CN113072645A - Cancer vaccine targeting EphA2 antigen - Google Patents

Abstract

The present invention relates to the field of immunotherapy and prevention of cancer, in particular to a tumor vaccine targeting EPHA 2. The invention improves the efficiency of the EPHA2 protein phagocytosis, processing and presentation by DC cells and improves the effect of inhibiting the tumor growth by fusing and expressing the EPHA2 protein and the DC cell ligand XCL 1. Experiments prove that the protein expressed by the nucleic acid vaccine can effectively induce an EPHA2 specific T cell response and obviously inhibit the growth of a tumor with high expression of EPHA2 in an animal tumor model.

Description

Cancer vaccine targeting EphA2 antigen

Technical Field

The invention relates to the technical field of immunotherapy, and particularly relates to an antigen vaccine targeting EphA2 and a preparation method thereof.

Background

Cancer is a major public health problem worldwide, and the incidence rate thereof is increasing, and by 2025, there are 4.2 billion new cancer cases each year around the world. Despite the great progress made in the clinical treatment of cancer, millions of people die from cancer every year worldwide, and improvements in cancer treatment remain elusive.

EphA2 is abundantly expressed in a variety of cancers, such as prostate, lung, oesophageal, colorectal, cervical, ovarian, breast and skin cancers, but is relatively poorly expressed in most normal adult tissues, and thus EphA2 is an ideal potential therapeutic target for cancer. The EphA2 receptor is a 130 kDa transmembrane glycoprotein, and EphA2-ephrin A1 signaling plays an important role in tumorigenesis. Furthermore, expression of EphA2 is associated with poor prognosis, increased metastatic potential and decreased survival in tumor patients. The mechanism of action of EphA2 has become clear after more than 30 years of research, and some drugs targeting EphA2 have been used in clinical studies of cancer treatment. Current treatments for EphA2 are: reducing EphA2 expression, promoting EphA2 degradation, blocking endogenous EphA2 activation, EphA2 as a drug delivery target, EphA 2-based immunotherapy and EphA 2-based combination therapy. Among them, immunotherapy against tumor cells by means of enhancing the immune defenses of patients has become a promising new therapy in the treatment of cancer. For example, when an animal model of colorectal cancer is immunized with a Dendritic Cell (DC) carrying an EphA 2-derived short peptide, the activity and antitumor activity of EphA 2-specific cytotoxic T Cell (CTL) can be improved; adoptive infusion of EphA 2-specific T cells killed EphA 2-positive tumor cells and inhibited the development of lung cancer in vivo. Although the above therapies are capable of activating anti-tumor immune responses, their efficacy is limited and insufficient to provide effective anti-tumor protection.

Most immunotherapeutic approaches rely on mature DCs presenting tumor-associated antigens for CTL activation. Among them, conventional DC of type I (convental DC1, cDC 1) can take up antigen from dead cells by cross-presentation and present it via MHC-I molecules, thereby activating and amplifying specific CTLs. CD103+ DC1 is a very important cDC1, which has migratory capacity, carries tumor antigens to the draining lymph nodes for cross presentation, and expresses XCL1 receptor (XCR1) on its surface, which is attracted to XCL1 molecule. Therefore, the fusion of EphA2 protein with XCL1 can increase the contact of CD103+ DC and EphA2 antigen, thereby enhancing the antigen presentation and immune activation of cDC1 to natural killer cells (NK) and CD8+ CTL, enhancing immune response and providing more effective anti-tumor protection.

Disclosure of Invention

In view of the above, the present invention provides a novel antigen vaccine targeting EPHA2 and a preparation method thereof; specifically, the invention provides a nucleic acid and protein vaccine targeting EPHA2, which improves the phagocytosis and presentation efficiency of the EPHA2 protein by DC, enhances the specific CD8+ CTL response of EPHA2 and improves the inhibition effect of tumors highly expressing EPHA2 by fusing and expressing cDC1 ligand XCL1 and EPHA2 protein.

The invention provides a fusion protein comprising the amino acid sequence of the extracellular region of EPHA2 capable of eliciting a specific CD8+ T response or an optimized form of its mhc class i molecule binding epitope; a joint; and human or murine XCL1 protein that specifically binds DC cells with cross-antigen presentation capability;

wherein:

the extracellular region of EPHA2 capable of inducing a specific CD8+ T response or an optimized form of its MHC class I molecule binding epitope has an amino acid sequence as shown in SEQ ID No.1 or 2; the human or mouse XCL1 specifically binding DC cells with antigen cross-presentation capability is shown as SEQ ID No.3 or 4;

or

(II) an amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I);

or

(III) an amino acid sequence which has at least 90% sequence identity with the sequence of (I) or (II) and which is functionally identical or similar to the amino acid sequence of (I).

In some embodiments of the invention, the fusion protein comprises an amino acid sequence as shown in SEQ ID No.9 or SEQ ID No. 10.

Based on the above, the present invention also provides a nucleic acid molecule encoding the fusion protein, wherein the nucleic acid molecule comprises DNA and/or mRNA, and the sequence of the nucleic acid molecule has:

(I) a nucleotide sequence as shown in any of SEQ ID Nos. 17-32;

or

(II) as shown in the complementary nucleotide sequence of the nucleotide sequence shown in any SEQ ID No. 15-30; or

(III) as represented by a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code;

or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III);

or

(V) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of (I), (II), (III) or (IV).

The invention also provides a recombinant expression vector, which comprises a vector and the fusion protein or the nucleic acid molecule.

In some embodiments of the invention, the vector comprises pcDNA3.1(+), pcDNA3.1(-), pFastbac1-dual-MBP, pVAX 1.

The invention also provides a recombinant strain or cell comprising said fusion protein or said nucleic acid molecule.

The invention also provides application of the fusion protein, the nucleic acid molecule, the recombinant expression vector or the recombinant strain or cell in preparation of a vaccine for a tumor with high expression of EPHA2 and/or in preparation of a medicament for preventing and/or treating the tumor with high expression of EPHA 2.

In some embodiments of the invention, the metastatic cancer comprises a tumor that highly expresses EPHA2, including prostate cancer, lung cancer, esophageal cancer, colorectal cancer, cervical cancer, ovarian cancer, breast cancer, and skin cancer.

The invention also provides a tumor vaccine targeting EPHA2, which comprises the fusion protein, the nucleic acid molecule, the recombinant expression vector or the recombinant strain or cell and a pharmaceutically acceptable carrier, excipient and/or adjuvant.

The invention also provides a medicament for preventing and/or treating tumors with high expression of EPHA2, which comprises the fusion protein, the nucleic acid molecule, the recombinant expression vector or the recombinant strain or cell and pharmaceutically acceptable auxiliary materials.

The invention provides a tumor vaccine targeting EPHA2, which consists of an amino acid sequence of an extracellular region of EPHA2 and human or mouse XCL1 protein of which the N end is fused and expressed by a linker and is specifically combined with DC cells with antigen cross presentation capacity, nucleic acid containing the fusion protein and a carrier containing the nucleic acid, and application of the fusion protein, the nucleic acid and the carrier in preventing and treating tumors with high expression of EPHA 2. The invention improves the efficiency of the EPHA2 protein phagocytosis, processing and presentation by DC cells and improves the effect of inhibiting the tumor growth by fusing and expressing the EPHA2 protein and the DC cell ligand XCL 1. Experiments prove that the protein expressed by the nucleic acid vaccine can effectively induce an EPHA2 specific T cell response and obviously inhibit the growth of a tumor with high expression of EPHA2 in a tumor animal model.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a map of a vector encoding a fusion protein nucleotide; wherein FIG. 1A shows the CMV promoter followed by the XCL1 secretion signal peptide and then the EPHA2 extracellular region of the nucleic acid vaccine coding sequence; FIG. 1B shows the coding sequence for a nucleic acid vaccine with the CMV promoter followed by the XCL1 sequence and then linked to the extracellular region of EPHA 2;

FIG. 2 is a graph showing the results of prediction of the three-dimensional structure of a fusion protein; inputting a nucleotide sequence for coding the fusion protein into http:// raptorx. uchicago. edu/website to predict the three-dimensional spatial structure of the fusion protein, and the spatial structure diagram of the XCL1-EPHA2 fusion protein is shown;

FIG. 3 shows the detection of expression of a nucleic acid encoding a fusion protein in HEK293T cells; transfecting HEK292T cells by a plasmid vector carrying nucleic acid for encoding the fusion protein for 48 hours, and detecting the expression condition of the fusion protein encoding nucleotide with Flag tag at the C end by using a western blot technology (Westernblot);

FIG. 4 is a graph showing the ratio of EPHA2 specific tetramer + CD8a + T cells in peripheral blood of mice immunized with the fusion gene encoding the extracellular domain of EPHA2 or the XCL1-EPHA2 fusion protein;

FIG. 5 shows the growth of mouse Hepa1-6 after immunization of mice with the fusion gene encoding the extracellular domain of EPHA2 or the XCL1-EPHA2 fusion protein; FIG. 5A shows a diagram of the immunization method and the immunization process of the fusion gene; FIG. 5B shows a tumor growth plot of tumor volume measurements taken after inoculation with Hepa 1-6.

Detailed Description

The invention discloses a tumor vaccine targeting EPHA2, which can be realized by appropriately modifying process parameters by the skilled person with reference to the content in the specification. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention adopts the amino acid sequence of the EPHA2 extracellular region as immunogen to facilitate the purification of soluble protein vaccine, the vaccine of the invention can effectively induce specific T cell immunoreaction aiming at EPHA2 in mice or human bodies, and breaks the immune tolerance of the original tumor-associated antigen EPHA2, thereby effectively playing a role of directly eliminating tumor cells expressing EPHA2, and inhibiting the growth of tumor.

