together with Gelonin mutant mGEL, it inhibits neovascularization and destroys neovascularization. The fusion toxin can be used as an active component to prepare medicines for treating various neovascular dependent diseases such as malignant tumor, neovascular eye diseases and the like, and can play an important role in the fields of medicine and biological pharmacy.

Description

FusionToxin VEGF 165b mGEL and its coding gene and application

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to fusion toxin VEGF _165b /mGEL and coding gene and application thereof, in particular to VEGF ₁₆₅ An inhibitory splice isomer and gelonin mutant fusion toxin, a coding gene, a preparation method and application thereof, in particular to application in treating neovascular dependence diseases such as malignant tumor, neovascular eye disease and the like.

Background

The angiogenesis-dependent diseases are a general term for a series of diseases including malignant tumors (e.g., lung cancer, gastrointestinal cancer, etc.), ocular neovascular diseases (e.g., wet macular degeneration, etc.), and the like. Malignant tumor growth depends on neovascularization, and if nutrition is obtained only by diffusion, the size of the tumor can not exceed 2mm ³ And thus will be at rest. However, tumor cells promote peripheral neovascularization during growth by autocrine of various cytokines. Before the new blood vessels grow in, the tumor grows linearly; after the new blood vessels grow in, the tumor grows exponentially. Therefore, inhibiting angiogenesis or destroying existing neovascular networks is equivalent to cutting off the nutrient supply of tumors, stopping the growth, keeping the tumors in a 'dormant' state, and finally eliminating the tumors by organisms after atrophy, apoptosis and necrosis.

Ocular neovascular diseases mainly include corneal neovascular hyperplasia and iris neovascular hyperplasia: (1) normal cornea has no blood vessels, and under the pathological conditions of wearing corneal contact lenses, ocular trauma, infectious and immune eye diseases and the like, blood capillaries invade from a corneal limbus vascular network to form corneal neovessels. Corneal neovascularization is structurally fragile, permeable and often blinding due to bleeding, exudation and secondary fibrosis. (2) Iris neovascularization is often followed by diseases of other parts of the eye such as central retinal vein occlusion, diabetes and the like and systemic diseases, and is a common cause of blindness and eyeball removal. Retinopathy is complicated by about 50% of diabetic patients with a history of more than ten years. Therefore, the eye disease of the neovascular eye can be effectively controlled and treated by inhibiting the generation of the neovascular or destroying the existing neovascular network.

Vascular endothelial cell growth factor (VEGF) and its receptor (VEGFR) family play an important role in the process of neovascularization, and are important targets for drug design. Human VEGF produces several different isoforms due to mRNA splicing, i.e., VEGF ₁₂₁ 、VEGF ₁₆₅ 、VEGF ₁₈₉ And VEGF ₂₀₈ Wherein VEGF ₁₂₁ And VEGF ₁₆₅ Is soluble cytokine, and has effects in promoting proliferation of vascular endothelial cells, angiogenesis, increasing vascular permeability, and accelerating blood flow. VEGFR, particularly VEGFR2, is a biomarker for neovascular endothelial cells with high specificity, and is highly expressed on the surface of neovascular endothelial cells, but very low or almost undetectable in the expression level of vascular endothelial cells in normal tissues.

It has been shown that differential splicing of the VEGF gene results in the production of VEGF that promotes angiogenesis _xxx And VEGF inhibiting angiogenesis _xxxb . In VEGF _xxxb In family, VEGF _165b Most typically, it antagonizes VEGF on the one hand by binding with high affinity to VEGFR ₁₆₅ The binding of the vascular endothelial growth factor and the receptor thereof can not activate the signal transduction pathway in the vascular endothelial cells, so that the vascular endothelial growth factor becomes a VEGF antagonist and plays a role in inhibiting the proliferation and angiogenesis of the neovascular endothelial cells.

Currently, the targeted drugs of vascular endothelial growth factor and its receptor are mainly divided into two categories: one is angiogenesis inhibitor, which only inhibits angiogenesis but can not destroy existing new vessels, such as bevacizumab and engdu; another class is neovascular disruptors, i.e., agents that treat diseases such as tumors by recognizing and disrupting existing neovascular networks, e.g., VEGFR-targeted fusion toxins, reported to be predominantly VEGF ₁₆₅ /DT390、VEGF ₁₂₁ /DT390 and VEGF ₁₂₁ rGel et al, wherein VEGF ₁₂₁ And VEGF ₁₆₅ Only plays the role of a targeting molecule, can not inhibit the generation of new blood vessels, and releases VEGF ₁₂₁ And VEGF ₁₆₅ Also has the effect of promoting neogenesisAngiogenesis; in addition, DT390 toxin has strong immunogenicity, poor curative effect after multiple administrations and causes severe vascular leakage reaction; the used rGEL toxin is wild type Gelonin, and has low solubility and biological activity, so that the method has a plurality of defects. The angiogenesis inhibitor and the angiogenesis disruptor both play a role by inhibiting angiogenesis or disrupting existing angiogenesis through a single mechanism, are also single targets, and do not play dual roles of the angiogenesis inhibitor and the disruptor.

Disclosure of Invention

In view of one or more of the problems of the prior art, one aspect of the present invention is to provide a fusion toxin VEGF _165b /mGEL, the fusion toxin VEGF _165b /mGEL comprises VEGF ₁₆₅ Inhibitory splice isoforms VEGF _165b And the Gelonin mutant mGEL can simultaneously inhibit the generation of new vessels and destroy a new vessel network aiming at new vessel dependent diseases.

The difference between the amino acid sequence of the mGEL of the Gelonin mutant and the amino acid sequence of the natural Gelonin is as follows: lys10, Cys44 and Cys50 of the amino acid sequence of the natural Gelonin are mutated into Ser10, Ala44 and Ala50 respectively in the amino acid sequence of the Gelonin mutant mGEL, and the amino acid residue of the Gelonin mutant mGEL is shown as the amino acid sequence from the 171-417(247aa) position of the amino terminal of the amino acid sequence shown as SEQ ID NO:1 in the sequence table.

The fusion toxin VEGF _165b the/mGEL further comprises an endoplasmic reticulum localization sequence located at the carboxy terminus of the Gelonin mutant mGEL; optionally, the amino acid residue sequence of the endoplasmic reticulum positioning sequence is shown as a sequence SEQ ID NO: 5, respectively.

The fusion toxin VEGF _165b /mGEL further comprises a linker peptide or analogue thereof for linking to the VEGF ₁₆₅ Inhibitory splice isoforms VEGF _165b And Gelonin mutant mGEL; optionally, the amino acid residue sequence of the connecting peptide is shown as SEQ ID NO: 3, respectively.

The fusion toxin VEGF _165b The amino acid residue sequence of/mGEL is one of the following amino acid residue sequences:

1) SEQ ID NO: 1;

2) and (2) mixing the sequences shown in SEQ ID NO:1 through substitution, deletion or addition of one to ten amino acid residues, and has the functions of VEGFR specificity, angiogenesis inhibition and endothelial cell killing of new blood vessels.

In another aspect of the invention, there is provided the fusion toxin VEGF _165b Encoding gene of/mGEL (VEGF) _165b /mGEL), in particular, the coding gene (VEGF) _165b /mGEL) is one of the following nucleotide sequences:

1) SEQ ID NO: 2;

2) (ii) encoding the amino acid sequence of SEQ ID NO: 1;

3) and SEQ ID NO: 2 has more than 90 percent of homology, VEGFR specificity, and the nucleotide sequence has the functions of inhibiting the generation of new vessels and killing endothelial cells of the new vessels;

4) under high stringency conditions, the polypeptide can be compared with SEQ ID NO: 2, and 2, or a nucleotide sequence which hybridizes to the nucleotide sequence shown in the figure.

The invention also provides a recombinant expression vector pDSS-VEGF _165b /mGEL, the recombinant expression vector pDSS-VEGF _165b /mGEL comprises the above-mentioned coding gene (VEGF) _165b /mGEL) for expressing the fusion toxin VEGF _165b /mGEL。

In still another aspect, the present invention provides a transgenic cell line or a host bacterium comprising the above-mentioned coding gene (VEGF) _165b /mGEL) or the recombinant expression vector described above for the fusion toxin VEGF described above _165b /mGEL。

In still another aspect of the present invention, there is provided a therapeutic agent for a neovascular dependent disease comprising the fusion toxin VEGF described above _165b /mGEL or the above-mentioned coding gene (VEGF) _165b /mGEL)；

Optionally, the therapeutic agent for a neovascular dependence disease further comprises one or more of: pharmaceutically acceptable carrier, diluent, excipient, filler, adhesive, wetting agent, disintegrant, absorption enhancer, and surfactant.

The above-mentioned neovascular dependent diseases include, but are not limited to, malignant tumors, ocular neovascular diseases.

