Tumor nucleic acid vaccine targeting egfr

Abstract

The present application relates to the technical field of tumor immunotherapy and prevention, and particularly relates to a tumor nucleic acid vaccine targeting EGFR. The fusion protein provided by the present application comprises: an XCL1 chemotactic factor and an extracellular region of EGFR. The present application expresses the EGFR protein and the DC cell ligand XCL1 in a fusion manner, uses XCL1 as a carrier to transport the tumor target protein EGFR to the cross-presentation DC cells, improves the efficiency of phagocytosis, processing and presentation of the EGFR protein by the DC cells, and improves the effect of inhibiting tumor growth, and can be used for preventing and treating EGFR high-expression tumors. Through experimental verification, the fusion protein encoded by the tumor nucleic acid vaccine of the present application can effectively bind to DC cells and significantly inhibit the tumor growth of lung cancer with high EGFR expression in an animal model.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy and prevention technology, and in particular to tumor nucleic acid vaccines targeting EGFR. Background Technology

[0002] For decades, the incidence and mortality rates of cancer have been rising, becoming a major global public health problem and seriously threatening human life and health. In recent years, significant progress has been made in cancer treatment, with many new treatment strategies showing marked improvements in disease control and patient survival. These therapies largely benefit from important targeted molecules that promote tumor progression, with the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, widely considered an effective tumor target (Yarden Y, Nat Rev Cancer 2012). EGFR (also known as HER1 / ErbB1) is a member of the EGFR family, a cell surface receptor for extracellular protein ligands, and an important transmembrane receptor. During embryonic growth and development, EGFR signaling is crucial for epidermal development, proliferation, and organogenesis. In mature tissues, EGFR activation stimulates cell proliferation to replace damaged or necrotic cells and maintains cell numbers by inhibiting programmed cell death or apoptosis. EGFR also stimulates cells to secrete other growth factors, such as vascular endothelial growth factor (VEGF), promoting angiogenesis during tissue remodeling. Numerous studies have shown that EGFR plays a crucial role in regulating tumor phenotypes such as abnormal proliferation and survival (Seshacharyulu P. et al. Expert Opin TherTargets 2012). Abnormal expression of the EGFR gene can activate genes related to tumor proliferation and differentiation, playing an important role in the formation and development of tumors. Many malignant tumors (including non-small cell lung cancer, breast cancer, colorectal cancer, esophageal cancer, prostate cancer, ovarian cancer, head and neck squamous cell carcinoma, etc.) have EGFR overexpression or dysfunction (Tawbi H. et al. Oncologist 2008; Yatabe Y. et al. Cancer Res 2008; Tse GM. Breast Cancer Res Treat 2008).

[0003] With in-depth research into the structure and function of the EGFR family, EGFR-targeting strategies are receiving increasing attention in the field of cancer treatment research. Currently, EGFR targeting mainly involves two aspects: monoclonal antibodies (Mabs) and small molecule tyrosine kinase inhibitors (TKIs). TKIs mainly include gefitinib, afatinib, and osimertinib, while monoclonal antibodies include cetuximab, panitumumab, and nimotuzumab. Both types of drugs have entered clinical application in many regions worldwide. However, due to the heterogeneity and plasticity of tumors, a high relapse rate still exists even after several months of disease control in cancer patients. Therefore, new and effective treatment strategies targeting EGFR are still urgently needed.

[0004] Tumor immunotherapy has received increasing attention as a novel approach to cancer treatment. It can inhibit tumor proliferation and induce cell death, activate specific cytotoxic T lymphocytes (CTLs), and generate memory to maintain long-term immune efficacy (Tagliamonte M. et al. Hum Vaccin Immunother 2014). In the field of tumor immunotherapy, there are many forms of tumor vaccines, among which tumor nucleic acid vaccines are developing rapidly, with several nucleic acid vaccines targeting human tumors currently undergoing animal experiments or clinical trials (Greten TF. et al. J Clin Onco 1999). Due to the complexity and diversity of tumor development, enhancing the immunogenicity of tumor vaccines, overcoming tumor immune escape, breaking the body's own immune tolerance mechanisms, and inducing targeted cellular immunity will become core issues in improving the efficacy of tumor vaccines (Said D. et al. British Medical Bulletin 2002; Berzofsky JA. et al. J Clin Invest 2004).

[0005] Tumor vaccines induce effector T cell function in patients by enhancing existing anti-tumor responses or activating naïve T cells. Antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in the anti-tumor process (Shankaran V. et al. Nature 2001). Dendritic cells (DCs) are the only professional antigen-presenting cells capable of activating naïve CD8+ T cells. They take up, process, and cross-present extracellular tumor antigens via MHC-I, which is essential for generating effective CTLs (Romero P. et al. Annu Rev Immunol 2017). Therefore, delivering tumor antigens to DCs via coupling with DC surface molecules is an effective tumor therapy strategy for inducing CD8+ T cell immune responses.