The invention relates to a fusion protein vaccine of a tumor highly expressing EPHA2, which consists of 1) an amino acid sequence of an EPHA2 extracellular region and 2) human or mouse XCL1 protein of which the N end is fused and expressed by a linker and is specifically combined with DC cells with antigen cross presentation capacity. For example, mouse or human EPHA2, which is composed of the sequences shown in SEQ ID No.1 and SEQ ID No.2, and whose N-terminal is fused by a linker to express human or mouse XCL1 that specifically binds to DC cells having antigen cross-presenting ability, and is composed of the sequences shown in amino acids 1 to 114 of SEQ ID No.3 and SEQ ID No. 4.

In some embodiments, the vaccine for a tumor that highly expresses EPHA2 comprises any of SEQ ID No.9 and SEQ ID No. 10.

Another aspect of the invention relates to a nucleic acid encoding the fusion protein vaccine, which consists of 1) the amino acid sequence of the extracellular domain of EPHA2, and 2) a nucleotide sequence of human or murine XCL1 protein, which expresses specific binding to DC cells having cross-antigen presenting ability at the N-terminus by linker fusion. Namely, the amino acid sequences shown in SEQ ID No.1 and SEQ ID No.2 and the human or mouse XCL1 sequence which is specifically combined with DC cells with antigen cross-presentation capacity, namely the amino acid sequences from 1 to 114 in SEQ ID No.3 and SEQ ID No. 4.

In some embodiments, the amino acid sequence encoding the extracellular region of EPHA2 and the sequence of human or murine XCL1 that specifically binds DC cells with cross-antigen presentation capability comprise the nucleotide sequences of SEQ ID No.23 and SEQ ID No. 24.

The invention also provides a recombinant expression vector loaded with the nucleic acid vaccine, which comprises the fusion protein vaccine and the nucleotide sequence and the vector of the corresponding nucleic acid vaccine. The vector can be a mammalian cell expression vector or an insect rod cell expression vector, and concretely can be pcDNA3.1(+), pcDNA3.1(-), pFastbac1-dual-MBP and pVAX 1.

The invention finally provides a vaccine for tumors with high expression of EPHA2, which comprises the fusion protein vaccine, the nucleic acid vaccine and the recombinant expression vector.

The vaccine of the invention provides a method for preventing relapse or treating tumors with high EPHA2 expression. Including administering the vaccines of the invention for other indications.

The invention also provides application of the vaccine, the nucleic acid vaccine and the vector in preparing a vaccine for a tumor with high expression of EPHA 2.

The invention also provides the vaccine, the nucleic acid vaccine and the vector for treating tumors with high expression of EPHA2, including prostate cancer, lung cancer, esophageal cancer, colorectal cancer, cervical cancer, ovarian cancer, breast cancer and skin cancer.

The vaccine or medicament of the invention comprises the fusion protein, the nucleic acid, the recombinant expression vector and optionally pharmaceutically acceptable carriers, excipients and/or adjuvants.

In conclusion, the invention provides a tumor vaccine targeting EPHA2, which consists of an amino acid sequence of an extracellular region of EPHA2, human or mouse XCL1 protein of which the N end is fused and expressed by a linker and is specifically combined with DC cells with antigen cross-presentation capacity, nucleic acid containing the fusion protein, a carrier containing the nucleic acid, and application of the fusion protein, the nucleic acid and the carrier in preventing and treating tumors with high expression of EPHA 2. The invention improves the efficiency of the EPHA2 protein phagocytosis, processing and presentation by DC cells and improves the effect of inhibiting the tumor growth by fusing and expressing the EPHA2 protein and the DC cell ligand XCL 1. Experiments prove that the protein expressed by the nucleic acid vaccine can effectively induce an EPHA2 specific T cell response and obviously inhibit the growth of a tumor with high expression of EPHA2 in a tumor animal model.

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

The invention is further illustrated by the following examples:

example 1 design of fusion protein vaccine antigens and construction and preparation of mammalian cell expression plasmids

(1) Construction of mammalian cell expression vector of fusion gene:

the fusion protein XCL1-EPHA2 is constructed from the amino acid sequences of mouse or human EPHA2 (SEQ ID No.1 and SEQ ID No.2) and mouse or human XCL1 (SEQ ID No.3 and SEQ ID No.4) proteins, and we retained the secretory signal peptides of XCL1 protein (SEQ ID No.5 and SEQ ID No.6) while selecting the extracellular domains of EPHA2 (SEQ ID No.7 and SEQ ID No.8) after removal of the transmembrane-localized signal peptides in order to promote efficient secretion of the fusion protein by nucleic acids expressing the fusion protein outside the cells and chemotaxis of MHC-II + CD11c + CD8A + antigen cross-presenting DC cells in vivo. The amino acid sequences of the resulting fusion proteins (SEQ ID No.9 and SEQ ID No. 10). For the single EPHA2 protein, the self-secretion signal peptide (SEQ ID No.11 and SEQ ID No.12) is reserved to be connected with the EPHA2 extracellular region (SEQ ID No.7 and SEQ ID No.8) so as to ensure that the EPHA2 protein expressed by the single EPHA2 expression vector can be secreted outside the cell. The nucleotide sequence corresponding to the amino acid sequence was codon optimized for mammalian cell expression preference, synthesized by Oncomelania, and ligated into pVAX1 expression vectors (SEQ ID No.13 and SEQ ID No. 14).

The nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.1 has the nucleotide sequence shown in SEQ ID No. 15;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.2 has the nucleotide sequence shown in SEQ ID No. 16;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.3 has the nucleotide sequence shown in SEQ ID No. 17;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.4 has the nucleotide sequence shown in SEQ ID No. 18;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.5 has the nucleotide sequence shown in SEQ ID No. 19;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.6 has the nucleotide sequence shown in SEQ ID No. 20;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.7 has the nucleotide sequence shown in SEQ ID No. 21;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.8 has the nucleotide sequence shown in SEQ ID No. 22;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.9 has the nucleotide sequence shown in SEQ ID No. 23;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.10 has the nucleotide sequence shown in SEQ ID No. 24;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.11 has the nucleotide sequence shown in SEQ ID No. 25;

the nucleic acid molecule which codes the amino acid sequence shown in SEQ ID No.12 has the nucleotide sequence shown in SEQ ID No. 26;

FIG. 1 shows a map of a vector encoding a fusion protein nucleotide; wherein FIG. 1A shows the CMV promoter followed by the XCL1 secretion signal peptide and then the EPHA2 extracellular region of the nucleic acid vaccine coding sequence; FIG. 1B shows the coding sequence for a nucleic acid vaccine with the CMV promoter followed by the XCL1 sequence and then linked to the extracellular region of EPHA 2.

(2) Amplification of fusion gene mammalian cell expression vectors

And (3) transforming bacteria, namely putting the competent bacteria frozen at the temperature of-80 ℃ on ice for thawing, adding 100 ng of plasmid when the competent bacteria are nearly completely thawed, gently and uniformly mixing, and putting the competent bacteria on ice for 30 mins. The competence was placed in a 42 ℃ water bath for 60 s with heat shock and immediately after removal placed on ice for 2 mins. And adding 500 mu L of LB culture medium without antibiotics into the tube, and carrying out shaking culture in a shaking table at 37 ℃ for 1 h. The bacteria were centrifuged at 4000 rpm for 2 mins at room temperature, a portion of the supernatant (about 450 μ L) was discarded and the bacteria were resuspended, and an appropriate amount of the bacterial suspension was spread onto a petri dish containing the corresponding antibiotic. The petri dish was placed face down and incubated overnight in an incubator at 37 ℃. After the clone size was appropriate (about 16 h), the clone was picked up with a gun tip into LB medium supplemented with antibiotics and shake-cultured in a shaker at 37 ℃ until turbid. Taking 15 mL of bacterial liquid cultured to a proper concentration, centrifuging at 4000 rpm for 5mins at room temperature, and discarding the supernatant. Bacterial DNA was extracted according to the instructions of the plasmid Mini kit (DP 103) of Beijing Tiangen. First, 250 μ L of RNase-containing cell resuspension P1 was added to thoroughly resuspend the bacterial pellet and the pellet was transferred to a 1.5 mL EP tube. Adding 250 mu L of alkaline cell lysate P2, and gently inverting and mixing until the liquid is clear. 350 mu L of neutralizing liquid P3 is added, and the mixture is inverted and mixed evenly until flocculent precipitates appear. The suspension was centrifuged at 12000 rpm for 10 mins at room temperature. And putting the DNA adsorption column into a recovery tube, adding 500 muL of equilibrium liquid to activate the adsorption membrane, centrifuging at 12000 rpm for 1min, and then discarding the liquid. The supernatant obtained by centrifugation in step 4 was transferred to a DNA adsorption column, centrifuged at 12000 rpm for 1min, and the liquid was discarded. And adding 600 mu L of cleaning solution into the adsorption column, centrifuging at 12000 rpm for 1min, then discarding the solution, and repeatedly washing once. The tube was evacuated at 12000 rpm for 2 mins. The collection tube was replaced with a new 1.5 mL EP tube and the column was allowed to air dry at room temperature for 5 mins. Adding 70 mu L of elution buffer preheated at 65 ℃ or purified water subjected to high pressure, standing at room temperature for 5mins to fully dissolve DNA, centrifuging at 12000 rpm at room temperature for 3 mins, and collecting liquid. And (5) after the adsorption column is discarded, carrying out concentration measurement on the plasmids in the tube and marking the name, the concentration and the extraction date of the plasmids.