In still another aspect, the invention provides a recombinant expression of the fusion toxin VEGF _165b A method of/mGEL comprising the steps of:

1) constructing the transgenic cell line or the host bacterium, and culturing the transgenic cell line or the host bacterium;

2) separating and purifying protein from culture medium or cells of transgenic cell line or host bacteria to obtain fusion toxin VEGF _165b /mGEL。

The method specifically comprises the following steps:

a) construction of a vector containing the above-described coding Gene (VEGF) _165b /mGEL) recombinant expression vector pDSS-VEGF _165b /mGEL；

b) Subjecting the recombinant expression vector pDSS-VEGF _165b the/mGEL is transformed into a transgenic cell line or a host bacterium and is cultured and expressed to obtain a fusion protein DSS-VEGF _165b /mGEL；

c) Excision of the fusion protein DSS-VEGF with the tool enzyme ULP1 _165b DSS label of/mGEL, purifying to obtain fusion toxin VEGF _165b /mGEL；

Preferably, the tool enzyme ULP1 and the fusion protein DSS-VEGF in step c) _165b The dosage proportion relation of/mGEL is as follows: 1: (100- > 150); further preferably 1: 100.

Fusion toxin VEGF provided based on the technical scheme _165b mGEL has double functions of a neovascular inhibitor and a destructive agent, and is prepared from a targeting molecule-recombinant human vascular endothelial growth factor VEGF ₁₆₅ Inhibitory splice isoforms VEGF _165b And an effector molecule, Gelonin mutant (mGEL), wherein VEGF _165b Is not only a VEGF antagonistAnd VEGFR targeting molecules: inhibition of VEGF function and neovascularization by competitive binding to VEGFR; meanwhile, the fusion toxin is brought into the endothelial cells of the new vessels by identifying VEGFR and endocytosis mechanism, the toxin molecule mGEL is released and is positioned to the rough endoplasmic reticulum through a C-terminal KDEL motif, and the synthesis of cell protein is inhibited, thereby destroying the existing new vessel network. Moreover, the fusion toxin has simple expression condition, is easy to purify and is suitable for industrial production. The fusion toxin can be used as an active ingredient to prepare medicines for treating various angiogenesis-dependent diseases such as malignant tumors (such as lung cancer, gastrointestinal cancer and the like) and neovascular eye diseases (such as macular degeneration), can play an important role in the fields of medicine and biological pharmacy, and has wide application prospect.

Drawings

FIG. 1 shows DSS-VEGF _165b GEL electrophoresis of GEL expression and purification in E.coli Origami (DE 3);

FIG. 2 shows DSS-VEGF _165b The expression and purification of the gel electrophoretogram of/mGEL in E.coli Origami (DE 3);

FIG. 3 is a diagram of the expression and purification gel electrophoresis of GST-ULP1 in E.coli;

FIG. 4 shows DSS-VEGF _165b The expression, purification and enzyme digestion of/mGEL identify the gel electrophoresis picture;

FIG. 5 shows DSS tag excision and VEGF _165b GEL electrophoresis image of GEL purification;

FIG. 6 shows DSS-VEGF _165b Expression purification of/mGEL, DSS tag excision and VEGF _165b a/mGEL purified gel electrophoresis image;

FIG. 7 shows VEGF _165b The cytotoxicity curve of GEL fusion toxin to PAE/KDR;

FIG. 8 shows VEGF _165b Cytotoxicity profiles of/mGEL fusion toxin versus PAE/KDR.

Detailed Description

The invention aims to provide a multi-target solution for treating the angiogenesis-dependent diseases, so that the multi-target solution has the dual functions of inhibiting angiogenesis and destroying a angiogenesis network.

An object of the present invention is toProvides a recombinant human vascular endothelial growth factor VEGF _165b Inhibitory splice isoforms VEGF _165b The fusion toxin with Gelonin mutant (mGEL) of Gelonin is named VEGF _165b /mGEL comprising VEGF ₁₆₅ Inhibitory splice isoforms VEGF _165b And the Gelonin mutant mGEL can simultaneously inhibit the generation of new blood vessels and destroy new blood vessel networks aiming at new blood vessel dependent diseases.

Specifically, the recombinant human vascular endothelial growth factor VEGF is connected to the amino terminal (N-terminal) of the Gelonin mutant through a connecting peptide ₁₆₅ Inhibitory splice isoforms VEGF _165b The fusion toxin is obtained, and the carboxyl terminal (C-terminal) of the fusion toxin has an endoplasmic reticulum localization sequence. The fusion toxin is a novel protein with double functions of a neovascular inhibitor and a neovascular damaging agent. Wherein:

VEGF _165b is a known recombinant human vascular endothelial growth factor VEGF ₁₆₅ The inhibitory splice isomer is a VEGF antagonist and a VEGFR targeting molecule, and has the functions of inhibiting the proliferation of neovascular endothelial cells and the generation of new blood vessels and targeting VEGFR.

The Gelonin mutant (mGEL) is a mutant protein of natural Gelonin, which is an I-type ribosome inactivating protein existing in white tree seeds, can act on 28S rRNA on the ribosome large subunit of a mammal nucleus, generates an adenine-removing effect, destroys a ribosome structure, inhibits protein translation, and participates in the regulation of apoptosis. According to the invention, Lys10, Cys44 and Cys50 of natural Gelonin amino acid are respectively mutated into Ser10, Ala44 and Ala50 to reduce protein disulfide mismatch, improve soluble expression and improve the killing effect of fusion toxin on target cells, so that mGEL can be used as a toxin molecule to inhibit cell protein synthesis and destroy the existing new blood vessel network.

In order to position the toxin molecule mGEL to the rough endoplasmic reticulum of the endothelial cells of the new blood vessels, an endoplasmic reticulum positioning sequence is arranged at the carboxyl end (C-end) of the mGEL, the endoplasmic reticulum positioning sequence is a KDEL motif, and the amino acid residue sequence of the endoplasmic reticulum positioning sequence can be shown as a sequence SEQ ID NO: 5, the nucleotide sequence of the coding gene can be shown as SEQ ID NO: and 6.

Use of linker peptides for VEGF _165b The carboxy terminus (C-terminus) of (C) is linked to the amino terminus (N-terminus) of the mGEL. The choice of linker peptide is manifold, for example, the amino acid residue sequence may be as shown in SEQ ID NO: 3, the nucleotide sequence of the coding gene can be shown as SEQ ID NO: 4, respectively. The linker peptide may be replaced by an analogue thereof, which differs from the linker peptide by either an amino acid sequence, a modified form which does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biology techniques. Analogs also include analogs having amino acid residues other than the naturally occurring L-amino acid residues (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids.

In particular, the fusion toxin VEGF _165b the/mGEL is one of the following amino acid residue sequences:

1) SEQ ID NO: 1;

2) and (2) mixing the sequences shown in SEQ ID NO:1 through the substitution, deletion or addition of one to ten amino acid residues, and has the functions of VEGFR specificity, angiogenesis inhibition and angiogenesis network disruption.

SEQ ID NO:1 consists of 421 amino acid residues, and the 1 st to 165 th (165aa) amino acid residues from the amino terminal are recombinant human vascular endothelial growth factor VEGF ₁₆₅ Inhibitory splice isoforms VEGF _165b The 166-170(5aa) amino acid residue from the amino terminal is a connecting peptide, the 171-417(247aa) amino acid residue from the amino terminal is a Gelonin mutant mGEL, and the 417 (4aa) amino acid residue from the amino terminal is a rough endoplasmic reticulum positioning signal sequence.

Further, in one aspect, the fusion toxin VEGF _165b Polypeptide fragments, derivatives and analogues of/mGEL also belong to the invention, which interact with VEGF _165b the/mGEL has the same biological function or activity, wherein the polypeptide fragment is defined as: 1) polypeptides substituted with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code; 2) polypeptides having a substituent group in one or more amino acid residues; 3) a polypeptide formed by fusing a mature polypeptide to another compound; 4) fusion polypeptides can be produced by fusing an additional amino acid sequence to the polypeptide sequence (e.g., to purify the sequence of the polypeptide, or to a fusion toxin of an antibody fragment or other antigen-ligand sequence), or by fusing a nucleic acid sequence (or portion thereof) encoding another polypeptide to the nucleic acid sequence (or portion thereof) of the invention to obtain the coding sequence for the fusion polypeptide, and expressing the coding sequence for the fusion polypeptide. Such techniques for producing fusion polypeptides are well known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator.

Fusion toxin VEGF _165b Derivatives of/mGEL refer to the use of VEGF in the invention _165b And fusion toxin constructed by fusing other toxin molecules.

VEGF _165b Analogues of/mGEL with VEGF _165b The difference in/mGEL may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both. The analogs include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having amino acid residues other than the naturally occurring L-amino acid residues (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids. It is to be understood that the amino acid residue sequence of the fusion toxin of the present invention is not limited to the representative sequences exemplified above.