[0006] Different subsets of dendritic cells (DCs) have varying abilities to induce antigen-specific immune responses. XCL1 is the sole ligand for XCR1, a member of the G protein-coupled receptor family. XCR1 is a chemokine receptor expressed only in a specific subset of DCs (i.e., "cross-presenting" DCs, also known as XCR1+DCs or cDC1). XCR1+DCs are highly effective in antigen cross-presentation, meaning that exogenous antigens are not presented by MHC class II CD4+ T cells, but rather by MHC class I antigens to CD8+ T cells (Kurts C. et al. J Exp Med 1997; den Haan JM. J Exp Med 2000; Pooley JL. et al. J Immunol 2001). XCR1+DCs are the most effective antigen cross-presenting cells for inducing CD8+ T cell activation. Therefore, XCR1 is an ideal target for inducing the body to produce cellular immunotoxicity to fight tumors (Evelyn Hartung. et al. JImmunol 2015). Delivering the target antigen to XCR1+DC by fusing XCL1 is a feasible strategy for tumor immunotherapy.

[0007] While there have been reports on using XCR1 in combination with targeted factors to improve the efficacy of tumor treatment, the improvement in delivery has varied. Therefore, the field is still exploring ways to improve the efficacy of tumor treatment. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a tumor nucleic acid vaccine that targets EGFR high expression.

[0009] The fusion protein provided by this invention comprises: XCL1 chemokine and EGFR extracellular domain;

[0010] The XCL1 chemokine includes a signal peptide;

[0011] The extracellular region of EGFR is an EGFR protein that does not contain a signal peptide;

[0012] The XCL1 chemokine and the extracellular region of EGFR are linked by a linker with the amino acid sequence (G5S)n.

[0013] In some embodiments, the amino acid sequence of the extracellular region of the EGFR described in this invention is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0014] In some embodiments, the amino acid sequence of the XCL1 chemokine described in this invention is shown in SEQ ID NO:3.

[0015] In this embodiment of the invention, the N-terminus of the fusion protein is an XCL1 chemokine, and the C-terminus is the extracellular region of EGFR. The n in (G5S)n is 1~10. In this embodiment of the invention, the linker sequence is GGGGGSGGGGG.

[0016] The amino acid sequence of the fusion protein described in this invention is shown in SEQ ID NO:4 or SEQ ID NO:5.

[0017] The present invention also provides a nucleic acid encoding the fusion protein.

[0018] The nucleotide sequence encoding the fusion protein described in this invention is shown in SEQ ID NO:6 or SEQ ID NO:7.

[0019] The present invention also provides a transcriptional unit containing the encoding the fusion protein.

[0020] The transcription unit includes a promoter and nucleic acid encoding the fusion protein.

[0021] In some embodiments, the promoter is a CMV promoter. The transcription unit also includes a FLAG tag. The transcript unit has CMV, nucleic acid encoding the fusion protein, and a 3×FLAG tag at its 5' to 3' ends, respectively.

[0022] The present invention also provides a recombinant vector comprising a backbone vector and a nucleic acid encoding the fusion protein.

[0023] In this invention, the backbone vector is selected from pcDNA3.1(-) or pcDNA3.1(+). In some embodiments, the backbone vector is a pcDNA3.1 / zeo(-) vector.

[0024] The present invention also provides a recombinant host for transforming or transfecting the recombinant vector.

[0025] The host cell of the recombinant host described in this invention is a bacterial or mammalian cell.

[0026] The bacteria are Escherichia coli; the mammalian cells are HEK293T cells.

[0027] The method for preparing the fusion protein is characterized by culturing the recombinant host of the present invention to obtain a culture containing the fusion protein.

[0028] The application of the fusion protein, nucleic acid, recombinant vector, recombinant host and / or the fusion protein prepared by the method described in this invention in the preparation of drugs for the prevention and treatment of tumors.

[0029] The tumor described in this invention is a malignant tumor. In some embodiments, the malignant tumor is a tumor that highly expresses EGFR. The tumor that highly expresses EGFR includes non-small cell lung cancer, breast cancer, colorectal cancer, esophageal cancer, prostate cancer, ovarian cancer, and head and neck squamous cell carcinoma. In some specific embodiments, the tumor is lung cancer. The lung cancer includes non-small cell lung cancer or small cell lung cancer.