Example 2 prediction of three-dimensional Structure of fusion protein

Based on the nucleotide sequence of the fusion protein, we used http:// raptorx. uchicago. edu/website to predict the three-dimensional structure of XCL1 and EPHA2 fusion proteins. XCL1 belongs to the chemokine, and its chemotactic function needs to maintain an intact spatial structure. In order to ensure that XCL1 still has a spatial structure after the fusion of XCL1 and EPHA2, the three-dimensional structure of the fused amino acid sequence is predicted on http:// raptorx. uchicago. edu/website, and the result is shown in FIG. 2, after the fusion of XCL1 and EPHA2, the XCL1 and EPHA2 still have the original spatial structures respectively and do not influence the chemotactic function of XCL 1.

Example 3 detection of expression Effect of mammalian cell expression vector encoding XCL1-EPHA2 fusion protein

24 hours prior to transfection, 6-well cell culture plates were inoculated with 1 x 10⁶HEK293T cells, the transfection assay was started when the cell density reached 70% -80%. Cell culture medium and serum-free Opti-MEM medium were pre-warmed in a 37 ℃ water bath at the time of transfection. 5 micrograms of empty Vector (Vector), EPHA2 expression Vector alone, XCL1-EPHA2 wild-type fusion gene plasmid, and 20. mu.L PEI transfection reagent were added to 200. mu.L of serum-free Opti-MEM in sequence for transfection, mixed well, and then allowed to stand at room temperature for 10 minutes. The cells to be transfected are replaced by fresh culture medium, and the cells are added into the transfection system gently and shaken up. The cells were returned to the cell incubator and incubated for 6 hours before changing the medium. Cells are harvested after 48 hours of transfer, and expression effect of XCL1-EPHA2 fusion gene plasmid HEK293T cells is detected by Western Blot.

To facilitate detection of expression effect of fusion gene, a Flag tag consisting of 5 amino acids of DDDDK is connected to C end of fusion protein, so as to detect expression of fusion protein by using Flag tag antibody. Cells were harvested and 60 μ L of 0.5% NP40 lysis buffer containing PMSF or Cocktail protease inhibitor was added. Resuspend the cells well and spin lyse the cells at 4 ℃ for 30 min. The lysate was centrifuged at 12000 rpm for 10min at 4 ℃ and the supernatant was collected in a fresh 1.5 mL EP tube and the pellet discarded. Adding a 5 xSDS-PAGE protein loading buffer according to the actual volume of a sample, uniformly mixing, placing the sample in an air bath at 100 ℃ for heating for 10 minutes, immediately carrying out Western blot, and detecting by using a Flag tag antibody (Sigma, F3165), wherein the result is shown in figure 3 that an empty Vector (Vector) has no protein expression, an EPHA2 expression Vector alone and an XCL1-EPHA2 wild-type fusion gene plasmid can effectively express and have equivalent expression quantity.

Example 4 exploration of whether fusion gene immunization induced a stronger cell-specific T cell response

We extracted EPHA2 and XCL1-EPHA2 wild-type plasmids alone and injected the mice with the immunizing plasmid using the gene gun (GDS-80) from Wealtec. Mouse peripheral blood EPHA 2-specific T cell responses were detected after 14 days using EPHA 2-specific tetramer flow antibodies.

Specifically, the inner and outer peripheral blood of the canthus of the mouse are taken firstly, added with heparin sodium anticoagulant and mixed evenly, centrifuged for 10 minutes at 200g, the supernatant is discarded, and the peripheral blood is treated with the red blood cell lysate (Cat. No. WL 2000) of the company R & D. Diluting the lysate A by 10 times with distilled water to prepare working solution, adding 2mL of working solution into each peripheral blood sample for resuspension, standing at room temperature for 10min, and diluting the neutralizing solution B by 10 times with distilled water to prepare the working solution. Ten minutes later, 10mL of the neutralization solution B was added to the lysate for neutralization, followed by centrifugation at 200g for 10 min. The cell pellet was washed once with 1% inactivated FBS in PBS buffer and flow stained: EPHA2-tetramer antibody-FITC, CD8 α -PE. After 2 hours, flow detection was carried out. The results are shown in fig. 4, the specific T cells of mice immunized by XCL1-EPHA2 wild-type plasmid against EPHA2 in peripheral blood are significantly increased compared with the group immunized by EPHA2 alone, which indicates that the XCL1-EPHA2 wild-type plasmid immunization really induces the specific response of CD8A cytotoxic T lymphocytes.

Table 1 figure 4 data

Example 5 Effect of the fusion Gene on intervention in transplantation tumorigenesis of CT26 cells overexpressing EPHA2

We extracted EPHA2 and XCL1-EPHA2 wild-type plasmids alone and injected the mice with the immunizing plasmid using the gene gun (GDS-80) from Wealtec. After the hepa1-6 high expression EPHA2 cells are transplanted into the same species, the inhibition of the fusion gene immunity on the growth of CT26 tumor cells over expressing EPHA2 is observed.

We performed particle gun plasmid injection into mice following the immunization strategy outlined by the time axis of fig. 5A. C57B6 (purchased from Wintolite, China) 6-week-old male mice were divided into a control group, 3 groups injected with EPHA2 and XCL1-EPHA2 wild-type plasmid, and five mice per group were depilatory-treated with depilatory cream at the right side of the mice near the inguinal lymph node. Then, 50 μ g of plasmid was injected into the site of hair removal using a gene gun, once a week, four times a total, and the CT26 cells overexpressing EPHA2 were investigated for tumor formation conditions one week after the last injection and prior to inoculation, and the tumor formation time was observed and the major and minor diameters a and b of the tumor were measured every two days, and tumor volume calculation was performed according to a b/2 to plot the tumor growth curve. As shown in FIGS. 5A and 5B, the injection of XCL1-EPHA2 wild-type plasmid was effective in delaying the onset and growth rate of tumors. The therapeutic effect of the fusion gene immunity is proved to be obvious and effective.

TABLE 2 FIG. 5B data

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Pro Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu

900 905 910

Trp Leu Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu His Phe Met Val

915 920 925

Ala Gly Tyr Thr Ala Ile Glu Lys Val Val Gln Met Ser Asn Glu Asp

930 935 940

Ile Lys Arg Ile Gly Val Arg Leu Pro Gly His Gln Lys Arg Ile Ala

945 950 955 960

Tyr Ser Leu Leu Gly Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro

965 970 975

Ile

<210> 2

<211> 976

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Glu Leu Gln Ala Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys

1 5 10 15

Ala Leu Ala Ala Ala Ala Ala Ala Gln Gly Lys Glu Val Val Leu Leu

20 25 30

Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr

35 40 45

Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met Asn Asp Met Pro Ile

50 55 60

Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp

65 70 75 80

Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile

85 90 95

Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala

100 105 110

Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu

115 120 125

Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr

130 135 140

Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His

145 150 155 160

Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys

165 170 175

Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu

180 185 190

Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu

195 200 205

Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala

210 215 220

Thr Val Ala Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly

225 230 235 240

Glu Glu Pro Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro

245 250 255

Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala

260 265 270

Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser

275 280 285

Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala

290 295 300

Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro

305 310 315 320

Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr

325 330 335

Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln

340 345 350

Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln

355 360 365

Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg

370 375 380

Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser

385 390 395 400

Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn

405 410 415

Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val

420 425 430

Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser

435 440 445

Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser

450 455 460

Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn

465 470 475 480

Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp

485 490 495

Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln

500 505 510

Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu Phe Gln Thr Leu Ser

515 520 525

Pro Glu Gly Ser Gly Asn Leu Ala Val Ile Gly Gly Val Ala Val Gly

530 535 540

Val Val Leu Leu Leu Val Leu Ala Gly Val Gly Phe Phe Ile His Arg

545 550 555 560

Arg Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe

565 570 575

Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His

580 585 590

Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile

595 600 605

His Pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe

610 615 620

Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu

625 630 635 640

Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln

645 650 655

Arg Val Asp Phe Leu Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His

660 665 670

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

675 680 685

Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu

690 695 700

Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gln Leu Val Gly Met Leu

705 710 715 720

Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val

725 730 735

His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val

740 745 750

Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro

755 760 765

Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr

770 775 780

Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val

785 790 795 800

Trp Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg

805 810 815

Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn Asp

820 825 830

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

835 840 845

Leu Met Met Gln Cys Trp Gln Gln Glu Arg Ala Arg Arg Pro Lys Phe

850 855 860

Ala Asp Ile Val Ser Ile Leu Asp Lys Leu Ile Arg Ala Pro Asp Ser

865 870 875 880

Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Leu Pro

885 890 895

Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp

900 905 910

Leu Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu His Phe Met Ala Ala

915 920 925

Gly Tyr Thr Ala Ile Glu Lys Val Val Gln Met Thr Asn Asp Asp Ile

930 935 940

Lys Arg Ile Gly Val Arg Leu Pro Gly His Gln Lys Arg Ile Ala Tyr

945 950 955 960

Ser Leu Leu Gly Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro Ile

965 970 975

<210> 3

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro

1 5 10 15

Trp Val Val Glu Gly Val Gly Thr Glu Val Leu Glu Glu Ser Ser Cys

20 25 30

Val Asn Leu Gln Thr Gln Arg Leu Pro Val Gln Lys Ile Lys Thr Tyr

35 40 45

Ile Ile Trp Glu Gly Ala Met Arg Ala Val Ile Phe Val Thr Lys Arg

50 55 60

Gly Leu Lys Ile Cys Ala Asp Pro Glu Ala Lys Trp Val Lys Ala Ala

65 70 75 80

Ile Lys Thr Val Asp Gly Arg Ala Ser Thr Arg Lys Asn Met Ala Glu

85 90 95

Thr Val Pro Thr Gly Ala Gln Arg Ser Thr Ser Thr Ala Ile Thr Leu

100 105 110

Thr Gly Gly

115

<210> 4

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala

1 5 10 15

Tyr Ile Val Glu Gly Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys

20 25 30

Val Ser Leu Thr Thr Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr

35 40 45

Thr Ile Thr Glu Gly Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg

50 55 60

Gly Leu Lys Val Cys Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val

65 70 75 80

Val Arg Ser Met Asp Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln

85 90 95

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

100 105 110

Thr Gly

<210> 5

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro

1 5 10 15

Trp Val Val Glu Gly

20

<210> 6

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala

1 5 10 15

Tyr Ile Val Glu Gly

20

<210> 7

<211> 513

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Lys Glu Val Val Leu Leu Asp Phe Ala Ala Met Lys Gly Glu Leu Gly

