CN113354738A - Fusion toxin VEGF165bmGEL and its coding gene and application - Google Patents

Abstract

The invention discloses a fusion toxin VEGF_165bThe mGEL and its coding gene and application belong to the field of biological pharmacy. Lifting deviceFusion toxin VEGF supplied_165b/mGEL comprises VEGF₁₆₅Inhibitory splice isoforms VEGF_165bTogether 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

Fusion toxin VEGF165bmGEL and its coding gene and application

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to fusion toxin VEGF_165bmGEL and its coding gene and application, in particular to VEGF₁₆₅An 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 2mm³And thus will be at rest. However, tumor cells promote peripheral neovascularization during growth by autocrine of various cytokines. Before the new blood vessel grows 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. The neovascular structure of the cornea is fragile and permeable, and often causes blindness due to bleeding, exudation, secondary fibrosis and the like. (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., VEGF₁₂₁、VEGF₁₆₅、VEGF₁₈₉And VEGF₂₀₈Wherein VEGF₁₂₁And VEGF₁₆₅Is 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_xxxAnd VEGF inhibiting angiogenesis_xxxb. In VEGF_xxxbIn family, VEGF_165bMost typically, it antagonizes VEGF on the one hand by binding with high affinity to VEGFR₁₆₅The 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 angiogenesisBut can not destroy the existing new blood vessels, such as bevacizumab, Endotimod and the like; 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 VEGF₁₆₅/DT390、VEGF₁₂₁/DT390 and VEGF₁₂₁rGel et al, wherein VEGF₁₂₁And VEGF₁₆₅Only plays the role of a targeting molecule, can not inhibit the generation of new blood vessels, and releases VEGF₁₂₁And VEGF₁₆₅Can also promote angiogenesis; 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 the method has a plurality of defects. The two products, namely the angiogenesis inhibitor and the angiogenesis disruptor, play a role by a single mechanism of inhibiting angiogenesis or destroying existing angiogenesis, are also single targets, and do not have double functions of the angiogenesis inhibitor and the disruptor.

Disclosure of Invention

In response to one or more of the problems of the prior art, one aspect of the present invention provides a fusion toxin VEGF_165b/mGEL, the fusion toxin VEGF_165b/mGEL comprises VEGF₁₆₅Inhibitory splice isoforms VEGF_165bAnd 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 Gelonin mutant mGEL 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 respectively mutated into Ser10, Ala44 and Ala50 in the amino acid sequence of a Gelonin mutant mGEL, and the amino acid residue of the Gelonin mutant mGEL is shown as the amino acid sequence from the 171 th-417 (247aa) amino acid of the amino terminal of the amino acid sequence shown as SEQ ID NO:1 in the sequence table.

The fusion toxin VEGF_165bthe/mGEL also contains an endoplasmic reticulum localization sequence, which is located in the GeloninThe carboxy terminus of 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_165bthe/mGEL also comprises a linker peptide or analogue thereof for linking the VEGF₁₆₅Inhibitory splice isoforms VEGF_165bAnd 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_165bThe amino acid residue sequence of/mGEL is one of the following amino acid residue sequences:

1) SEQ ID NO: 1;

2) and (3) mixing the amino acid sequence 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_165bCoding gene for/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) encoding the amino acid sequence shown in 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_165bThe mGEL contains the above-mentioned coding gene (VEGF)_165b/mGEL) for expressing the fusion toxin V described aboveEGF_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 dependent 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_165bA 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_165bthe/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/mGEL DSS label, purifying to obtain fusion toxin VEGF_165b/mGEL；

Preferably, the step ofc) Middle tool enzyme ULP1 and fusion protein DSS-VEGF_165bThe dosage proportion relation of the/mGEL is as follows: 1: (100- > 150); further preferably 1: 100.