[0030] The prevention and treatment methods described in this invention include: inhibiting tumor growth, reducing tumor volume, or slowing down the growth rate of tumors.

[0031] The present invention also provides a drug for preventing and treating tumors, comprising: a fusion protein, a nucleic acid, a recombinant vector, a recombinant host, and / or a fusion protein prepared by the preparation method described above.

[0032] The drug described in this invention also includes pharmaceutically acceptable excipients or carriers.

[0033] The present invention also provides a method for preventing and treating tumors, which involves administering the drug described herein. The administration methods include injection, oral administration, or gene gun administration.

[0034] The fusion protein provided by this invention comprises XCL1 chemokine and the extracellular domain of EGFR. This invention expresses EGFR protein by fusing it with the DC cell ligand XCL1, utilizing XCL1 as a carrier to transport the tumor target protein EGFR to cross-presenting DC cells. This improves the efficiency of EGFR protein phagocytosis, processing, and presentation by DC cells, thereby enhancing its tumor-inhibiting effect and making it suitable for the prevention and treatment of EGFR-overexpressing tumors. Experimental verification shows that the fusion protein encoded by the tumor nucleic acid vaccine of this invention can effectively bind to DC cells and significantly inhibit the tumor growth of lung cancer with high EGFR expression in animal models. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings will be described below:

[0036] Figure 1 The vector map of the nucleic acid vaccine encoding the fusion protein is shown; among them Figure 1 The A in the diagram indicates that the extracellular region of mouse EGFR wild-type is attached after the CMV promoter for use as a nucleic acid vaccine coding sequence; Figure 1 The B in the diagram represents the CMV promoter followed by the mouse XCL1 sequence, which is then linked to the mouse EGFR wild-type extracellular region for use as a nucleic acid vaccine coding sequence. Figure 1 The C in the figure represents the coding sequence for a mouse EGFR MHCI-like molecule that binds to the CMV promoter followed by the mouse XCL1 sequence and then to the optimized extracellular region of the mutant nucleic acid vaccine.

[0037] Figure 2 The image shows a predicted three-dimensional structure of the fusion protein encoded by the nucleic acid vaccine. The three-dimensional spatial structure of the fusion protein is predicted by inputting the sequence of the fusion protein encoded by the nucleic acid vaccine into the URL http: / / raptorx.uchicago.edu / . The image shows the tertiary structure of the XCL1-EGFR fusion protein.

[0038] Figure 3 The expression of the nucleic acid vaccine encoding the fusion protein in HEK293T cells was detected. After transfecting HEK292T cells with a plasmid vector carrying the nucleic acid encoding the fusion protein for 48 hours, the expression of the fusion protein (with a Flag tag at the C-terminus) encoded by the nucleic acid vaccine was detected by Western blot.

[0039] Figure 4 The results showed that the nucleic acid vaccine can effectively bind to MHCII+CD11c+CD8α+ antigen cross-presenting DC cells; HEK293T cell lysate transfected with nucleic acid vaccine plasmid was incubated with spleen cells enriched with CD11c magnetic beads, and the binding of the fusion protein to MHCII+CD11c+CD8α+ antigen cross-presenting DC cells was detected by Flag fluorescent antibody.

[0040] Figure 5 The tumor growth of lung cancer in mice after immunization with a nucleic acid vaccine encoding the XCL1-EGFR wild-type / XCL1-EGFR mutant fusion protein is shown. Figure 5 A in the diagram represents the immunization method and process of nucleic acid vaccines; Figure 5 B in the figure represents a tumor growth curve showing the tumor volume measured after inoculation with lung cancer cells. Figure 5 The figure in C shows the weight analysis of the dissected tumors after the mice were sacrificed when the largest tumor reached an ethical endpoint of 2000 mm3. Detailed Implementation

[0041] This invention provides a tumor nucleic acid vaccine targeting EGFR-high expression. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the vaccine. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0042] The fusion protein of this invention is formed by fusing XCL1 chemokine and the extracellular region of EGFR. The XCL1 chemokine and the EGFR extracellular region can be mouse-derived fragments or human-derived fragments. In this application, a mouse-derived fragment was used as the experimental subject to verify the enhanced presentation effect of XCL1 on EGFR. The connection order of the XCL1 chemokine and the EGFR extracellular region is not limited in this invention. The N-terminus of the fusion protein can be either XCL1 chemokine or the EGFR extracellular region. Previous studies have shown that the N-terminus of XCL1 chemokine is more conducive to protein expression, thereby improving the therapeutic effect on tumors.