1 5 10 15

Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln Asn Ile

20 25 30

Met Asp Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn Val Val Ser

35 40 45

Gly Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Glu Glu

50 55 60

Ala Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg Asp Cys Asn

65 70 75 80

Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr

85 90 95

Tyr Ala Glu Ser Asp Val Asp Tyr Gly Thr Asn Phe Gln Lys Arg Gln

100 105 110

Phe Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr Val Ser Ser

115 120 125

Asp Phe Glu Ala Arg Asn Val Lys Leu Asn Val Glu Glu Arg Met Val

130 135 140

Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly

145 150 155 160

Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro

165 170 175

Glu Met Leu Gln Ser Leu Ala Arg Phe Pro Glu Thr Ile Ala Val Ala

180 185 190

Val Ser Asp Thr Gln Pro Leu Ala Thr Val Ala Gly Thr Cys Val Asp

195 200 205

His Ala Val Val Pro Tyr Gly Gly Glu Gly Pro Leu Met His Cys Thr

210 215 220

Val Asp Gly Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Glu

225 230 235 240

Gly Tyr Glu Lys Val Glu Asp Ala Cys Arg Ala Cys Ser Pro Gly Phe

245 250 255

Phe Lys Ser Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His

260 265 270

Thr Leu Pro Ser Thr Glu Gly Ala Thr Ser Cys Gln Cys Glu Glu Gly

275 280 285

Tyr Phe Arg Ala Pro Glu Asp Pro Leu Ser Met Ser Cys Thr Arg Pro

290 295 300

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

305 310 315 320

Glu Leu Arg Trp Thr Ala Pro Lys Asp Thr Gly Gly Arg Gln Asp Ile

325 330 335

Val Tyr Ser Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu Cys

340 345 350

Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Ala Leu

355 360 365

Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr

370 375 380

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

385 390 395 400

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

405 410 415

Lys Val Arg Leu Glu Asp Arg Ser Thr Thr Ser Leu Ser Val Thr Trp

420 425 430

Ser Ile Pro Val Ser Gln Gln Ser Arg Val Trp Lys Tyr Glu Val Thr

435 440 445

Tyr Arg Lys Lys Gly Asp Ala Asn Ser Tyr Asn Val Arg Arg Thr Glu

450 455 460

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

465 470 475 480

Val Gln Val Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser Lys

485 490 495

Val His Glu Phe Gln Thr Leu Ser Thr Glu Gly Ser Ala Asn Met Ala

500 505 510

Val

<210> 8

<211> 514

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ala Gln Gly Lys Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly

1 5 10 15

Glu Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met

20 25 30

Gln Asn Ile Met Asn Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn

35 40 45

Val Met Ser Gly Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr

50 55 60

Arg Gly Glu Ala Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg

65 70 75 80

Asp Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe

85 90 95

Asn Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gln

100 105 110

Lys Arg Leu Phe Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr

115 120 125

Val Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu

130 135 140

Arg Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln

145 150 155 160

Asp Ile Gly Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys

165 170 175

Lys Cys Pro Glu Leu Leu Gln Gly Leu Ala His Phe Pro Glu Thr Ile

180 185 190

Ala Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val

195 200 205

Asp His Ala Val Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys

210 215 220

Ala Val Asp Gly Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln

225 230 235 240

Ala Gly Tyr Glu Lys Val Glu Asp Ala Cys Gln Ala Cys Ser Pro Gly

245 250 255

Phe Phe Lys Phe Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu

260 265 270

His Thr Leu Pro Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu

275 280 285

Gly Phe Phe Arg Ala Pro Gln Asp Pro Ala Ser Met Pro Cys Thr Arg

290 295 300

Pro Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys

305 310 315 320

Val Glu Leu Arg Trp Thr Pro Pro Gln Asp Ser Gly Gly Arg Glu Asp

325 330 335

Ile Val Tyr Ser Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu

340 345 350

Cys Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly

355 360 365

Leu Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn

370 375 380

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

385 390 395 400

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

405 410 415

Pro Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser

420 425 430

Trp Ser Ile Pro Pro Pro Gln Gln Ser Arg Val Trp Lys Tyr Glu Val

435 440 445

Thr Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr

450 455 460

Glu Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr

465 470 475 480

Leu Val Gln Val Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser

485 490 495

Lys Val His Glu Phe Gln Thr Leu Ser Pro Glu Gly Ser Gly Asn Leu

500 505 510

Ala Val

<210> 9

<211> 638

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro

1 5 10 15

Trp Val Val Glu Gly Val Gly Thr Glu Val Leu Glu Glu Ser Ser Cys

20 25 30

Val Asn Leu Gln Thr Gln Arg Leu Pro Val Gln Lys Ile Lys Thr Tyr

35 40 45

Ile Ile Trp Glu Gly Ala Met Arg Ala Val Ile Phe Val Thr Lys Arg

50 55 60

Gly Leu Lys Ile Cys Ala Asp Pro Glu Ala Lys Trp Val Lys Ala Ala

65 70 75 80

Ile Lys Thr Val Asp Gly Arg Ala Ser Thr Arg Lys Asn Met Ala Glu

85 90 95

Thr Val Pro Thr Gly Ala Gln Arg Ser Thr Ser Thr Ala Ile Thr Leu

100 105 110

Thr Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Lys Glu Val

115 120 125

Val Leu Leu Asp Phe Ala Ala Met Lys Gly Glu Leu Gly Trp Leu Thr

130 135 140

His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met Asp Asp

145 150 155 160

Met Pro Ile Tyr Met Tyr Ser Val Cys Asn Val Val Ser Gly Asp Gln

165 170 175

Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Glu Glu Ala Glu Arg

180 185 190

Ile Phe Ile Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro

195 200 205

Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu

210 215 220

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

225 230 235 240

Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp Phe Glu

245 250 255

Ala Arg Asn Val Lys Leu Asn Val Glu Glu Arg Met Val Gly Pro Leu

260 265 270

Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Val

275 280 285

Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Met Leu

290 295 300

Gln Ser Leu Ala Arg Phe Pro Glu Thr Ile Ala Val Ala Val Ser Asp

305 310 315 320

Thr Gln Pro Leu Ala Thr Val Ala Gly Thr Cys Val Asp His Ala Val

325 330 335

Val Pro Tyr Gly Gly Glu Gly Pro Leu Met His Cys Thr Val Asp Gly

340 345 350

Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Glu Gly Tyr Glu

355 360 365

Lys Val Glu Asp Ala Cys Arg Ala Cys Ser Pro Gly Phe Phe Lys Ser

370 375 380

Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro

385 390 395 400

Ser Thr Glu Gly Ala Thr Ser Cys Gln Cys Glu Glu Gly Tyr Phe Arg

405 410 415

Ala Pro Glu Asp Pro Leu Ser Met Ser Cys Thr Arg Pro Pro Ser Ala

420 425 430

Pro Asn Tyr Leu Thr Ala Ile Gly Met Gly Ala Lys Val Glu Leu Arg

435 440 445

Trp Thr Ala Pro Lys Asp Thr Gly Gly Arg Gln Asp Ile Val Tyr Ser

450 455 460

Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys

465 470 475 480

Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Ala Leu Thr Arg Thr

485 490 495

Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr Thr Phe Ala

500 505 510

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

515 520 525

Arg Thr Ala Ser Val Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg

530 535 540

Leu Glu Asp Arg Ser Thr Thr Ser Leu Ser Val Thr Trp Ser Ile Pro

545 550 555 560

Val Ser Gln Gln Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys

565 570 575

Lys Gly Asp Ala Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser

580 585 590

Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val

595 600 605

Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu

610 615 620

Phe Gln Thr Leu Ser Thr Glu Gly Ser Ala Asn Met Ala Val

625 630 635

<210> 10

<211> 638

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala

1 5 10 15

Tyr Ile Val Glu Gly Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys

20 25 30

Val Ser Leu Thr Thr Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr

35 40 45

Thr Ile Thr Glu Gly Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg

50 55 60

Gly Leu Lys Val Cys Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val

65 70 75 80

Val Arg Ser Met Asp Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln

85 90 95

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

100 105 110

Thr Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ala Gln Gly Lys

115 120 125

Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp

130 135 140

Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met

145 150 155 160

Asn Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly

165 170 175

Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala

180 185 190

Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser

195 200 205

Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr

210 215 220

Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe

225 230 235 240

Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp

245 250 255

Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly

260 265 270

Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala

275 280 285

Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu

290 295 300

Leu Leu Gln Gly Leu Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp

305 310 315 320

Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val Asp His Ala Val

325 330 335

Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys Ala Val Asp Gly

340 345 350

Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu

355 360 365

Lys Val Glu Asp Ala Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys Phe

370 375 380

Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro

385 390 395 400

Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg

405 410 415

Ala Pro Gln Asp Pro Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala

420 425 430

Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg

435 440 445

Trp Thr Pro Pro Gln Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser

450 455 460

Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys

465 470 475 480

Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr

485 490 495

Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr

500 505 510

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

515 520 525

Arg Thr Ala Ser Val Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg

530 535 540

Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro

545 550 555 560

Pro Pro Gln Gln Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys

565 570 575

Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser

580 585 590

Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val

595 600 605

Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu

610 615 620

Phe Gln Thr Leu Ser Pro Glu Gly Ser Gly Asn Leu Ala Val

625 630 635

<210> 11

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Glu Leu Arg Ala Val Gly Phe Cys Leu Ala Leu Leu Trp Gly Cys