Fusion toxin VEGF provided based on the technical scheme_165bmGEL has double functions of a neovascular inhibitor and a destructive agent, and is prepared from a targeting molecule-recombinant human vascular endothelial growth factor VEGF₁₆₅Inhibitory splice isoforms VEGF_165bAnd an effector molecule, Gelonin mutant (mGEL), wherein VEGF_165bIs both a VEGF antagonist and a VEGFR targeting molecule: inhibition of VEGF function and neovascularization by competitive binding to VEGFR; meanwhile, the fusion toxin is brought into the endothelial cells of the new blood vessels by identifying VEGFR and endocytosis mechanism, the toxin molecule mGEL is released and 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 blood vessel network. Moreover, the fusion toxin has simple expression conditions, 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_165bGEL electrophoresis of GEL expression and purification in E.coli Origami (DE 3);

FIG. 2 shows DSS-VEGF_165bThe expression and purification of the gel electrophoretogram of/mGEL in E.coli Origami (DE 3);

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

FIG. 4 shows DSS-VEGF_165bThe expression, purification and enzyme digestion of/mGEL identify the gel electrophoresis picture;

FIG. 5 shows DSS tag excision and VEGF_165bGEL electrophoresis image of GEL purification;

FIG. 6 shows DSS-VEGF_165bExpression purification of/mGEL, DSS tag excision and VEGF_165ba/mGEL purified gel electrophoresis image;

FIG. 7 shows VEGF_165bThe cytotoxicity curve of GEL fusion toxin to PAE/KDR;

FIG. 8 shows VEGF_165bCytotoxicity 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 to provide a recombinant human vascular endothelial growth factor VEGF_165bInhibitory splice isoforms VEGF_165bThe fusion toxin with Gelonin mutant (mGEL) of Gelonin is named VEGF_165b/mGEL comprising VEGF₁₆₅Inhibitory splice isoforms VEGF_165bAnd 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 peptide₁₆₅Inhibitory splice isoforms VEGF_165bThe 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_165bis a known recombinant human vascular endothelial growth factor VEGF₁₆₅The 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_165bThe carboxy terminus (C-terminus) of (C) is linked to the amino terminus (N-terminus) of 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 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.

In particular, the fusion toxin VEGF_165bthe/mGEL is one of the following amino acid residue sequences:

1) SEQ ID NO: 1;

2) and (3) mixing the amino acid sequence 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 new blood vessel network destruction.

In the sequence listingSEQ 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 VEGF₁₆₅Inhibitory splice isoforms VEGF_165bThe 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_165bPolypeptide fragments, derivatives and analogues of/mGEL also belong to the invention, which interact with VEGF_165bthe/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 polypeptide sequence, 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 a nucleic acid sequence (or portion thereof) of the invention to obtain a coding sequence for a 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_165bDerivatives of/mGEL refer to the use of VEGF in the invention_165bAnd fusion toxin constructed by fusing other toxin molecules.

VEGF_165bAnalogues of/mGEL with VEGF_165bThe difference in/mGEL may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both. Said likeIncluded are 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_165bthe/mGEL may also be a VEGF modified, or modified to increase its anti-proteolytic or solubility-optimized properties_165ba/mGEL polypeptide. Modified (generally without altering primary structure) forms include: 1) chemically derivatized forms of the polypeptide in vivo or in vitro, such as acetylation or carboxylation; 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;

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 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' end₁₆₅Inhibitory splice isoforms VEGF_165bThe 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 1252-1263 th base from the 5 ' end encodes the endoplasmic reticulum localization signal.

Encoding the fusion toxin VEGF of the invention_165bthe/mGEL polynucleotide may be in the form of DNA or RNA. 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.

Furthermore, VEGF, a fusion toxin of the invention, is encoded_165bVariants of the polynucleotides of the/mGEL, which encode and fuse the toxin VEGF_165bthe/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)_165bmGEL), transgenic cell line and host bacteria.