[0043] In this invention, the XCL1 chemokine includes a signal peptide, while the transmembrane-mediating signal peptide in EGFR is removed. In some specific embodiments, the amino acid sequence of the XCL1 chemokine is shown in SEQ ID NO:3. This invention validates the efficacy of wild-type and optimized fragments of EGFR. The wild-type fragment is denoted as EGFR WT, and its amino acid sequence is shown in SEQ ID NO:1. The artificially optimized sequence is denoted as EGFR Mutation, and its amino acid sequence is shown in SEQ ID NO:2. Studies have shown that, compared with the wild-type EGFR sequence, the fusion protein formed by the artificially optimized sequence and XCL1 has a more significant effect on inhibiting tumor volume (p<0.05), but no statistically significant difference was observed in the effect of the fusion protein formed by XCL1 and the wild-type sequence on inhibiting tumor volume.

[0044] In this invention, the linker sequence ensures that the extracellular region of EGFR and XCL1 can fold smoothly without steric hindrance after expression. The linker sequence used in this invention is GGGGGSGGGGG. In some embodiments, the fragment obtained by fusing a wild-type EGFR fragment with XCL1 is denoted as XCL1-EGFR WT, and its amino acid sequence is shown in SEQ ID No. 4; the fragment obtained by fusing an artificially optimized EGFR fragment with XCL1 is denoted as XCL1-EGFR Mutation, and its amino acid sequence is shown in SEQ ID No. 5.

[0045] In order to enable good detection of proteins, this invention adds a flag tag sequence (SEQ ID No. 13) immediately after the C-terminus of the EGFR extracellular region sequence.

[0046] The nucleic acid encoding the protein described in this invention can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be, for example, a portion of a vector (e.g., an expression or cloning vector) or a fragment. The nucleic acid can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques.

[0047] In this invention, the nucleic acid sequence for expressing the fusion protein is optimized. These optimizations include, but are not limited to: codon usage bias, elimination of secondary structures that are unfavorable to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, mRNA secondary structure, hidden splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning.

[0048] This invention also provides a transcription unit for a fusion protein, wherein the transcription unit refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included flanking or between the promoter and terminator. These regulatory fragments may include a promoter operatively linked to the nucleic acid sequence, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site, such as an enhancer of the promoter, a poly(A) signal, etc. The transcription unit provided by this invention includes a CMV promoter, a CMV enhancer, and a nucleic acid fragment encoding the fusion protein.

[0049] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences essential for the expression of an operatively linked coding gene in a specific host organism. The nucleic acid sequences essential for expression in prokaryotic cells include a promoter, optionally including an operator gene sequence, a ribosome binding site, and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions, which are known in the art or will become known, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0050] The host cell described in this invention is a prokaryotic or eukaryotic host containing a nucleic acid vector and / or a target gene. The host cell is transformed or transfected with a vector constructed using recombinant DNA technology. Such transformed host cells are capable of replicating the protein-encoding vector or expressing the desired protein.

[0051] In this embodiment of the invention, the fusion protein is prepared by inducing recombinant host expression, and the culture can be bacterial cells, cell bodies, culture medium, or substances obtained by extraction and / or purification from the above-mentioned cultures.

[0052] In this invention, the prevention and treatment of tumors includes both prevention and / or treatment. Prevention, as described in this invention, refers to administering the drug described in this invention before the onset of a tumor, thereby reducing the risk of tumor development. Treatment, as described in this invention, refers to administering the drug described in this invention after the onset of a tumor, thereby inhibiting tumor growth, reducing tumor volume, or slowing the growth rate of the tumor.

[0053] The tumor referred to in this invention refers to a malignant tumor. The malignant tumor is selected from bladder cancer, leukemia, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, external genital cancer, urogenital tract cancer, head cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, muscle tissue cancer, cervical cancer, oral or nasal mucosal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer, and / or thyroid cancer. In some embodiments, the malignant tumor is a tumor that highly expresses EGFR. The tumor that highly expresses EGFR is non-small cell lung cancer, breast cancer, colorectal cancer, esophageal cancer, prostate cancer, ovarian cancer, and head and neck squamous cell carcinoma. In some specific embodiments, the tumor is lung cancer. The lung cancer includes non-small cell lung cancer or small cell lung cancer. This invention uses LL2 lung cancer cells as the test subject to verify the activity of the drug described in this invention.

[0054] In this embodiment of the invention, the amino acid sequences of the involved fragments and the encoded nucleic acid fragments are shown in Table 1:

[0055] Table 1 shows the amino acid sequence and encoded nucleic acid fragment of the fragment.