Further, in another aspect, the fusion toxin VEGF _165b the/mGEL may also be a VEGF modified or modified to increase its anti-proteolytic or solubility properties _165b a/mGEL polypeptide. Modified (generally without altering primary structure) forms include: 1) chemically derivatized forms of the polypeptide in vivo or in vitro, such as acetylated or carboxylated; 2) glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps, which can be accomplished by exposing the polypeptide to an enzyme that effects glycosylation (e.g., mammalian glycosylase or deglycosylase); 3) having a sequence of phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

Gene encoding the above fusion toxin (VEGF) _165b /mGEL) also belongs to the invention, the gene is one of the following nucleotide sequences:

1) SEQ ID NO: 2;

2) encoding the amino acid sequence shown in SEQ ID NO: 1;

4) under high stringency conditions with SEQ ID NO: 2, and 2, or a nucleotide sequence which hybridizes to the nucleotide sequence shown in the figure.

The high stringency conditions are those in which the membrane is washed after hybridization with a solution containing 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.

SEQ ID NO: 2 consists of 1263 bases, the coding sequence of which is the 1 st to 1131 st bases from the 5' end, and codes the nucleotide sequence of SEQ ID NO:1, coding a recombinant human vascular endothelial growth factor VEGF from 1 to 495 th base of 5' end ₁₆₅ Inhibitory splice isoforms VEGF _165b The 496-510 th base from the 5 ' end encodes the connecting peptide, the 511-1251 th base from the 5 ' end encodes the gelonin mGEL, and the 12 th base from the 5 ' end encodes the gelonin mGELBases 52-1263 encode signals for localization of endoplasmic reticulum.

Encoding the fusion toxin VEGF of the invention _165b the/mGEL polynucleotide may be in DNA form or RNA form. The form of DNA includes cDNA or synthetic DNA, which may be single-stranded or double-stranded, and may be either the coding strand or the non-coding strand.

Further, encoding the fusion toxin VEGF of the present invention _165b Variants of the polynucleotides of the/mGEL, which encode and fuse the toxin VEGF _165b the/mGEL has the same amino acid sequence of the polypeptide or polypeptide fragments, analogues and derivatives. The variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants, and may include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

Containing the Gene of the invention (VEGF) _165b /mGEL), transgenic cell lines and host bacteria.

Another objective of the invention is to provide a method for recombinant expression of the fusion toxin VEGF _165b The preparation method of/mGEL is to use VEGF containing fusion toxin _165b /mGEL coding gene recombinant expression vector transformation or transduction host cell, culture of host cell, separation and purification of protein from culture medium or cell, obtaining fusion toxin VEGF _165b /mGEL。

The method comprises the following steps: VEGF containing fusion toxin _165b The recombinant expression vector of the coding gene of mGEL is fusion toxin VEGF _165b The coding gene of/mGEL or its variant gene is inserted into a recombinant expression vector, Xba I restriction site is added at the upstream of the coding gene, and Not I restriction site is added at the downstream. The starting vector for constructing the recombinant expression vector can be any one of bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors which are well known in the art and can express foreign genes.The starting vectors include, but are not limited to: expression vectors based on the T7 promoter for expression in bacteria, vectors for expression in mammalian cells and baculovirus-derived vectors for expression in insect cells. In general, any plasmid and vector can be used as long as they can replicate and are stable in the host. An important feature of the starting vector is that it usually contains a replication site, a promoter, a marker gene and translation control elements. The starting carrier preferably comprises an expression carrier containing disulfide bond isomerase DsbC-ubiquitin-like protein modification molecule SUMO fusion protein DsbC-SUMO (DSS for short), named as pDSS carrier, the carrier is based on pET carrier, and the amino acid residue sequence of the DsbC-SUMO fusion protein is shown as SEQ ID NO: 7, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: shown in fig. 8. DsbC-SUMO can promote the formation of protein disulfide bonds and the repair of mismatched disulfide bonds in bacterial cytoplasm, and promote the correct folding and soluble expression of fusion proteins.

VEGF containing fusion toxin constructed by taking pDSS carrier as starting carrier _165b The recombinant expression vector of the coding gene of the mGEL is named as pDSS-VEGF _165b and/mGEL. Recombinant expression vector pDSS-VEGF _165b The fusion protein expressed by the mGEL in the host cell or host bacterium has a DSS label, and the fusion protein is named DSS-VEGF _165b The amino acid sequence of the/mGEL is shown as SEQ ID NO. 9 in the sequence table, and the nucleotide sequence for coding the amino acid sequence is shown as SEQ ID NO. 10 in the sequence table. The DSS tag can be excised by using a tool enzyme ULP1 to finally obtain fusion toxin VEGF with natural N-terminal _165b /mGEL。

The recombinant expression vector pDSS-VEGF can be constructed by methods well known to those skilled in the art _165b mGEL, such as in vitro recombinant DNA technology, DNA synthesis technology and in vivo recombinant technology (Sambrook, et al Molecular cloning, a Laboratory Manual. Cold spring harbor Laboratory. New York, 1989). The fusion toxin VEGF _165b The DNA sequence of the gene encoding/mGEL can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The promoter may be: lac or trp promoter of Escherichia coli, phageSomatic promoters, retroviruses and other known promoters which control the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the recombinant expression vector pDSS-VEGF _165b the/mGEL may also contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as the dihydrofolate reductase gene, neomycin resistance gene, and the Green Fluorescent Protein (GFP) gene for eukaryotic cells, or the tetracycline or ampicillin resistance gene for E.coli, among others. Fusion toxin VEGF of the invention _165b When the gene encoding mGEL is expressed in a higher eukaryotic cell, the transcription can be enhanced by using a recombinant expression vector pDSS-VEGF _165b An enhancer sequence is inserted into/mGEL. Enhancers are cis-acting elements of DNA, usually 10-300 base pairs in length, that act on a promoter to increase transcription of a gene. Such as the SV40 enhancer on the late side of the replication origin at about 100-270 bp in length, the polyoma enhancer or adenovirus enhancer on the late side of the replication origin, and the like.

In this method, the transformed or transduced host cell may be a prokaryotic cell, such as a bacterial cell; lower eukaryotic cells, such as yeast cells; higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomycete; bacterial cells of salmonella typhimurium; eukaryotic cells such as yeast, plant cells; insect cells such as Drosophila S2 or Sf 9; CHO, COS, 293 cells or Bowes melanoma cells.

The recombinant expression vector pDSS-VEGF can be expressed by conventional techniques well known to those skilled in the art _165b mGEL transformation of host cells, culture of transformants, inducible expression of the protein of interest (i.e., fusion toxin VEGF) _165b /mGEL) and separating and purifying the target protein.

Culturing the fusion toxin VEGF with the dual functions of inhibiting the generation of new blood vessels and destroying the network of the new blood vessels _165b The culture medium and culture conditions of the host cell of the encoding gene of the mGEL can be both culture initiationThe culture medium and the culture conditions of the host.

Use of fusion toxin VEGF _165b The invention also provides a medicament for treating angiogenesis-dependent diseases, including malignant tumors (such as lung cancer and gastrointestinal cancer) and neovascular eye diseases (such as wet macular degeneration). The active component of the medicine comprises the fusion toxin VEGF _165b /mGEL or a gene encoding the same.

When the active ingredient in the medicament for treating the new vessel dependent diseases is fusion toxin VEGF _165b When encoding gene of mGEL, the fusion toxin VEGF _165b The gene encoding/mGEL may be present in a eukaryotic expression vector.

If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier includes diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, etc. which are conventional in the pharmaceutical field.

The medicine of the present invention may be prepared into injection, freeze dried powder, etc. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

The percentage concentration is a mass/volume (W/V) percentage concentration or a volume/volume (V/V) percentage concentration unless otherwise specified.

The primers, DNA sequence synthesis and DNA sequence determination were performed by Nanjing Kingsrey Biotech.

The various biomaterials described in the examples are obtained by way of experimental acquisition for the specific purpose disclosed and should not be construed as limiting the source of the biomaterials of the present invention. In fact, the sources of the biological materials used are wide, and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples; in industrial practice, various cells derived from mammals such as rat, mouse, pig or human are isolated, and include those obtained from cell banks or commercially available, prepared according to the teachings of the prior art, and induced by known methods from a variety of commercially available stem cells.

The embodiments are provided to assist understanding of the present invention, but the scope of the present invention is not limited to the following embodiments.

Example 1 fusion toxin VEGF _165b Expression and purification of mGEL in Escherichia coli

Recombinant expression vector pDSS-VEGF _165b GEL and pDSS-VEGF _165b Construction of/mGEL

The DSS-VEGF was synthesized in whole gene by Nanjing Kingsrey Biotech Co., Ltd, with codon optimization according to the preference of Escherichia coli _165b GEL and DSS-VEGF _165b mGEL gene and construction of expression vector pDSS-VEGF _165b GEL and pDSS-VEGF _165b /mGEL。

DSS-VEGF _165b The nucleotide sequence of mGEL is shown as SEQ ID NO:10, SEQ ID NO:10 consists of 2342 bases and encodes a polypeptide having the sequence shown in SEQ ID NO:9, wherein the 1 st to 6 th bases from the 5 ' -end are Xba I enzyme cutting sites, the 43 th to 1065 th bases from the 5 ' -end encode DSS labels, and the 1066-1560 th bases from the 5 ' -end encode recombinant human vascular endothelial growth factor VEGF ₁₆₅ Inhibitory splice isoforms VEGF _165b The 1561-1575 base from the 5 ' -end encodes the connecting peptide, the 1576-2316 base from the 5 ' -end encodes the gelonin mutant mGEL, the 2317-2328 base from the 5 ' -end encodes the endoplasmic reticulum localization signal, the 2329-2334 base from the 5 ' -end is the stop codon, and the 2335-2342 base from the 5 ' -end is the Not I restriction site.