Another objective of the invention is to provide a method for recombinant expression of the fusion toxin VEGF_165bThe preparation method of/mGEL is to prepare 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_165bThe recombinant expression vector of the coding gene of mGEL is fusion toxin VEGF_165bThe 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 vector preferably comprises an expression vector containing disulfide bond isomerase DsbC-ubiquitin-like protein modified molecule SUMO fusion protein DsbC-SUMO (DSS for short), and is named as a pDSS vector, the vector is based on a pET vector, and the amino acid residue sequence of the DsbC-SUMO fusion protein is shown as SEQ ID NO: 7, 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 (vascular endothelial growth factor) containing fusion toxin constructed by taking pDSS (plasmid DNA sequence) vector as starting vector_165bThe recombinant expression vector of the coding gene of the mGEL is named as pDSS-VEGF_165band/mGEL. Recombinant expression vector pDSS-VEGF_165bThe fusion protein expressed by the mGEL in the host cell or the host bacterium has a DSS label, and is named as DSS-VEGF_165bThe 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_165bmGEL, 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_165bThe 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: the lac or trp promoter of E.coli, phage promoters, retroviruses and other known promoters which control the expression of genes in prokaryotic or eukaryotic cells or their 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_165bthe/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_165bWhen the gene encoding mGEL is expressed in a higher eukaryotic cell, the transcription can be enhanced by using a recombinant expression vector pDSS-VEGF_165bAn 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_165bmGEL 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_165bThe culture medium and culture conditions of the host cell of the encoding gene of/mGEL can be the culture medium and culture conditions for culturing the original host.

Use of fusion toxin VEGF_165bThe 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_165bWhen encoding gene of mGEL, the fusion toxin VEGF_165bThe 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 present invention is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are given, and the examples will help understanding the present invention, but the scope of the present invention is not limited to the following examples.

Example 1 fusion toxin VEGF_165bExpression and purification of/mGEL in E.coli

Recombinant expression vector pDSS-VEGF_165bGEL and pDSS-VEGF_165bConstruction 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_165bGEL and DSS-VEGF_165bmGEL gene and construction of expression vector pDSS-VEGF_165bGEL and pDSS-VEGF_165b/mGEL。

DSS-VEGF_165bThe 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 cleavage sites, and the 43 rd bases from the 5' -end are Xba I cleavage sitesThe 1065-bit base codes a DSS label, and the 1066-th and 1560-th bases from the 5' -end code the recombinant human vascular endothelial growth factor VEGF₁₆₅Inhibitory splice isoforms VEGF_165bThe 1561-1575 th base from the 5 ' -end encodes a connecting peptide, the 1576-2316 th base from the 5 ' -end encodes a gelonin mutant mGEL, the 2317-2328 th base from the 5 ' -end encodes an endoplasmic reticulum localization signal, the 2329-2334 th base from the 5 ' -end is a stop codon, and the 2335-2342 th base from the 5 ' -end is a Not I restriction site.

DSS-VEGF_165bThe 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 VEGF₁₆₅Inhibitory splice isoforms VEGF_165bThe 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.

Second, construction of recombinant expression vector pGEX-ULP1

The ULP1 gene was synthesized in its entirety by Nanjing Kingsrey Biotech according to the codon preference of E.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_165bExpression 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_165bThe 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 the overnight culture was transferred to 1L of LB medium (containing carbenicillin 200. mu.g/mL, kanamycin 50. mu.g/mL and tetracycline 25. mu.g/mL), and cultured at 37 ℃ and 200rpm to OD₆₀₀≈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_165bGEL and VEGF_165bPurification of/mGEL

(1) Purification of DSS-VEGF Using Ni-Sepharose FF_165b/GEL、DSS-VEGF_165bThe 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_165bGEL and DSS-VEGF_165bThe 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_165bResults 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 is molecular weight standard; lane 2 is whole ultrasonic lysis; lanes 3, 4 are ultrasonication supernatants; lane 5 is a crossLiquid; lane 6 is eluted DSS-VEGF_165ba/GEL; FIG. 2 shows DSS-VEGF_165bThe 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_165bGEL and DSS-VEGF_165bThe molecular weight of the/mGEL target protein is about 85kD, and the soluble DSS-VEGF in the thalli lysis supernatant_165bThe expression level of/mGEL is obviously higher than that of DSS-VEGF_165bThe expression level of GEL.