1 5 10 15

Ala Leu Ala Ala Ala Ala Ala Gln Gly

20 25

<210> 12

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Glu Leu Gln Ala Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys

1 5 10 15

Ala Leu Ala Ala Ala Ala Ala Ala

20

<210> 13

<211> 4910

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccgaatt cgccaccatg agacttctcc tcctgacttt cctgggagtc 780

tgctgcctca ccccatgggt tgtggaaggt gtggggactg aagtcctaga agagagtagc 840

tgtgtgaact tacaaaccca gcggctgcca gttcaaaaaa tcaagaccta tatcatctgg 900

gagggggcca tgagagctgt aatttttgtc accaaacgag gactaaaaat ttgtgctgat 960

ccagaagcca aatgggtgaa agcagcgatc aagactgtgg atggcagggc cagtaccaga 1020

aagaacatgg ctgaaactgt tcccacagga gcccagaggt ccaccagcac agcgataacc 1080

ctgactgggg gcggaggcgg aggatcaggg ggagggggag gaaaggaagt tgttttgttg 1140

gacttcgcag caatgaaggg agagctcggc tggctcacgc acccctatgg caaagggtgg 1200

gacctgatgc agaacatcat ggacgacatg cctatctaca tgtactcggt gtgcaacgtg 1260

gtatccggcg accaggacaa ctggctccgc accaactggg tgtaccggga ggaggccgag 1320

cgcatcttta ttgagctcaa gttcacggtg cgagactgta acagcttccc gggtggcgcc 1380

agctcatgca aagagacctt caacctctac tatgcagagt cagatgtgga ctatggcacc 1440

aacttccaga agcgccagtt caccaagatt gacaccatcg cccctgacga gatcacggtc 1500

agcagtgact tcgaggctcg caatgtcaag ctgaacgtag aggagcgcat ggtggggccc 1560

cttacccgga agggcttcta cctggccttc caggacatcg gcgcctgcgt ggcgctgctc 1620

tccgttcgcg tctactacaa gaagtgtccc gagatgctgc agagcttggc tcgcttcccc 1680

gagaccattg ctgtcgctgt ctccgataca caacccctgg ccacggtggc cggtacctgc 1740

gtggaccatg ccgtggtgcc ttatgggggc gaggggcctc tcatgcactg cacggtggat 1800

ggcgagtggc tggtgcccat cgggcagtgc ctgtgccagg aaggctacga gaaggtcgag 1860

gatgcctgcc gagcctgttc tccaggattc ttcaagtctg aggcatctga gagcccttgc 1920

ctggagtgtc cagagcatac cctgccatcc acagagggtg ccacctcctg ccagtgtgaa 1980

gaaggctatt tcagggcacc tgaggaccca ctgtccatgt cttgcacacg tccaccctct 2040

gcccccaact acctcacggc cattggcatg ggtgccaaag tagaactgcg ttggacagct 2100

cccaaggaca ctggtggccg ccaggacatt gtctacagtg tcacttgcga acagtgctgg 2160

ccagagtctg gcgagtgtgg gccctgtgag gcgagcgtgc gctattcaga acctcctcac 2220

gccctgaccc gcacgagtgt gacagtcagt gacctggagc cccacatgaa ctataccttc 2280

gctgtcgaag cacgcaatgg tgtctcaggc ctggtgacta gccgaagctt ccggactgcc 2340

agcgtcagta ttaaccaaac agagcccccc aaagtgaggc tggaggaccg aagcaccacc 2400

tccctgagtg tcacctggag catcccggtg tcacagcaga gccgtgtgtg gaagtacgaa 2460

gtcacctacc gcaagaaggg ggatgccaac agctataatg tgcgccgcac ggaaggcttc 2520

tccgtgaccc tggatgacct tgctccggat accacgtacc tggtgcaggt gcaggcgctg 2580

acgcaggagg gccagggagc cggcagcaaa gtgcacgagt tccagacact gtccacggaa 2640

ggatctgcca acatggcggt ggattacaag gatgacgacg ataagtaagc ggccgcctcg 2700

agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 2760

catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 2820

tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 2880

tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 2940

ctggggatgc ggtgggctct atggcttcta ctgggcggtt ttatggacag caagcgaacc 3000

ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat 3060

ggctttctcg ccgccaagga tctgatggcg caggggatca agctctgatc aagagacagg 3120

atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 3180

ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 3240

cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 3300

tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt 3360

tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 3420

cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 3480

catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca 3540

ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 3600

ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 3660

ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 3720

tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 3780

ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 3840

atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 3900

cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt tcctgatgcg 3960

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacaggtgg cacttttcgg 4020

ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 4080

ctcatgagac aataaccctg ataaatgctt caataatagc acgtgctaaa acttcatttt 4140

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 4200

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 4260

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 4320

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 4380

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 4440

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 4500

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 4560

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 4620

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 4680

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 4740

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 4800

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 4860

gcctttttac ggttcctggg cttttgctgg ccttttgctc acatgttctt 4910

<210> 14

<211> 4910

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccgaatt cgccaccatg agacttctca tcctggccct ccttggcatc 780

tgctctctca ctgcatacat tgtggaaggt gtagggagtg aagtctcaga taagaggacc 840

tgtgtgagcc tcactaccca gcgactgccg gttagcagaa tcaagaccta caccatcacg 900

gaaggctcct tgagagcagt aatttttatt accaaacgtg gcctaaaagt ctgtgctgat 960

ccacaagcca catgggtgag agacgtggtc aggagcatgg acaggaaatc caacaccaga 1020

aataacatga tccagaccaa gccaacagga acccagcaat cgaccaatac agctgtgact 1080

ctgactggcg gaggcggagg atcaggggga gggggaggag cgcagggcaa ggaagtggta 1140

ctgctggact ttgctgcagc tggaggggag ctcggctggc tcacacaccc gtatggcaaa 1200

gggtgggacc tgatgcagaa catcatgaat gacatgccga tctacatgta ctccgtgtgc 1260

aacgtgatgt ctggcgacca ggacaactgg ctccgcacca actgggtgta ccgaggagag 1320

gctgagcgta tcttcattga gctcaagttt actgtacgtg actgcaacag cttccctggt 1380

ggcgccagct cctgcaagga gactttcaac ctctactatg ccgagtcgga cctggactac 1440

ggcaccaact tccagaagcg cctgttcacc aagattgaca ccattgcgcc cgatgagatc 1500

accgtcagca gcgacttcga ggcacgccac gtgaagctga acgtggagga gcgctccgtg 1560

gggccgctca cccgcaaagg cttctacctg gccttccagg atatcggtgc ctgtgtggcg 1620

ctgctctccg tccgtgtcta ctacaagaag tgccccgagc tgctgcaggg cctggcccac 1680

ttccctgaga ccatcgccgg ctctgatgca ccttccctgg ccactgtggc cggcacctgt 1740

gtggaccatg ccgtggtgcc accggggggt gaagagcccc gtatgcactg tgcagtggat 1800

ggcgagtggc tggtgcccat tgggcagtgc ctgtgccagg caggctacga gaaggtggag 1860

gatgcctgcc aggcctgctc gcctggattt tttaagtttg aggcatctga gagcccctgc 1920

ttggagtgcc ctgagcacac gctgccatcc cctgagggtg ccacctcctg cgagtgtgag 1980

gaaggcttct tccgggcacc tcaggaccca gcgtcgatgc cttgcacacg acccccctcc 2040

gccccacact acctcacagc cgtgggcatg ggtgccaagg tggagctgcg ctggacgccc 2100

cctcaggaca gcgggggccg cgaggacatt gtctacagcg tcacctgcga acagtgctgg 2160

cccgagtctg gggaatgcgg gccgtgtgag gccagtgtgc gctactcgga gcctcctcac 2220

ggactgaccc gcaccagtgt gacagtgagc gacctggagc cccacatgaa ctacaccttc 2280

accgtggagg cccgcaatgg cgtctcaggc ctggtaacca gccgcagctt ccgtactgcc 2340

agtgtcagca tcaaccagac agagcccccc aaggtgaggc tggagggccg cagcaccacc 2400

tcgcttagcg tctcctggag catccccccg ccgcagcaga gccgagtgtg gaagtacgag 2460

gtcacttacc gcaagaaggg agactccaac agctacaatg tgcgccgcac cgagggtttc 2520

tccgtgaccc tggacgacct ggccccagac accacctacc tggtccaggt gcaggcactg 2580

acgcaggagg gccagggggc cggcagcaag gtgcacgaat tccagacgct gtccccggag 2640

ggatctggca acttggcggt ggattacaag gatgacgacg ataagtaagc ggccgcctcg 2700

agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 2760

catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 2820

tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 2880

tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 2940

ctggggatgc ggtgggctct atggcttcta ctgggcggtt ttatggacag caagcgaacc 3000

ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat 3060

ggctttctcg ccgccaagga tctgatggcg caggggatca agctctgatc aagagacagg 3120

atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 3180

ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 3240

cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 3300

tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt 3360

tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 3420

cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 3480

catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca 3540

ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 3600

ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 3660

ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 3720

tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 3780

ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 3840

atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 3900

cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt tcctgatgcg 3960

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacaggtgg cacttttcgg 4020

ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 4080

ctcatgagac aataaccctg ataaatgctt caataatagc acgtgctaaa acttcatttt 4140

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 4200

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 4260

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 4320

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 4380

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 4440

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 4500

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 4560

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 4620

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 4680

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 4740

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 4800

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 4860

gcctttttac ggttcctggg cttttgctgg ccttttgctc acatgttctt 4910

<210> 15

<211> 2931

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggagctcc gggcagtcgg tttctgcctg gcgctgctgt ggggttgcgc gctggcggcc 60