DSS-VEGF _165b The nucleotide sequence of the/GEL is shown as SEQ ID NO: 14, SEQ ID NO: 14 consists of 2342 bases and encodes a polypeptide having the sequence shown in SEQ ID NO: 13, wherein the 1 st to 6 th bases from the 5 ' -end are Xba I enzyme cutting sites, the 43 th to 1065 th bases from the 5 ' -end encode DSS labels, and the 1066 th and 1560 th bases from the 5 ' -end encode recombinant human vascular endothelial growth factor VEGF ₁₆₅ Inhibitory splice isoforms VEGF _165b The 1561-1575 th base from the 5 ' -end encodes the connecting peptide, 1576-2316 th base from the 5 ' -end encodes the wild type gelonin GEL, 2317-2328 th base from the 5 ' -end encodes the endoplasmic reticulum localization signal, 2329-2334 th base from the 5 ' -end is the stop codon, and 2335-2342 th base from the 5 ' -end is the Not I restriction site.

Secondly, construction of recombinant expression vector pGEX-ULP1

The ULP1 gene was synthesized from the entire gene by Nanjing Kinsley Biotechnology company according to the codon preference of Escherichia coli, and an expression vector pGEX-ULP1 was constructed. The nucleotide sequence is shown as SEQ ID NO: 12, SEQ ID NO: 12 consists of 677 bases and encodes a polypeptide having the sequence shown in SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof. The 1-6 bases from the 5 'end are BamH I enzyme cutting sites, the 7-663 bases from the 5' end encode ULP1, the 664-669 bases from the 5 'end are stop codons, and the 670-677 bases from the 5' end are Not I enzyme cutting sites.

Three, DSS-VEGF _165b /GEL、DSS-VEGF _165b Expression of/mGEL and ULP1 in E.coli

The specific expression method comprises the following steps:

(1) pDSS-VEGF constructed by the first step and the second step _165b /GEL、pDSS-VEGF _165b The plasmid mGEL and pGEX-ULP1 were transformed into Origimi (DE3) competent bacteria, respectively, and cultured at 37 ℃ for 16-20 hours. A single colony was inoculated into 10mL of LB medium (containing carbenicillin 200. mu.g/mL, kanamycin 50. mu.g/mL and tetracycline 25. mu.g/mL), and cultured overnight at 37 ℃ at 200 rpm.

(2) 10mL of overnight culture was transferred to 1L of LB medium (containing carbenicillin 200. mu.g/mL, Calif.)Natamycin 50. mu.g/mL and tetracycline 25. mu.g/mL), cultured at 37 ℃ and 200rpm to OD ₆₀₀ ≈0.4～0.6。

(3) IPTG was added to a final concentration of 0.25mM (250. mu.l of 1M IPTG solution was added), the culture was continued overnight at 16 ℃ and 180rpm, and the bacteria were harvested by centrifugation at 6000rpm for 5 minutes.

Four, VEGF _165b GEL and VEGF _165b Purification of/mGEL

(1) Purification of DSS-VEGF Using Ni-Sepharose FF _165b /GEL、DSS-VEGF _165b The mGEL fusion protein: the bacterial pellets of both were resuspended in binding buffer (50mM Tris. HCl, 300mM NaCl, 20mM imidazole, pH8.0) at a ratio of 1:20, sonicated in ice bath (power 600W, 5 seconds each, 10 seconds apart, 20 minutes), centrifuged at 12000rpm for 60 minutes at 4 ℃ and the supernatant was collected. The HisTrapFF nickel column (1ml pre-packed column) was equilibrated with 5 column volumes of binding buffer (50mM Tris. HCl, 300mM NaCl, 20mM imidazole, pH 8.0). Loading on a column at a flow rate of 1 mL/min; the heteroproteins were eluted with 5 column volumes of wash solution (50mM Tris. HCl, pH8.0, 300mM NaCl, 40mM imidazole). The target protein was eluted with 50mM Tris-HCl, 300mM NaCl, 200mM imidazole (pH 8.0).

For DSS-VEGF respectively _165b GEL and DSS-VEGF _165b The expression and purification of/mGEL in E.coli Origami (DE3) was examined by gel electrophoresis (12% SDS-PAGE), the results are shown in FIGS. 1 and 2, where FIG. 1 is DSS-VEGF _165b Results of detection of the expression and purification of GEL in Escherichia coli Origami (DE3), lanes 1-5 in FIG. 1 are respectively shown as: lane 1 molecular weight standards; lane 2 is whole ultrasonic lysis;

lanes

3, 4 are ultrasonication supernatants; lane 5 is the passage; lane 6 is eluted DSS-VEGF _165b a/GEL; FIG. 2 shows DSS-VEGF _165b The results of the detection of the expression and purification of/mGEL in E.coli Origami (DE3), lanes 1-5 in FIG. 2 are shown as: lane 1 is whole ultrasonic lysis; lane 2 is the lysis supernatant; lane 3 is the permeate; lane 4 shows eluted DSS-VEGF _165b (ii)/mGEL; lane 5 is molecular weight standard. Visible, DSS-VEGF _165b GEL and DSS-VEGF _165b The molecular weight of the/mGEL target protein is about 85kD, and the soluble DSS-VE in the thalli lysis supernatantGF _165b The expression level of/mGEL is obviously higher than that of DSS-VEGF _165b The expression level of GEL.

(2) GST-ULP1 purification Using GST HP: the bacterial pellet was resuspended in buffer A (10mM Na) at 1:20 ₂ HPO ₄ ， 1.8mM KH ₂ PO ₄ 140mM NaCl, 2.7mM KCl) was sonicated in an ice bath (power 600W, 5 seconds each, 10 seconds apart, 20 minutes), centrifuged at 12000rpm for 60 minutes at 4 ℃ and the supernatant was collected. After the 5 times column volume buffer A column is balanced, 1ml/min sample is loaded, buffer A washes impurities, the flow rate is 5ml/min, 5 column volumes. ULP1 protein was eluted with buffer B (50mM TrisCl, 10mM reduced glutathione, pH8.0) at a flow rate of 5ml/min, 3 bed volumes were collected and examined by gel electrophoresis, as shown in FIG. 3, wherein each lane is represented as: lane 1 molecular weight standards; lane 2 is whole ultrasonic lysis; lane 3 is the lysis supernatant; lane 4 is the crossing fluid; lane 5 is eluted GST-ULP 1.

(3) Determination of enzyme digestion system: (iii) use of GST-ULP1 obtained in step (2) above to DSS-VEGF obtained in step (1) above _165b The enzyme was cleaved with the aid of a different cleavage system (50mM Tris-HCl, 300mM NaCl, 200mM imidazole (pH8.0) buffer) at 8 ℃ overnight. As shown in FIG. 4, for DSS-VEGF _165b The expression, purification and cleavage results of/mGEL were examined by gel electrophoresis (12% SDS-PAGE), in which lanes 1-4 represent DSS-VEGF _165b Expression purification of/mGEL, lanes 5-13 show different cleavage systems (where approximately DSS-VEGF is contained per 100. mu.l cleavage system) _165b 120 μ g/mGEL fusion protein) of DSS-VEGF _165b The enzyme cutting identification result of/mGEL, wherein M represents a molecular weight standard. Visible, DSS-VEGF _165b the/mGEL is cut by ULP1 enzyme to obtain two fragments: fusion protein of interest VEGF _165b the/mGEL and DSS labels, and the different enzyme systems have different enzyme cutting effects, as can be seen from FIG. 4, when the tool enzyme ULP1 is compared with DSS-VEGF _165b When the dosage ratio of the mGEL fusion protein is about 1:150, the enzyme digestion efficiency can reach more than 90 percent; when the dosage ratio is 1:100, the enzyme digestion effect is better, and DSS-VEGF can be fully utilized _165b The DSS tag is excised from the/mGEL fusion protein.