(2) GST-ULP1 was purified using GST HP: the bacterial pellet was resuspended in buffer A (10mM Na) at 1:20₂HPO₄，1.8mM KH₂PO₄140mM 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 is molecular weight standard; 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: (ii) the DSS-VEGF obtained in the above step (1) was treated with GST-ULP1 obtained in the above step (2)_165bThe enzyme/mGEL was cleaved with a different cleavage system (50mM Tris-HCl, 300mM NaCl, 200mM imidazole (pH8.0) buffer) overnight at 8 ℃. As shown in FIG. 4, for DSS-VEGF_165bThe expression, purification and cleavage results of/mGEL were examined by gel electrophoresis (12% SDS-PAGE), in which lanes 1-4 represent DSS-VEGF_165bThe results of purification of the/mGEL expression, lanes 5-13 represent different digestion systems (where approximately DSS-VEGF was contained per 100. mu.l digestion system)_165b120 μ g/mGEL fusion protein) of DSS-VEGF_165bThe enzyme cutting identification result of/mGEL, wherein M represents a molecular weight standard. Visible, DSS-VEGF_165bthe/mGEL is cut by ULP1 enzyme to obtain two fragments: fusion protein of interest VEGF_165bthe/mGEL and DSS labels have different enzyme cutting effects in different enzyme cutting systems, and as can be seen from FIG. 4, when the tool enzymes ULP1 and DSS-VEGF_165bWhen 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 the DSS-VEGF can be fully used_165bThe DSS tag is excised from the/mGEL fusion protein.

(4) DSS tag excision and VEGF_165bGEL and VEGF_165bPurification of mGEL: each 1. mu.g of ULP1 was digested with 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_165bGEL and VEGF_165band/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_165bThe results of the GEL purification assay, shown in FIG. 5 as lanes 1-3: lane 1 is DSS-VEGF_165bThe GEL is subjected to enzyme digestion by ULP 1; lane 2 is VEGF_165bThe result after GEL reduction electrophoresis (the loading buffer solution contains beta-mercaptoethanol); lane 3 is VEGF_165bResults of GEL non-reduction (no beta-mercaptoethanol in loading buffer); m is a molecular weight standard. FIG. 6 shows DSS-VEGF_165bExpression purification of/mGEL, DSS tag excision and VEGF_165bThe results of the/mGEL purification assay, shown in FIG. 6 as lanes 1-10: lane 1 is molecular weight standard; 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 crossing results; lane 9 is SP column crossing results; lane 10 shows VEGF eluted from SP column_165band/mGEL. Visible DSS-VEGF_165bGEL and DSS-VEGF_165bAfter Ni-affinity chromatography, label removal and ion exchange chromatography are carried out on/mGEL, higher purity is respectively obtainedVEGF of degree_165bGEL and VEGF_165bthe/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_165bThe yield of the/mGEL fusion toxin is far higher than that of VEGF_165bThe 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 VEGFR₂Cytotoxicity of/KDR Positive cells PAE/KDR (present in professor Chun of university of Beijing university institute of Life sciences)

This example uses the VEGF obtained in example 1 above_165bGEL and VEGF_165bThe 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 10⁴Perml, add to 96-well plates, 200. mu.l per well (3000 cells/well) (first row without cells), 37 ℃ 5% CO₂Incubate overnight. VEGF dilution in 96-well plates at 1:5 gradient_165bThe 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% CO₂Culturing 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_165bToxic effects of GEL fusion toxin on PAE/KDR cells.