gcggcggcac agggaaagga agttgttttg ttggacttcg cagcaatgaa gggagagctc 120

ggctggctca cgcaccccta tggcaaaggg tgggacctga tgcagaacat catggacgac 180

atgcctatct acatgtactc ggtgtgcaac gtggtatccg gcgaccagga caactggctc 240

cgcaccaact gggtgtaccg ggaggaggcc gagcgcatct ttattgagct caagttcacg 300

gtgcgagact gtaacagctt cccgggtggc gccagctcat gcaaagagac cttcaacctc 360

tactatgcag agtcagatgt ggactatggc accaacttcc agaagcgcca gttcaccaag 420

attgacacca tcgcccctga cgagatcacg gtcagcagtg acttcgaggc tcgcaatgtc 480

aagctgaacg tagaggagcg catggtgggg ccccttaccc ggaagggctt ctacctggcc 540

ttccaggaca tcggcgcctg cgtggcgctg ctctccgttc gcgtctacta caagaagtgt 600

cccgagatgc tgcagagctt ggctcgcttc cccgagacca ttgctgtcgc tgtctccgat 660

acacaacccc tggccacggt ggccggtacc tgcgtggacc atgccgtggt gccttatggg 720

ggcgaggggc ctctcatgca ctgcacggtg gatggcgagt ggctggtgcc catcgggcag 780

tgcctgtgcc aggaaggcta cgagaaggtc gaggatgcct gccgagcctg ttctccagga 840

ttcttcaagt ctgaggcatc tgagagccct tgcctggagt gtccagagca taccctgcca 900

tccacagagg gtgccacctc ctgccagtgt gaagaaggct atttcagggc acctgaggac 960

ccactgtcca tgtcttgcac acgtccaccc tctgccccca actacctcac ggccattggc 1020

atgggtgcca aagtagaact gcgttggaca gctcccaagg acactggtgg ccgccaggac 1080

attgtctaca gtgtcacttg cgaacagtgc tggccagagt ctggcgagtg tgggccctgt 1140

gaggcgagcg tgcgctattc agaacctcct cacgccctga cccgcacgag tgtgacagtc 1200

agtgacctgg agccccacat gaactatacc ttcgctgtcg aagcacgcaa tggtgtctca 1260

ggcctggtga ctagccgaag cttccggact gccagcgtca gtattaacca aacagagccc 1320

cccaaagtga ggctggagga ccgaagcacc acctccctga gtgtcacctg gagcatcccg 1380

gtgtcacagc agagccgtgt gtggaagtac gaagtcacct accgcaagaa gggggatgcc 1440

aacagctata atgtgcgccg cacggaaggc ttctccgtga ccctggatga ccttgctccg 1500

gataccacgt acctggtgca ggtgcaggcg ctgacgcagg agggccaggg agccggcagc 1560

aaagtgcacg agttccagac actgtccacg gaaggatctg ccaacatggc ggtgatcggc 1620

ggtgtggctg taggtgttgt tttgcttctg gtactggcag gagttggcct cttcatccat 1680

cgaaggagga ggaacctgcg ggctcgccag tcctctgagg atgtccgttt ttccaagtca 1740

gaacaactaa agcccctgaa gacctatgtg gatcctcaca cttacgaaga ccccaaccag 1800

gctgtactca agtttaccac cgagatccac ccatcctgtg tggcaaggca gaaggtcatt 1860

ggagcaggag agtttggaga ggtctataaa gggacgctga aggcatcctc ggggaagaag 1920

gagataccgg tggccatcaa gacactgaaa gcgggctaca ctgagaagca gcgggtggac 1980

ttcctgagcg aggccagcat catgggccag tttagccacc acaatatcat ccgcctggag 2040

ggcgtggtct ctaaatacaa acccatgatg attatcacag agtacatgga gaatggagcg 2100

ctagacaagt tccttaggga gaaggatggt gagttcagtg tacttcagtt ggtgggcatg 2160

ctgaggggta tcgcatccgg catgaagtac ctggccaaca tgaactacgt gcaccgggac 2220

ctggccgccc gcaacatcct cgtcaacagc aacctggtgt gcaaggtgtc cgattttggc 2280

ctgtcgcgtg tgctggaaga tgaccccgag gccacctaca ccacaagtgg cggcaagatc 2340

cctattcgat ggacagcccc agaggccatt tcctaccgca agttcacctc agccagcgat 2400

gtgtggagct acggcattgt catgtgggaa gtgatgactt atggcgaacg gccctactgg 2460

gaactgtcaa accacgaggt catgaaagcc atcaacgacg gcttccggct ccctacgccc 2520

atggactgcc cttcagccat ttaccagctc atgatgcagt gctggcagca agagcgctcc 2580

cgccgaccca agtttgccga catcgttagc atcctggaca agctcatccg agcccccgac 2640

tccctcaaga cgctggctga ctttgatccc cgagtgtcca tccggctgcc cagcaccagc 2700

ggctcggagg gagtcccctt ccgtacggtg tccgagtggc tggagagcat caagatgcaa 2760

cagtacacgg aacacttcat ggtggctggc tacacggcca tcgagaaggt ggtacagatg 2820

tccaacgaag acatcaaaag gatcggagtg cgtcttcctg gccaccagaa gcgtattgcc 2880

tacagcctgc tgggactcaa ggaccaggtc aacacagtgg ggattcctat c 2931

<210> 16

<211> 2928

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atggagctcc aggcagcccg cgcctgcttc gccctgctgt ggggctgtgc gctggccgcg 60