(4) DSS labelExcision and VEGF _165b GEL and VEGF _165b Purification of mGEL: each 1. mu.g of ULP1 enzyme-digested 100. mu.g of fusion protein (DSS-VEGF) _165b /GEL or DSS-VEGF _165b /mGEL), 8 ℃ overnight. The cleavage products were diluted 1:3 with 50mM Tris Cl, pH8.0, applied to QFF column (50mM Tris HCl, 100mM NaCl, pH8.0 equilibrated), the permeate was collected, applied to SPFF column (50mM Tris HCl, 100mM NaCl, pH8.0 equilibrated) and washed for 5 column volumes. 50mM Tris-HCl, 300mM NaCl, pH8.0 respectively elute the fusion protein of interest VEGF _165b GEL and VEGF _165b and/mGEL. The results of 12% SDS-PAGE electrophoresis of the two samples are shown in FIGS. 5 and 6, where FIG. 5 shows the DSS tag excision and VEGF _165b The results of the GEL purification assay, shown in FIG. 5 as lanes 1-3: lane 1 is DSS-VEGF _165b The result of enzyme digestion of GEL by ULP 1; lane 2 is VEGF _165b The result after GEL reduction electrophoresis (the loading buffer solution contains beta-mercaptoethanol); lane 3 is VEGF _165b Results of GEL non-reduction (no beta-mercaptoethanol in loading buffer); m is a molecular weight standard. FIG. 6 shows DSS-VEGF _165b Expression purification of/mGEL, DSS tag excision and VEGF _165b The results of the/mGEL purification assay, shown in FIG. 6 as lanes 1-10: lane 1 molecular weight standards; lane 2 is whole ultrasonic lysis; lane 3 is the sonicated supernatant; lane 4 is the crossing fluid; lane 5 eluted protein; lane 6 shows the cleavage result; lane 7 is 1:3 dilution result; lane 8 is Q-column pass results; lane 9 is SP column crossing results; lane 10 shows VEGF eluted from SP column _165b and/mGEL. Visible DSS-VEGF _165b GEL and DSS-VEGF _165b After Ni-affinity chromatography, label removal and ion exchange chromatography are carried out on/mGEL, VEGF with higher purity is respectively obtained _165b GEL and VEGF _165b the/mGEL fusion toxin has a molecular weight of about 48kD, and VEGF is shown by the results of the enzyme cleavage in FIGS. 5 and 6 _165b The yield of the/mGEL fusion toxin is far higher than that of VEGF _165b The amount of/GEL fusion toxin obtained indicates that mutations in the mGEL sequence do increase soluble expression.

Example 2 VEGF _165b /GEL、VEGF _165b /mGEL fusion toxin vs VEGFR ₂ KDR positive cell PACytotoxicity of E/KDR (proffered by professor Chunchun of university of Beijing university's institute of Life sciences)

This example used VEGF obtained in example 1 above _165b GEL and VEGF _165b The mGEL fusion toxin is used for respectively detecting the toxic effect of the mGEL fusion toxin on PAE/KDR cells, and the specific method comprises the following steps:

1) dilution of PAE/KDR cells in logarithmic growth phase to 1.5X 10 ⁴ Perml, add to 96-well plates, 200. mu.l per well (3000 cells/well) (first row without cells), 37 ℃ 5% CO ₂ The incubation was carried out overnight. VEGF dilution in 96-well plates at 1:5 gradient _165b The initial concentration was 1. mu.M/mGEL, and the final volume was 200. mu.l/well. Pouring out the culture medium from the culture well, adding fusion toxin (first row with culture medium and second row without toxin) at 37 deg.C and 5% CO ₂ Culturing for 24 and 48 hours respectively.

2) The medium was decanted from the cell culture wells and 100 μ l of 0.5% crystal violet solution (in 20% methanol) was added, incubated for 30 minutes at room temperature, washed off the dye and air dried.

3) Mu.l of Sorenson's buffer (0.1M sodium citrate, pH 4.2, 50% ethanol) was added, incubated at room temperature for 60-90 minutes, and the absorbance was measured at 630 nm.

4) And (3) measuring the absorbance value (OD value) of each hole, and calculating the survival rate according to a formula: (mean OD value of experimental wells/mean OD value of negative control wells) × 100%, and a cell viability curve was plotted.

Synchronous detection of VEGF in the same manner as above _165b Toxic effects of GEL fusion toxin on PAE/KDR cells.

As shown in FIGS. 7 and 8 (the abscissa represents the concentration of fusion toxin and the ordinate represents the cell viability), VEGF was found from the respective curves _165b GEL and VEGF _165b /mGEL vs VEGFR ₂ IC50 of/KDR positive cell PAE/KDR is 0.56nM and 0.1nM respectively, VEGF can be seen _165b GEL and VEGF _165b /mGEL fusion toxin vs VEGFR ₂ The vascular endothelial cells PAE/KDR with high KDR expression have specific cytotoxic effect, and VEGF _165b The cytotoxicity of/mGEL is obviously higher than that of VEGF _165b Cytotoxicity of GEL.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Ningbo Defanghua Biotechnology Ltd

Changeable grandchild

Shao Shuai

<120> fusion toxin VEGF165b/mGEL and coding gene and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 421

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1 5 10 15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

20 25 30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

35 40 45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

50 55 60

Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile

65 70 75 80

Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe

85 90 95

Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg

100 105 110

Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe

115 120 125

Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser

130 135 140

Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Ser

145 150 155 160

Leu Thr Arg Lys Asp Gly Gly Gly Gly Ser Gly Leu Asp Thr Val Ser

165 170 175

Phe Ser Thr Ser Gly Ala Thr Tyr Ile Thr Tyr Val Asn Phe Leu Asn

180 185 190

Glu Leu Arg Val Lys Leu Lys Pro Glu Gly Asn Ser His Gly Ile Pro

195 200 205

Leu Leu Arg Lys Lys Ala Asp Asp Pro Gly Lys Ala Phe Val Leu Val

210 215 220

Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala Glu Ile Ala Ile Asp Val

225 230 235 240

Thr Ser Val Tyr Val Val Gly Tyr Gln Val Arg Asn Arg Ser Tyr Phe

245 250 255

Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu Gly Leu Phe Lys Asn Thr

260 265 270

Ile Lys Thr Arg Leu His Phe Gly Gly Ser Tyr Pro Ser Leu Glu Gly

275 280 285

Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu Gly Ile Glu Pro Leu Arg

290 295 300

Ile Gly Ile Lys Lys Leu Asp Glu Asn Ala Ile Asp Asn Tyr Lys Pro

305 310 315 320

Thr Glu Ile Ala Ser Ser Leu Leu Val Val Ile Gln Met Val Ser Glu

325 330 335

Ala Ala Arg Phe Thr Phe Ile Glu Asn Gln Ile Arg Asn Asn Phe Gln

340 345 350

Gln Arg Ile Arg Pro Ala Asn Asn Thr Ile Ser Leu Glu Asn Lys Trp

355 360 365

Gly Lys Leu Ser Phe Gln Ile Arg Thr Ser Gly Ala Asn Gly Met Phe

370 375 380

Ser Glu Ala Val Glu Leu Glu Arg Ala Asn Gly Lys Lys Tyr Tyr Val

385 390 395 400

Thr Ala Val Asp Gln Val Lys Pro Lys Ile Ala Leu Leu Lys Phe Val

405 410 415

Asp Lys Asp Glu Leu

420

<210> 2

<211> 1263

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcgccgatgg cggagggtgg cggccagaac catcacgagg ttgtgaaatt tatggacgtt 60

tatcaacgta gctattgcca cccgatcgaa accctggtgg acattttcca ggaatatccg 120

gatgagatcg aatacatctt taaaccgagc tgcgtgccgc tgatgcgctg cggtggctgc 180

tgcaatgacg agggtctgga atgcgttccg accgaagaga gcaacatcac catgcagatt 240

atgcgtatta agccgcatca aggccagcat atcggtgaaa tgagcttcct gcagcacaac 300

aaatgcgagt gccgtccgaa gaaagaccgt gcgcgtcaag agaacccgtg cggtccgtgc 360

agcgagcgtc gcaagcacct gttcgttcaa gacccgcaga cctgcaaatg cagctgcaag 420

aacaccgaca gccgttgcaa agcgcgtcaa ctggaactga atgaacgtac ctgccgtagc 480

ctgacccgta aggacggcgg cggcggtagc ggtctggaca ccgtgagctt cagcaccagc 540

ggcgcgacct atatcaccta cgttaacttc ctgaatgagc tgcgtgttaa gctgaaaccg 600

gaaggtaata gccacggtat tccgctgctg cgcaaaaagg cggacgatcc gggcaaagcg 660

tttgttctgg tggcgctgag caatgacaac ggccaactgg cggagattgc gatcgacgtt 720

accagcgtgt atgtggtggg ttaccaagtt cgcaatcgta gctacttctt caaggatgcg 780

ccggatgcgg cgtatgaggg tctgttcaag aacaccatca agacccgtct gcattttggc 840

ggtagctacc cgagcctgga aggtgaaaaa gcgtaccgtg aaaccaccga cctgggtatc 900

gaaccgctgc gtattggcat caagaagctg gacgagaacg cgatcgacaa ctacaaaccg 960

accgagattg cgagcagcct gctggttgtg atccagatgg ttagcgaggc ggcgcgtttt 1020

accttcattg agaaccagat ccgcaacaat ttccaacaac gcattcgccc ggcgaataac 1080

accatcagcc tggagaataa atggggtaaa ctgagcttcc aaatccgtac cagcggtgcg 1140

aacggcatgt ttagcgaagc ggtggaactg gagcgtgcga atggcaagaa gtattacgtg 1200

accgcggtgg accaagtgaa gccgaagatt gcgctgctga agtttgtgga taaagatgag 1260

ctg 1263

<210> 3

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gly Gly Gly Gly Ser

1 5

<210> 4

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggcggcggcg gtagc 15

<210> 5

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Lys Asp Glu Leu

1

<210> 6

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aaagatgagc tg 12

<210> 7

<211> 341

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Arg Gly Ser His His His His His His Ser Ser Gly Asp Asp Ala