FIG. 7 and FIG. 8 (the abscissa shows the concentration of the fusion toxin and the ordinate shows the concentration of the fusion toxinMarked as cell viability), VEGF was known from the respective curves_165bGEL and VEGF_165b/mGEL vs VEGFR₂IC50 of/KDR positive cell PAE/KDR is 0.56nM and 0.1nM respectively, VEGF can be seen_165bGEL and VEGF_165b/mGEL fusion toxin vs VEGFR₂The vascular endothelial cells PAE/KDR with high KDR expression have specific cytotoxic effect, and VEGF_165bThe cytotoxicity of/mGEL is obviously higher than that of VEGF_165bCytotoxicity 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 changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. 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 to sun-burn

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 (11)

1. Fusion toxin VEGF_165b/mGEL, characterized in that the fusion toxin VEGF_165b/mGEL comprises VEGF₁₆₅Inhibitory splice isoforms VEGF_165bAnd the Gelonin mutant mGEL can simultaneously inhibit the generation of new vessels and destroy a new vessel network aiming at new vessel dependent diseases.

2. The fusion toxin VEGF of claim 1_165bThe mGEL is characterized in that the amino acid sequence of the Gelonin mutant mGEL is different from the amino acid sequence of natural Gelonin by the following steps: lys10, Cys44 and Cys50 of the amino acid sequence of the natural Gelonin are respectively mutated into Ser10, Ala44 and Ala50 in the amino acid sequence of a Gelonin mutant mGEL, and the amino acid residue of the Gelonin mutant mGEL is shown as the amino acid sequence from the 171 th-417 (247aa) amino acid of the amino terminal of the amino acid sequence shown as SEQ ID NO:1 in the sequence table.

3. The fusion toxin VEGF of claim 1 or 2_165b/mGEL, characterized in that the fusion toxin VEGF_165bthe/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 is shown in the specification;

preferably, the fusion toxin VEGF_165bthe/mGEL also comprises a linker peptide or analogue thereof for linking the VEGF₁₆₅Inhibitory splice isoforms VEGF_165bAnd Gelonin mutant mGEL; optionally, the amino acid residue sequence of the connecting peptide is shown as SEQ ID NO: 3, respectively.

4. The fusion toxin VEGF of any of claims 1-3_165b/mGEL, characterized in that the fusion toxin VEGF_165bThe amino acid residue sequence of/mGEL is one of the following amino acid residue sequences:

1) SEQ ID NO: 1;

2) and (3) mixing the amino acid sequence 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.

5. The fusion toxin VEGF of any one of claims 1 to 4_165bCoding gene for/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) encoding the amino acid sequence shown in 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.

6. Recombinant expression vector pDSS-VEGF_165b/mGEL, characterized in that said recombinant expression vector pDSS-VEGF_165b/mGEL comprising the coding gene (VEGF) according to claim 5_165b/mGEL) for expressing the fusion toxin VEGF of any of claims 1 to 4_165b/mGEL。

7. A transgenic cell line or host bacterium comprising the coding gene (VEGF) of claim 5_165b/mGEL) or the recombinant expression vector of claim 6 for expressing the fusion toxin VEGF of any one of claims 1 to 4_165b/mGEL。

8. A therapeutic agent for a neovascular dependent disease, which comprises the compound of claim 1 to 4The fusion toxin VEGF of any one of_165b/mGEL or the coding gene (VEGF) according to claim 5_165b/mGEL)；

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

9. The agent for treating a neovascular-dependent disease according to claim 8, wherein the neovascular-dependent disease includes, but is not limited to, malignant tumor, ocular neovascular disease.

10. A method of recombinantly expressing the fusion toxin VEGF of any one of claims 1 to 4_165bA method of/mGEL comprising the steps of:

1) constructing a transgenic cell line or host bacterium according to claim 7 and culturing the transgenic cell line or 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。

11. The method according to claim 10, characterized in that it comprises in particular the steps of:

a) construction of a recombinant vector containing the coding gene (VEGF) according to claim 5_165b/mGEL) recombinant expression vector pDSS-VEGF_165b/mGEL；

b) Subjecting the recombinant expression vector pDSS-VEGF_165bthe/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/mGEL DSS label, purifying to obtain fusion toxin VEGF_165b/mGEL；

Preferably, the tool enzyme ULP1 is fused with the fusion protein DSS-VEGF in step c)_165bThe dosage proportion relation of the/mGEL is as follows: 1: (100-150) (ii) a Further preferably 1: 100.

Images