gccgcggcgg cgcagggcaa ggaagtggta ctgctggact ttgctgcagc tggaggggag 120

ctcggctggc tcacacaccc gtatggcaaa gggtgggacc tgatgcagaa catcatgaat 180

gacatgccga tctacatgta ctccgtgtgc aacgtgatgt ctggcgacca ggacaactgg 240

ctccgcacca actgggtgta ccgaggagag gctgagcgta tcttcattga gctcaagttt 300

actgtacgtg actgcaacag cttccctggt ggcgccagct cctgcaagga gactttcaac 360

ctctactatg ccgagtcgga cctggactac ggcaccaact tccagaagcg cctgttcacc 420

aagattgaca ccattgcgcc cgatgagatc accgtcagca gcgacttcga ggcacgccac 480

gtgaagctga acgtggagga gcgctccgtg gggccgctca cccgcaaagg cttctacctg 540

gccttccagg atatcggtgc ctgtgtggcg ctgctctccg tccgtgtcta ctacaagaag 600

tgccccgagc tgctgcaggg cctggcccac ttccctgaga ccatcgccgg ctctgatgca 660

ccttccctgg ccactgtggc cggcacctgt gtggaccatg ccgtggtgcc accggggggt 720

gaagagcccc gtatgcactg tgcagtggat ggcgagtggc tggtgcccat tgggcagtgc 780

ctgtgccagg caggctacga gaaggtggag gatgcctgcc aggcctgctc gcctggattt 840

tttaagtttg aggcatctga gagcccctgc ttggagtgcc ctgagcacac gctgccatcc 900

cctgagggtg ccacctcctg cgagtgtgag gaaggcttct tccgggcacc tcaggaccca 960

gcgtcgatgc cttgcacacg acccccctcc gccccacact acctcacagc cgtgggcatg 1020

ggtgccaagg tggagctgcg ctggacgccc cctcaggaca gcgggggccg cgaggacatt 1080

gtctacagcg tcacctgcga acagtgctgg cccgagtctg gggaatgcgg gccgtgtgag 1140

gccagtgtgc gctactcgga gcctcctcac ggactgaccc gcaccagtgt gacagtgagc 1200

gacctggagc cccacatgaa ctacaccttc accgtggagg cccgcaatgg cgtctcaggc 1260

ctggtaacca gccgcagctt ccgtactgcc agtgtcagca tcaaccagac agagcccccc 1320

aaggtgaggc tggagggccg cagcaccacc tcgcttagcg tctcctggag catccccccg 1380

ccgcagcaga gccgagtgtg gaagtacgag gtcacttacc gcaagaaggg agactccaac 1440

agctacaatg tgcgccgcac cgagggtttc tccgtgaccc tggacgacct ggccccagac 1500

accacctacc tggtccaggt gcaggcactg acgcaggagg gccagggggc cggcagcaag 1560

gtgcacgaat tccagacgct gtccccggag ggatctggca acttggcggt gattggcggc 1620

gtggctgtcg gtgtggtcct gcttctggtg ctggcaggag ttggcttctt tatccaccgc 1680

aggaggaaga accagcgtgc ccgccagtcc ccggaggacg tttacttctc caagtcagaa 1740

caactgaagc ccctgaagac atacgtggac ccccacacat atgaggaccc caaccaggct 1800

gtgttgaagt tcactaccga gatccatcca tcctgtgtca ctcggcagaa ggtgatcgga 1860

gcaggagagt ttggggaggt gtacaagggc atgctgaaga catcctcggg gaagaaggag 1920

gtgccggtgg ccatcaagac gctgaaagcc ggctacacag agaagcagcg agtggacttc 1980

ctcggcgagg ccggcatcat gggccagttc agccaccaca acatcatccg cctagagggc 2040

gtcatctcca aatacaagcc catgatgatc atcactgagt acatggagaa tggggccctg 2100

gacaagttcc ttcgggagaa ggatggcgag ttcagcgtgc tgcagctggt gggcatgctg 2160

cggggcatcg cagctggcat gaagtacctg gccaacatga actatgtgca ccgtgacctg 2220

gctgcccgca acatcctcgt caacagcaac ctggtctgca aggtgtctga ctttggcctg 2280

tcccgcgtgc tggaggacga ccccgaggcc acctacacca ccagtggcgg caagatcccc 2340

atccgctgga ccgccccgga ggccatttcc taccggaagt tcacctctgc cagcgacgtg 2400

tggagctttg gcattgtcat gtgggaggtg atgacctatg gcgagcggcc ctactgggag 2460

ttgtccaacc acgaggtgat gaaagccatc aatgatggct tccggctccc cacacccatg 2520

gactgcccct ccgccatcta ccagctcatg atgcagtgct ggcagcagga gcgtgcccgc 2580

cgccccaagt tcgctgacat cgtcagcatc ctggacaagc tcattcgtgc ccctgactcc 2640

ctcaagaccc tggctgactt tgacccccgc gtgtctatcc ggctccccag cacgagcggc 2700

tcggaggggg tgcccttccg cacggtgtcc gagtggctgg agtccatcaa gatgcagcag 2760

tatacggagc acttcatggc ggccggctac actgccatcg agaaggtggt gcagatgacc 2820

aacgacgaca tcaagaggat tggggtgcgg ctgcccggcc accagaagcg catcgcctac 2880

agcctgctgg gactcaagga ccaggtgaac actgtgggga tccccatc 2928

<210> 17

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60

ggtgtgggga ctgaagtcct agaagagagt agctgtgtga acttacaaac ccagcggctg 120

ccagttcaaa aaatcaagac ctatatcatc tgggaggggg ccatgagagc tgtaattttt 180

gtcaccaaac gaggactaaa aatttgtgct gatccagaag ccaaatgggt gaaagcagcg 240

atcaagactg tggatggcag ggccagtacc agaaagaaca tggctgaaac tgttcccaca 300

ggagcccaga ggtccaccag cacagcgata accctgactg ggggc 345

<210> 18

<211> 342

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60

ggtgtaggga gtgaagtctc agataagagg acctgtgtga gcctcactac ccagcgactg 120

ccggttagca gaatcaagac ctacaccatc acggaaggct ccttgagagc agtaattttt 180

attaccaaac gtggcctaaa agtctgtgct gatccacaag ccacatgggt gagagacgtg 240

gtcaggagca tggacaggaa atccaacacc agaaataaca tgatccagac caagccaaca 300

ggaacccagc aatcgaccaa tacagctgtg actctgactg gc 342

<210> 19

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60

ggt 63

<210> 20

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60

ggt 63

<210> 21

<211> 1539

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aaggaagttg ttttgttgga cttcgcagca atgaagggag agctcggctg gctcacgcac 60

ccctatggca aagggtggga cctgatgcag aacatcatgg acgacatgcc tatctacatg 120

tactcggtgt gcaacgtggt atccggcgac caggacaact ggctccgcac caactgggtg 180

taccgggagg aggccgagcg catctttatt gagctcaagt tcacggtgcg agactgtaac 240

agcttcccgg gtggcgccag ctcatgcaaa gagaccttca acctctacta tgcagagtca 300

gatgtggact atggcaccaa cttccagaag cgccagttca ccaagattga caccatcgcc 360

cctgacgaga tcacggtcag cagtgacttc gaggctcgca atgtcaagct gaacgtagag 420

gagcgcatgg tggggcccct tacccggaag ggcttctacc tggccttcca ggacatcggc 480

gcctgcgtgg cgctgctctc cgttcgcgtc tactacaaga agtgtcccga gatgctgcag 540

agcttggctc gcttccccga gaccattgct gtcgctgtct ccgatacaca acccctggcc 600

acggtggccg gtacctgcgt ggaccatgcc gtggtgcctt atgggggcga ggggcctctc 660

atgcactgca cggtggatgg cgagtggctg gtgcccatcg ggcagtgcct gtgccaggaa 720

ggctacgaga aggtcgagga tgcctgccga gcctgttctc caggattctt caagtctgag 780

gcatctgaga gcccttgcct ggagtgtcca gagcataccc tgccatccac agagggtgcc 840

acctcctgcc agtgtgaaga aggctatttc agggcacctg aggacccact gtccatgtct 900

tgcacacgtc caccctctgc ccccaactac ctcacggcca ttggcatggg tgccaaagta 960

gaactgcgtt ggacagctcc caaggacact ggtggccgcc aggacattgt ctacagtgtc 1020

acttgcgaac agtgctggcc agagtctggc gagtgtgggc cctgtgaggc gagcgtgcgc 1080

tattcagaac ctcctcacgc cctgacccgc acgagtgtga cagtcagtga cctggagccc 1140

cacatgaact ataccttcgc tgtcgaagca cgcaatggtg tctcaggcct ggtgactagc 1200

cgaagcttcc ggactgccag cgtcagtatt aaccaaacag agccccccaa agtgaggctg 1260

gaggaccgaa gcaccacctc cctgagtgtc acctggagca tcccggtgtc acagcagagc 1320

cgtgtgtgga agtacgaagt cacctaccgc aagaaggggg atgccaacag ctataatgtg 1380

cgccgcacgg aaggcttctc cgtgaccctg gatgaccttg ctccggatac cacgtacctg 1440

gtgcaggtgc aggcgctgac gcaggagggc cagggagccg gcagcaaagt gcacgagttc 1500

cagacactgt ccacggaagg atctgccaac atggcggtg 1539

<210> 22

<211> 1542

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gcgcagggca aggaagtggt actgctggac tttgctgcag ctggagggga gctcggctgg 60