1 5 10 15

Ala Ile Gln Gln Thr Leu Ala Lys Met Gly Ile Lys Ser Ser Asp Ile

20 25 30

Gln Pro Ala Pro Val Ala Gly Met Lys Thr Val Leu Thr Asn Ser Gly

35 40 45

Val Leu Tyr Ile Thr Asp Asp Gly Lys His Ile Ile Gln Gly Pro Met

50 55 60

Tyr Asp Val Ser Gly Thr Ala Pro Val Asn Val Thr Asn Lys Met Leu

65 70 75 80

Leu Lys Gln Leu Asn Ala Leu Glu Lys Glu Met Ile Val Tyr Lys Ala

85 90 95

Pro Gln Glu Lys His Val Ile Thr Val Phe Thr Asp Ile Thr Cys Gly

100 105 110

Tyr Cys His Lys Leu His Glu Gln Met Ala Asp Tyr Asn Ala Leu Gly

115 120 125

Ile Thr Val Arg Tyr Leu Ala Phe Pro Arg Gln Gly Leu Asp Ser Asp

130 135 140

Ala Glu Lys Glu Met Lys Ala Ile Trp Cys Ala Lys Asp Lys Asn Lys

145 150 155 160

Ala Phe Asp Asp Val Met Ala Gly Lys Ser Val Ala Pro Ala Ser Cys

165 170 175

Asp Val Asp Ile Ala Asp His Tyr Ala Leu Gly Val Gln Leu Gly Val

180 185 190

Ser Gly Thr Pro Ala Val Val Leu Ser Asn Gly Thr Leu Val Pro Gly

195 200 205

Tyr Gln Pro Pro Lys Asp Met Lys Glu Phe Leu Asp Glu His Gln Lys

210 215 220

Met Thr Ser Gly Lys Gly Ser Thr Ser Gly Ser Gly His His His His

225 230 235 240

His His Gly Gly Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu

245 250 255

Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser

260 265 270

Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu

275 280 285

Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp

290 295 300

Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr

305 310 315 320

Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg

325 330 335

Glu Gln Ile Gly Gly

340

<210> 8

<211> 1023

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgcgtggta gccatcatca tcatcatcat agcagcggcg acgacgcggc gattcaacaa 60

accctggcga aaatgggcat caagagcagc gacatccagc cggcgccggt tgcgggcatg 120

aaaaccgtgc tgaccaacag cggcgtgctg tacatcaccg acgatggcaa gcatatcatc 180

caaggcccga tgtatgacgt gagcggcacc gcgccggtga acgttaccaa taagatgctg 240

ctgaaacagc tgaacgcgct ggaaaaggag atgatcgttt acaaagcgcc gcaggaaaaa 300

cacgtgatta ccgtttttac cgacattacc tgcggctatt gccataaact gcatgagcaa 360

atggcggatt acaatgcgct gggtatcacc gttcgctacc tggcgtttcc gcgtcaaggt 420

ctggacagcg atgcggagaa agagatgaaa gcgatctggt gcgcgaaaga caaaaacaag 480

gcgttcgatg acgtgatggc gggcaaaagc gttgcgccgg cgagctgcga cgttgatatt 540

gcggaccatt acgcgctggg cgtgcaactg ggtgtgagcg gcaccccggc ggtggttctg 600

agcaacggca ccctggttcc gggttatcaa ccgccgaagg acatgaagga gtttctggac 660

gaacaccaga agatgaccag cggcaaaggt agcaccagcg gtagcggcca tcatcaccac 720

catcatggtg gcagcgatag cgaggtgaat caggaagcga agccggaagt gaaaccggaa 780

gtgaagccgg aaacccacat taacctgaaa gtgagcgatg gtagcagcga aatctttttc 840

aaaatcaaga aaaccacccc gctgcgtcgc ctgatggagg cgttcgcgaa gcgtcagggt 900

aaagaaatgg acagcctgcg tttcctgtac gacggtattc gcattcaggc ggaccaaacc 960

ccggaagacc tggacatgga agacaatgat atcattgagg cgcatcgtga gcagattggt 1020

ggt 1023

<210> 9

<211> 762

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Arg Gly Ser His His His His His His Ser Ser Gly Asp Asp Ala

1 5 10 15

Ala Ile Gln Gln Thr Leu Ala Lys Met Gly Ile Lys Ser Ser Asp Ile

20 25 30

Gln Pro Ala Pro Val Ala Gly Met Lys Thr Val Leu Thr Asn Ser Gly

35 40 45

Val Leu Tyr Ile Thr Asp Asp Gly Lys His Ile Ile Gln Gly Pro Met

50 55 60

Tyr Asp Val Ser Gly Thr Ala Pro Val Asn Val Thr Asn Lys Met Leu

65 70 75 80

Leu Lys Gln Leu Asn Ala Leu Glu Lys Glu Met Ile Val Tyr Lys Ala

85 90 95

Pro Gln Glu Lys His Val Ile Thr Val Phe Thr Asp Ile Thr Cys Gly

100 105 110

Tyr Cys His Lys Leu His Glu Gln Met Ala Asp Tyr Asn Ala Leu Gly

115 120 125

Ile Thr Val Arg Tyr Leu Ala Phe Pro Arg Gln Gly Leu Asp Ser Asp

130 135 140

Ala Glu Lys Glu Met Lys Ala Ile Trp Cys Ala Lys Asp Lys Asn Lys

145 150 155 160

Ala Phe Asp Asp Val Met Ala Gly Lys Ser Val Ala Pro Ala Ser Cys

165 170 175

Asp Val Asp Ile Ala Asp His Tyr Ala Leu Gly Val Gln Leu Gly Val

180 185 190

Ser Gly Thr Pro Ala Val Val Leu Ser Asn Gly Thr Leu Val Pro Gly

195 200 205

Tyr Gln Pro Pro Lys Asp Met Lys Glu Phe Leu Asp Glu His Gln Lys

210 215 220

Met Thr Ser Gly Lys Gly Ser Thr Ser Gly Ser Gly His His His His

225 230 235 240

His His Gly Gly Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu

245 250 255

Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser

260 265 270

Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu

275 280 285

Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp

290 295 300

Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr

305 310 315 320

Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg

325 330 335

Glu Gln Ile Gly Gly Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His

340 345 350

His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His

355 360 365

Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile

370 375 380

Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly

385 390 395 400

Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn

405 410 415

Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile

420 425 430

Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys

435 440 445

Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg

450 455 460

Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys

465 470 475 480

Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu

485 490 495

Arg Thr Cys Arg Ser Leu Thr Arg Lys Asp Gly Gly Gly Gly Ser Gly

500 505 510

Leu Asp Thr Val Ser Phe Ser Thr Ser Gly Ala Thr Tyr Ile Thr Tyr

515 520 525

Val Asn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly Asn

530 535 540

Ser His Gly Ile Pro Leu Leu Arg Lys Lys Ala Asp Asp Pro Gly Lys

545 550 555 560

Ala Phe Val Leu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala Glu

565 570 575

Ile Ala Ile Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val Arg

580 585 590

Asn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu Gly

595 600 605

Leu Phe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser Tyr

610 615 620

Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu Gly

625 630 635 640

Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu Asn Ala Ile

645 650 655

Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu Leu Val Val Ile

660 665 670

Gln Met Val Ser Glu Ala Ala Arg Phe Thr Phe Ile Glu Asn Gln Ile

675 680 685

Arg Asn Asn Phe Gln Gln Arg Ile Arg Pro Ala Asn Asn Thr Ile Ser

690 695 700

Leu Glu Asn Lys Trp Gly Lys Leu Ser Phe Gln Ile Arg Thr Ser Gly

705 710 715 720

Ala Asn Gly Met Phe Ser Glu Ala Val Glu Leu Glu Arg Ala Asn Gly

725 730 735

Lys Lys Tyr Tyr Val Thr Ala Val Asp Gln Val Lys Pro Lys Ile Ala

740 745 750

Leu Leu Lys Phe Val Asp Lys Asp Glu Leu

755 760

<210> 10

<211> 2342

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tctagaaata attttgttta actttaagaa ggagatatac atatgcgtgg tagccatcat 60

catcatcatc atagcagcgg cgacgacgcg gcgattcaac aaaccctggc gaaaatgggc 120

atcaagagca gcgacatcca gccggcgccg gttgcgggca tgaaaaccgt gctgaccaac 180

agcggcgtgc tgtacatcac cgacgatggc aagcatatca tccaaggccc gatgtatgac 240

gtgagcggca ccgcgccggt gaacgttacc aataagatgc tgctgaaaca gctgaacgcg 300

ctggaaaagg agatgatcgt ttacaaagcg ccgcaggaaa aacacgtgat taccgttttt 360

accgacatta cctgcggcta ttgccataaa ctgcatgagc aaatggcgga ttacaatgcg 420

ctgggtatca ccgttcgcta cctggcgttt ccgcgtcaag gtctggacag cgatgcggag 480

aaagagatga aagcgatctg gtgcgcgaaa gacaaaaaca aggcgttcga tgacgtgatg 540

gcgggcaaaa gcgttgcgcc ggcgagctgc gacgttgata ttgcggacca ttacgcgctg 600

ggcgtgcaac tgggtgtgag cggcaccccg gcggtggttc tgagcaacgg caccctggtt 660

ccgggttatc aaccgccgaa ggacatgaag gagtttctgg acgaacacca gaagatgacc 720

agcggcaaag gtagcaccag cggtagcggc catcatcacc accatcatgg tggcagcgat 780

agcgaggtga atcaggaagc gaagccggaa gtgaaaccgg aagtgaagcc ggaaacccac 840

attaacctga aagtgagcga tggtagcagc gaaatctttt tcaaaatcaa gaaaaccacc 900

ccgctgcgtc gcctgatgga ggcgttcgcg aagcgtcagg gtaaagaaat ggacagcctg 960

cgtttcctgt acgacggtat tcgcattcag gcggaccaaa ccccggaaga cctggacatg 1020

gaagacaatg atatcattga ggcgcatcgt gagcagattg gtggtgcgcc gatggcggag 1080

ggtggcggcc agaaccatca cgaggttgtg aaatttatgg acgtttatca acgtagctat 1140

tgccacccga tcgaaaccct ggtggacatt ttccaggaat atccggatga gatcgaatac 1200

atctttaaac cgagctgcgt gccgctgatg cgctgcggtg gctgctgcaa tgacgagggt 1260

ctggaatgcg ttccgaccga agagagcaac atcaccatgc agattatgcg tattaagccg 1320

catcaaggcc agcatatcgg tgaaatgagc ttcctgcagc acaacaaatg cgagtgccgt 1380

ccgaagaaag accgtgcgcg tcaagagaac ccgtgcggtc cgtgcagcga gcgtcgcaag 1440

cacctgttcg ttcaagaccc gcagacctgc aaatgcagct gcaagaacac cgacagccgt 1500

tgcaaagcgc gtcaactgga actgaatgaa cgtacctgcc gtagcctgac ccgtaaggac 1560

ggcggcggcg gtagcggtct ggacaccgtg agcttcagca ccagcggcgc gacctatatc 1620

acctacgtta acttcctgaa tgagctgcgt gttaagctga aaccggaagg taatagccac 1680

ggtattccgc tgctgcgcaa aaaggcggac gatccgggca aagcgtttgt tctggtggcg 1740

ctgagcaatg acaacggcca actggcggag attgcgatcg acgttaccag cgtgtatgtg 1800

gtgggttacc aagttcgcaa tcgtagctac ttcttcaagg atgcgccgga tgcggcgtat 1860

gagggtctgt tcaagaacac catcaagacc cgtctgcatt ttggcggtag ctacccgagc 1920

ctggaaggtg aaaaagcgta ccgtgaaacc accgacctgg gtatcgaacc gctgcgtatt 1980

ggcatcaaga agctggacga gaacgcgatc gacaactaca aaccgaccga gattgcgagc 2040

agcctgctgg ttgtgatcca gatggttagc gaggcggcgc gttttacctt cattgagaac 2100

cagatccgca acaatttcca acaacgcatt cgcccggcga ataacaccat cagcctggag 2160

aataaatggg gtaaactgag cttccaaatc cgtaccagcg gtgcgaacgg catgtttagc 2220

gaagcggtgg aactggagcg tgcgaatggc aagaagtatt acgtgaccgc ggtggaccaa 2280

gtgaagccga agattgcgct gctgaagttt gtggataaag atgagctgta atgagcggcc 2340

gc 2342

<210> 11

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Leu Val Pro Glu Leu Asn Glu Lys Asp Asp Asp Gln Val Gln Lys Ala

1 5 10 15

Leu Ala Ser Arg Glu Asn Thr Gln Leu Met Asn Arg Asp Asn Ile Glu

20 25 30

Ile Thr Val Arg Asp Phe Lys Thr Leu Ala Pro Arg Arg Trp Leu Asn

35 40 45

Asp Thr Ile Ile Glu Phe Phe Met Lys Tyr Ile Glu Lys Ser Thr Pro

50 55 60

Asn Thr Val Ala Phe Asn Ser Phe Phe Tyr Thr Asn Leu Ser Glu Arg

65 70 75 80

Gly Tyr Gln Gly Val Arg Arg Trp Met Lys Arg Lys Lys Thr Gln Ile

85 90 95

Asp Lys Leu Asp Lys Ile Phe Thr Pro Ile Asn Leu Asn Gln Ser His

100 105 110

Trp Ala Leu Gly Ile Ile Asp Leu Lys Lys Lys Thr Ile Gly Tyr Val

115 120 125

Asp Ser Leu Ser Asn Gly Pro Asn Ala Met Ser Phe Ala Ile Leu Thr

130 135 140

Asp Leu Gln Lys Tyr Val Met Glu Glu Ser Lys His Thr Ile Gly Glu

145 150 155 160

Asp Phe Asp Leu Ile His Leu Asp Cys Pro Gln Gln Pro Asn Gly Tyr

165 170 175

Asp Cys Gly Ile Tyr Val Cys Met Asn Thr Leu Tyr Gly Ser Ala Asp

180 185 190

Ala Pro Leu Asp Phe Asp Tyr Lys Asp Ala Ile Arg Met Arg Arg Phe

195 200 205

Ile Ala His Leu Ile Leu Thr Asp Ala Leu Lys

210 215

<210> 12

<211> 677

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggatccctgg tgccggagct gaacgaaaaa gacgatgacc aggttcaaaa ggcgctggcg 60

agccgtgaga acacccagct gatgaaccgt gataacatcg aaattaccgt gcgtgacttc 120

aaaaccctgg cgccgcgtcg ttggctgaac gataccatca tcgagttctt tatgaagtac 180

atcgaaaaga gcaccccgaa caccgtggcg tttaacagct tcttttacac caacctgagc 240

gagcgtggtt atcagggcgt tcgtcgttgg atgaagcgta agaaaaccca aatcgataaa 300

ctggacaaaa tcttcacccc gattaacctg aaccagagcc actgggcgct gggtatcatt 360

gatctgaaga aaaagaccat cggttacgtg gacagcctga gcaacggccc gaacgcgatg 420

agcttcgcga ttctgaccga tctgcaaaaa tatgttatgg aggaaagcaa gcacaccatc 480

ggtgaagatt ttgacctgat tcacctggat tgcccgcagc aaccgaacgg ttacgactgc 540

ggcatctatg tttgcatgaa caccctgtat ggcagcgcgg atgcgccgct ggatttcgac 600

tataaagacg cgattcgtat gcgtcgtttt atcgcgcacc tgattctgac cgacgcgctg 660

aagtaatgag cggccgc 677

<210> 13

<211> 762

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Met Arg Gly Ser His His His His His His Ser Ser Gly Asp Asp Ala