ctcacacacc cgtatggcaa agggtgggac ctgatgcaga acatcatgaa tgacatgccg 120

atctacatgt actccgtgtg caacgtgatg tctggcgacc aggacaactg gctccgcacc 180

aactgggtgt accgaggaga ggctgagcgt atcttcattg agctcaagtt tactgtacgt 240

gactgcaaca gcttccctgg tggcgccagc tcctgcaagg agactttcaa cctctactat 300

gccgagtcgg acctggacta cggcaccaac ttccagaagc gcctgttcac caagattgac 360

accattgcgc ccgatgagat caccgtcagc agcgacttcg aggcacgcca cgtgaagctg 420

aacgtggagg agcgctccgt ggggccgctc acccgcaaag gcttctacct ggccttccag 480

gatatcggtg cctgtgtggc gctgctctcc gtccgtgtct actacaagaa gtgccccgag 540

ctgctgcagg gcctggccca cttccctgag accatcgccg gctctgatgc accttccctg 600

gccactgtgg ccggcacctg tgtggaccat gccgtggtgc caccgggggg tgaagagccc 660

cgtatgcact gtgcagtgga tggcgagtgg ctggtgccca ttgggcagtg cctgtgccag 720

gcaggctacg agaaggtgga ggatgcctgc caggcctgct cgcctggatt ttttaagttt 780

gaggcatctg agagcccctg cttggagtgc cctgagcaca cgctgccatc ccctgagggt 840

gccacctcct gcgagtgtga ggaaggcttc ttccgggcac ctcaggaccc agcgtcgatg 900

ccttgcacac gacccccctc cgccccacac tacctcacag ccgtgggcat gggtgccaag 960

gtggagctgc gctggacgcc ccctcaggac agcgggggcc gcgaggacat tgtctacagc 1020

gtcacctgcg aacagtgctg gcccgagtct ggggaatgcg ggccgtgtga ggccagtgtg 1080

cgctactcgg agcctcctca cggactgacc cgcaccagtg tgacagtgag cgacctggag 1140

ccccacatga actacacctt caccgtggag gcccgcaatg gcgtctcagg cctggtaacc 1200

agccgcagct tccgtactgc cagtgtcagc atcaaccaga cagagccccc caaggtgagg 1260

ctggagggcc gcagcaccac ctcgcttagc gtctcctgga gcatcccccc gccgcagcag 1320

agccgagtgt ggaagtacga ggtcacttac cgcaagaagg gagactccaa cagctacaat 1380

gtgcgccgca ccgagggttt ctccgtgacc ctggacgacc tggccccaga caccacctac 1440

ctggtccagg tgcaggcact gacgcaggag ggccaggggg ccggcagcaa ggtgcacgaa 1500

ttccagacgc tgtccccgga gggatctggc aacttggcgg tg 1542

<210> 23

<211> 1914

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60

ggtgtgggga ctgaagtcct agaagagagt agctgtgtga acttacaaac ccagcggctg 120

ccagttcaaa aaatcaagac ctatatcatc tgggaggggg ccatgagagc tgtaattttt 180

gtcaccaaac gaggactaaa aatttgtgct gatccagaag ccaaatgggt gaaagcagcg 240

atcaagactg tggatggcag ggccagtacc agaaagaaca tggctgaaac tgttcccaca 300

ggagcccaga ggtccaccag cacagcgata accctgactg ggggcggagg cggaggatca 360

gggggagggg gaggaaagga agttgttttg ttggacttcg cagcaatgaa gggagagctc 420

ggctggctca cgcaccccta tggcaaaggg tgggacctga tgcagaacat catggacgac 480

atgcctatct acatgtactc ggtgtgcaac gtggtatccg gcgaccagga caactggctc 540

cgcaccaact gggtgtaccg ggaggaggcc gagcgcatct ttattgagct caagttcacg 600

gtgcgagact gtaacagctt cccgggtggc gccagctcat gcaaagagac cttcaacctc 660

tactatgcag agtcagatgt ggactatggc accaacttcc agaagcgcca gttcaccaag 720

attgacacca tcgcccctga cgagatcacg gtcagcagtg acttcgaggc tcgcaatgtc 780

aagctgaacg tagaggagcg catggtgggg ccccttaccc ggaagggctt ctacctggcc 840

ttccaggaca tcggcgcctg cgtggcgctg ctctccgttc gcgtctacta caagaagtgt 900

cccgagatgc tgcagagctt ggctcgcttc cccgagacca ttgctgtcgc tgtctccgat 960

acacaacccc tggccacggt ggccggtacc tgcgtggacc atgccgtggt gccttatggg 1020

ggcgaggggc ctctcatgca ctgcacggtg gatggcgagt ggctggtgcc catcgggcag 1080

tgcctgtgcc aggaaggcta cgagaaggtc gaggatgcct gccgagcctg ttctccagga 1140

ttcttcaagt ctgaggcatc tgagagccct tgcctggagt gtccagagca taccctgcca 1200

tccacagagg gtgccacctc ctgccagtgt gaagaaggct atttcagggc acctgaggac 1260

ccactgtcca tgtcttgcac acgtccaccc tctgccccca actacctcac ggccattggc 1320

atgggtgcca aagtagaact gcgttggaca gctcccaagg acactggtgg ccgccaggac 1380

attgtctaca gtgtcacttg cgaacagtgc tggccagagt ctggcgagtg tgggccctgt 1440

gaggcgagcg tgcgctattc agaacctcct cacgccctga cccgcacgag tgtgacagtc 1500

agtgacctgg agccccacat gaactatacc ttcgctgtcg aagcacgcaa tggtgtctca 1560

ggcctggtga ctagccgaag cttccggact gccagcgtca gtattaacca aacagagccc 1620

cccaaagtga ggctggagga ccgaagcacc acctccctga gtgtcacctg gagcatcccg 1680

gtgtcacagc agagccgtgt gtggaagtac gaagtcacct accgcaagaa gggggatgcc 1740

aacagctata atgtgcgccg cacggaaggc ttctccgtga ccctggatga ccttgctccg 1800

gataccacgt acctggtgca ggtgcaggcg ctgacgcagg agggccaggg agccggcagc 1860

aaagtgcacg agttccagac actgtccacg gaaggatctg ccaacatggc ggtg 1914

<210> 24

<211> 1914

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60

ggtgtaggga gtgaagtctc agataagagg acctgtgtga gcctcactac ccagcgactg 120

ccggttagca gaatcaagac ctacaccatc acggaaggct ccttgagagc agtaattttt 180

attaccaaac gtggcctaaa agtctgtgct gatccacaag ccacatgggt gagagacgtg 240

gtcaggagca tggacaggaa atccaacacc agaaataaca tgatccagac caagccaaca 300

ggaacccagc aatcgaccaa tacagctgtg actctgactg gcggaggcgg aggatcaggg 360

ggagggggag gagcgcaggg caaggaagtg gtactgctgg actttgctgc agctggaggg 420

gagctcggct ggctcacaca cccgtatggc aaagggtggg acctgatgca gaacatcatg 480

aatgacatgc cgatctacat gtactccgtg tgcaacgtga tgtctggcga ccaggacaac 540

tggctccgca ccaactgggt gtaccgagga gaggctgagc gtatcttcat tgagctcaag 600

tttactgtac gtgactgcaa cagcttccct ggtggcgcca gctcctgcaa ggagactttc 660

aacctctact atgccgagtc ggacctggac tacggcacca acttccagaa gcgcctgttc 720

accaagattg acaccattgc gcccgatgag atcaccgtca gcagcgactt cgaggcacgc 780

cacgtgaagc tgaacgtgga ggagcgctcc gtggggccgc tcacccgcaa aggcttctac 840

ctggccttcc aggatatcgg tgcctgtgtg gcgctgctct ccgtccgtgt ctactacaag 900

aagtgccccg agctgctgca gggcctggcc cacttccctg agaccatcgc cggctctgat 960

gcaccttccc tggccactgt ggccggcacc tgtgtggacc atgccgtggt gccaccgggg 1020

ggtgaagagc cccgtatgca ctgtgcagtg gatggcgagt ggctggtgcc cattgggcag 1080

tgcctgtgcc aggcaggcta cgagaaggtg gaggatgcct gccaggcctg ctcgcctgga 1140

ttttttaagt ttgaggcatc tgagagcccc tgcttggagt gccctgagca cacgctgcca 1200

tcccctgagg gtgccacctc ctgcgagtgt gaggaaggct tcttccgggc acctcaggac 1260

ccagcgtcga tgccttgcac acgacccccc tccgccccac actacctcac agccgtgggc 1320

atgggtgcca aggtggagct gcgctggacg ccccctcagg acagcggggg ccgcgaggac 1380

attgtctaca gcgtcacctg cgaacagtgc tggcccgagt ctggggaatg cgggccgtgt 1440

gaggccagtg tgcgctactc ggagcctcct cacggactga cccgcaccag tgtgacagtg 1500

agcgacctgg agccccacat gaactacacc ttcaccgtgg aggcccgcaa tggcgtctca 1560

ggcctggtaa ccagccgcag cttccgtact gccagtgtca gcatcaacca gacagagccc 1620

cccaaggtga ggctggaggg ccgcagcacc acctcgctta gcgtctcctg gagcatcccc 1680

ccgccgcagc agagccgagt gtggaagtac gaggtcactt accgcaagaa gggagactcc 1740

aacagctaca atgtgcgccg caccgagggt ttctccgtga ccctggacga cctggcccca 1800

gacaccacct acctggtcca ggtgcaggca ctgacgcagg agggccaggg ggccggcagc 1860

aaggtgcacg aattccagac gctgtccccg gagggatctg gcaacttggc ggtg 1914

<210> 25

<211> 75

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

atggagctcc gggcagtcgg tttctgcctg gcgctgctgt ggggttgcgc gctggcggcc 60

gcggcggcac aggga 75

<210> 26

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

atggagctcc aggcagcccg cgcctgcttc gccctgctgt ggggctgtgc gctggccgcg 60

gccgcggcg 69

Claims (10)

1. A fusion protein comprising the amino acid sequence of the extracellular domain of EPHA2 capable of eliciting a specific CD8+ T response or an mhc class i molecule-binding epitope-optimized form thereof; a joint; and human or murine XCL1 protein that specifically binds DC cells with cross-antigen presentation capability;

wherein:

the extracellular region of EPHA2 capable of inducing a specific CD8+ T response or an optimized form of its MHC class I molecule binding epitope has an amino acid sequence as shown in SEQ ID No.1 or 2; the human or mouse XCL1 specifically binding DC cells with antigen cross-presentation capability is shown as SEQ ID No.3 or 4;

or

(II) an amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I);

or

(III) an amino acid sequence which has at least 90% sequence identity with the sequence of (I) or (II) and which is functionally identical or similar to the amino acid sequence of (I).

2. The fusion protein of claim 1, wherein the fusion protein comprises an amino acid sequence as set forth in SEQ ID No.9 or SEQ ID No. 10.

3. A nucleic acid molecule encoding the fusion protein of claim 1 or 2, comprising DNA and/or mRNA, wherein the sequence of the nucleic acid molecule has:

(1) a nucleotide sequence as shown in any of SEQ ID No. 17-32;

or

(2) The nucleotide sequence shown as the complementary nucleotide sequence of the nucleotide sequence shown in any SEQ ID No. 15-30; or

(3) The nucleotide sequence which encodes the same protein as the nucleotide sequence of (1) or (2) but differs from the nucleotide sequence of (1) or (2) due to the degeneracy of the genetic code;

or

(4) A nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences with the nucleotide sequence shown in (1), (2) or (3), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in (1), (2) or (3);

or

(5) A nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of (1), (2), (3) or (4).

4. Recombinant expression vector comprising a vector and the fusion protein according to claim 1 or 2 or the nucleic acid molecule according to claim 3.

5. The recombinant expression vector of claim 4, wherein the vector comprises pcDNA3.1(+), pcDNA3.1(-), pFastgac 1-dual-MBP, pVAX 1.

6. A recombinant strain or cell comprising the fusion protein of claim 1 or 2 or the nucleic acid molecule of claim 3.

7. Use of the fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4 or 5, or the recombinant strain or cell according to claim 6 for the preparation of a vaccine for tumors highly expressing EPHA2 and/or for the preparation of a medicament for the prevention and/or treatment of tumors highly expressing EPHA 2.

8. The use of claim 7, wherein the metastatic cancer comprises tumors highly expressing EPHA2, including prostate, lung, esophageal, colorectal, cervical, ovarian, breast, and skin cancers.

9. A tumor vaccine targeting EPHA2, comprising the fusion protein of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4 or 5, or the recombinant strain or cell of claim 6, and a pharmaceutically acceptable carrier, excipient and/or adjuvant.

10. Medicament for preventing and/or treating tumors highly expressing EPHA2, comprising the fusion protein of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4 or 5 or the recombinant strain or cell of claim 6 and pharmaceutically acceptable excipients.

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