1 5 10 15

Ala Ile Gln Gln Thr Leu Ala Lys Met Gly Ile Lys Ser Ser Asp Ile

20 25 30

Gln Pro Ala Pro Val Ala Gly Met Lys Thr Val Leu Thr Asn Ser Gly

35 40 45

Val Leu Tyr Ile Thr Asp Asp Gly Lys His Ile Ile Gln Gly Pro Met

50 55 60

Tyr Asp Val Ser Gly Thr Ala Pro Val Asn Val Thr Asn Lys Met Leu

65 70 75 80

Leu Lys Gln Leu Asn Ala Leu Glu Lys Glu Met Ile Val Tyr Lys Ala

85 90 95

Pro Gln Glu Lys His Val Ile Thr Val Phe Thr Asp Ile Thr Cys Gly

100 105 110

Tyr Cys His Lys Leu His Glu Gln Met Ala Asp Tyr Asn Ala Leu Gly

115 120 125

Ile Thr Val Arg Tyr Leu Ala Phe Pro Arg Gln Gly Leu Asp Ser Asp

130 135 140

Ala Glu Lys Glu Met Lys Ala Ile Trp Cys Ala Lys Asp Lys Asn Lys

145 150 155 160

Ala Phe Asp Asp Val Met Ala Gly Lys Ser Val Ala Pro Ala Ser Cys

165 170 175

Asp Val Asp Ile Ala Asp His Tyr Ala Leu Gly Val Gln Leu Gly Val

180 185 190

Ser Gly Thr Pro Ala Val Val Leu Ser Asn Gly Thr Leu Val Pro Gly

195 200 205

Tyr Gln Pro Pro Lys Asp Met Lys Glu Phe Leu Asp Glu His Gln Lys

210 215 220

Met Thr Ser Gly Lys Gly Ser Thr Ser Gly Ser Gly His His His His

225 230 235 240

His His Gly Gly Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu

245 250 255

Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser

260 265 270

Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu

275 280 285

Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp

290 295 300

Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr

305 310 315 320

Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg

325 330 335

Glu Gln Ile Gly Gly Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His

340 345 350

His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His

355 360 365

Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile

370 375 380

Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly

385 390 395 400

Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn

405 410 415

Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile

420 425 430

Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys

435 440 445

Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg

450 455 460

Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys

465 470 475 480

Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu

485 490 495

Arg Thr Cys Arg Ser Leu Thr Arg Lys Asp Gly Gly Gly Gly Ser Gly

500 505 510

Leu Asp Thr Val Ser Phe Ser Thr Lys Gly Ala Thr Tyr Ile Thr Tyr

515 520 525

Val Asn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly Asn

530 535 540

Ser His Gly Ile Pro Leu Leu Arg Lys Lys Cys Asp Asp Pro Gly Lys

545 550 555 560

Cys Phe Val Leu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala Glu

565 570 575

Ile Ala Ile Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val Arg

580 585 590

Asn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu Gly

595 600 605

Leu Phe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser Tyr

610 615 620

Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu Gly

625 630 635 640

Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu Asn Ala Ile

645 650 655

Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu Leu Val Val Ile

660 665 670

Gln Met Val Ser Glu Ala Ala Arg Phe Thr Phe Ile Glu Asn Gln Ile

675 680 685

Arg Asn Asn Phe Gln Gln Arg Ile Arg Pro Ala Asn Asn Thr Ile Ser

690 695 700

Leu Glu Asn Lys Trp Gly Lys Leu Ser Phe Gln Ile Arg Thr Ser Gly

705 710 715 720

Ala Asn Gly Met Phe Ser Glu Ala Val Glu Leu Glu Arg Ala Asn Gly

725 730 735

Lys Lys Tyr Tyr Val Thr Ala Val Asp Gln Val Lys Pro Lys Ile Ala

740 745 750

Leu Leu Lys Phe Val Asp Lys Asp Glu Leu

755 760

<210> 14

<211> 2342

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tctagaaata attttgttta actttaagaa ggagatatac atatgcgtgg cagccaccac 60

caccaccacc acagcagcgg tgacgatgcg gcgattcagc aaaccctggc gaaaatgggc 120

atcaagagca gcgatattca accggcgccg gttgcgggta tgaagaccgt gctgaccaac 180

agcggtgttc tgtacatcac cgacgatggc aaacacatca ttcaaggtcc gatgtatgac 240

gtgagcggca ccgcgccggt gaacgttacc aacaaaatgc tgctgaagca gctgaacgcg 300

ctggagaagg aaatgatcgt ttacaaggcg ccgcaagaaa aacacgtgat caccgttttc 360

accgatatta cctgcggcta ttgccacaaa ctgcacgagc agatggcgga ctacaacgcg 420

ctgggcatta ccgtgcgtta tctggcgttc ccgcgtcaag gtctggacag cgatgcggag 480

aaagaaatga aggcgatctg gtgcgcgaag gataaaaaca aggcgtttga cgatgttatg 540

gcgggcaaga gcgttgcgcc ggcgagctgc gatgtggata ttgcggacca ctatgcgctg 600

ggtgttcaac tgggcgtgag cggcaccccg gcggtggttc tgagcaacgg caccctggtt 660

ccgggttatc agccgccgaa agacatgaag gagtttctgg atgaacacca aaagatgacc 720

agcggcaaag gtagcaccag cggtagcggt caccaccacc accaccacgg tggcagcgat 780

agcgaggtga accaggaagc gaaaccggaa gtgaagccgg aagtgaaacc ggaaacccac 840

atcaacctga aggttagcga cggtagcagc gaaatcttct ttaagattaa gaaaaccacc 900

ccgctgcgtc gtctgatgga agcgttcgcg aagcgtcaag gcaaagagat ggacagcctg 960

cgttttctgt acgatggtat ccgtattcag gcggaccaaa ccccggaaga cctggatatg 1020

gaggacaacg atatcattga agcgcatcgt gagcagatcg gtggtgcgcc gatggcggaa 1080

ggtggcggtc aaaaccacca cgaggtggtt aagttcatgg atgtgtacca gcgtagctat 1140

tgccacccga tcgaaaccct ggttgatatt ttccaagagt acccggacga gatcgaatat 1200

atttttaaac cgagctgcgt gccgctgatg cgttgcggcg gttgctgcaa cgacgagggt 1260

ctggaatgcg ttccgaccga ggaaagcaac attaccatgc agatcatgcg tattaagccg 1320

caccagggcc aacacatcgg tgaaatgagc ttcctgcagc acaacaaatg cgagtgccgt 1380

ccgaagaaag accgtgcgcg tcaagaaaac ccgtgcggtc cgtgcagcga gcgtcgtaaa 1440

cacctgtttg tgcaggaccc gcagacctgc aagtgcagct gcaaaaacac cgacagccgt 1500

tgcaaggcgc gtcagctgga gctgaacgaa cgtacctgcc gtagcctgac ccgtaaagat 1560

ggcggtggcg gatccggcct ggacaccgtt agcttcagca ccaaaggtgc gacctacatt 1620

acctatgtga actttctgaa cgaactgcgt gttaaactga agccggaggg caacagccac 1680

ggtatcccgc tgctgcgtaa gaaatgcgac gatccgggca agtgcttcgt gctggttgcg 1740

ctgagcaacg ataacggtca gctggcggaa atcgcgattg acgtgaccag cgtttacgtg 1800

gttggctatc aagtgcgtaa ccgtagctac ttctttaaag acgcgccgga tgcggcgtat 1860

gagggtctgt tcaagaacac cattaaaacc cgtctgcact ttggcggtag ctacccgagc 1920

ctggagggtg aaaaggcgta tcgtgaaacc accgatctgg gcatcgagcc gctgcgtatc 1980

ggtattaaga aactggacga aaacgcgatc gataactaca aaccgaccga gattgcgagc 2040

agcctgctgg ttgtgatcca gatggttagc gaagcggcgc gtttcacctt tatcgagaac 2100

caaattcgta acaacttcca gcaacgtatc cgtccggcga acaacaccat tagcctggaa 2160

aacaaatggg gcaagctgag cttccagatt cgtaccagcg gcgcgaacgg catgtttagc 2220

gaggcggtgg agctggaacg tgcgaacggt aagaaatact atgtgaccgc ggttgaccaa 2280

gtgaaaccga agatcgcgct gctgaagttt gttgacaaag atgagctgta atgagcggcc 2340

gc 2342

Claims

1. Fusion toxin VEGF _165b /mGEL, characterized in that the fusion toxin VEGF _165b /mGEL comprises VEGF ₁₆₅ Inhibitory splice isoforms VEGF _165b The Gelonin mutant mGEL can inhibit the generation of new vessels and destroy a new vessel network aiming at new vessel dependent diseases;

the fusion toxin VEGF _165b The amino acid sequence of/mGEL is shown as SEQ ID NO:1 is shown.

2. The fusion toxin VEGF of claim 1 _165b The coding gene of/mGEL is characterized in that the nucleotide sequence of the coding gene is one of the following nucleotide sequences:

1) SEQ ID NO: 2;

2) encoding the amino acid sequence of SEQ ID NO: 1.

3. A recombinant expression vector comprising the coding gene of claim 2 for expressing the fusion toxin VEGF of claim 1 _165b /mGEL。

4. A transgenic cell line or host bacterium comprising the coding gene of claim 2 or the recombinant expression vector of claim 3 for expressing the fusion toxin VEGF of claim 1 _165b /mGEL。

5. A therapeutic agent for a neovascular dependent disease, comprising the fusion toxin VEGF of claim 1 _165b The gene encoding/mGEL or the gene of claim 2.

6. The therapeutic agent for a neovascular dependence disease according to claim 5, further comprising one or more of: pharmaceutically acceptable carrier, diluent, excipient, filler, adhesive, wetting agent, disintegrant, absorption enhancer, and surfactant.

7. The agent for treating a neovascular-dependent disease according to claim 5 or 6, wherein the neovascular-dependent disease includes a malignant tumor and an ocular neovascular disease.

8. A method for recombinantly expressing the fusion toxin VE of claim 1GF _165b A method of/mGEL comprising the steps of:

1) constructing a transgenic cell line or a host bacterium according to claim 4, and culturing the transgenic cell line or the host bacterium;

9. The method according to claim 8, characterized in that it comprises in particular the steps of:

a) constructing a recombinant expression vector containing the coding gene of claim 2;

b) transforming the recombinant expression vector into a transgenic cell line or a host bacterium, and carrying out culture expression to obtain a fusion protein DSS-VEGF _165b /mGEL；

c) Excision of the fusion protein DSS-VEGF with the tool enzyme ULP1 _165b DSS label of/mGEL, purifying to obtain fusion toxin VEGF _165b /mGEL。

10. The method according to claim 9, wherein in step c) the tool enzyme ULP1 is fused to the fusion protein DSS-VEGF _165b The dosage proportion relation of/mGEL is 1: (100-150).

11. The method according to claim 10, wherein in step c) the tool enzyme ULP1 is fused to the fusion protein DSS-VEGF _165b The dosage proportion relation of/mGEL is 1: 100.