[0056]

[0057] The vaccine provided by this invention comprises an extracellular region encoding the epidermal growth factor receptor EGFR, capable of evoking a specific CD8+ T cell immune response, and an extracellular region of an optimized form of the MHCI-like molecule binding epitope, fused at its N-terminus with the base sequence of a DC protein XCL1 that specifically binds to and cross-presents antigens. The vaccine includes the nucleic acid of the fusion protein and a vector containing the nucleic acid. It also includes the application of the nucleic acid sequence and vector described in this invention in the prevention and treatment of EGFR-overexpressing tumors (including non-small cell lung cancer, breast cancer, colorectal cancer, esophageal cancer, prostate cancer, ovarian cancer, and head and neck squamous cell carcinoma). This invention improves the efficiency of EGFR protein phagocytosis, processing, and presentation by DC cells by fusing the EGFR protein with the DC cell ligand XCL1, using XCL1 as a vector to transport the tumor target protein EGFR to cross-presenting DC cells, thereby enhancing its tumor-inhibiting effect. Experimental verification shows that the fusion protein encoded by the tumor nucleic acid vaccine of this invention can effectively bind to DC cells and significantly inhibit the tumor growth of lung cancer with high EGFR expression in animal models.

[0058] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0059] Example 1: Design scheme of tumor nucleic acid vaccine and construction and preparation of mammalian cell expression plasmids

[0060] (1) Construction of mammalian cell expression vector for fusion gene

[0061] The fusion protein XCL1-EGFR was constructed based on the extracellular region of mouse EGFR and the amino acid sequence of mouse XCL1 protein.

[0062] Experiments were conducted using both the wild-type fragment (denoted as EGFR WT) and the optimized fragment (denoted as EGFR Mutation). To promote the efficient secretion of the fusion protein translated from the nucleic acid expressing the fusion protein into extracellular cells and its chemotaxis of MHC-II+CD11c+CD8α+ antigen-cross-presenting dendritic cells (DCs), we retained the secretion signal peptide of the XCL1 protein (SEQ ID No. 11) while removing the EGFR-mediated transmembrane signal peptide (SEQ ID No. 12). The amino acid sequence of EGFR WT used in the examples is shown in SEQ ID NO:1, and the amino acid sequence of EGFR Mutation is shown in SEQ ID NO:2. The amino acid sequence of the XCL1 protein is shown in SEQ ID NO:3.

[0063] To ensure that the EGFR extracellular region and XCL1 can fold smoothly without steric hindrance after expression, an amino acid sequence was added. Furthermore, to facilitate the detection of the expressed target sequence, a flag tag sequence (SEQ ID No. 13) was immediately appended after the EGFR extracellular region sequence, resulting in the amino acid sequence of the fusion protein:

[0064] XCL1-EGFR WT, the amino acid sequence of which is shown in SEQ ID No. 4;

[0065] XCL1-EGFR Mutation, whose amino acid sequence is shown in SEQ ID No. 5.

[0066] We synthesized the nucleotide sequences corresponding to the amino acid sequences using Beijing Qingke Biotechnology Co., Ltd., and ligated them into the pcDNA3.1 / zeo(-) expression vector, constructing a total of three vectors, with the ligated nucleic acid fragments as follows:

[0067] The nucleic acid encoding EGFR WT has the sequence shown in SEQ ID No. 8;

[0068] The nucleic acid encoding XCL1-EGFR WT has the sequence shown in SEQ ID No. 6;

[0069] The nucleic acid encoding XCL1-EGFR Mutation has the sequence shown in SEQ ID No. 7.

[0070] (2) Amplification of the E. coli expression vector for the fusion gene

[0071] For bacterial transformation, E. coli DH5α competent cells frozen at -80℃ were thawed on ice. When nearly completely thawed, 100 ng of plasmid was added, gently mixed, and incubated on ice for 30 mins. The competent cells were then heat-shocked in a 42℃ water bath for 60 s, and immediately placed on ice for 2 mins. 500 µL of antibiotic-free LB medium was added to the tubes, and the cells were incubated at 37℃ with shaking for 1 h. The bacteria were centrifuged at 4000 rpm at room temperature for 2 mins, and a portion of the supernatant (approximately 450 µL) was discarded. The bacteria were resuspended, and an appropriate amount of bacterial suspension was spread onto a culture dish containing the appropriate antibiotic. The culture dish was placed face down and incubated overnight at 37℃. When the clones reached a suitable size (approximately 16 h), the clones were transferred to LB medium containing antibiotics using a pipette tip and incubated at 37℃ with shaking until turbidity was reached. Take 15 mL of bacterial culture cultured to the appropriate concentration, centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant. Extract bacterial DNA according to the instructions of the Plasmid Mini-Prep Kit (DP103) from Beijing Tiangen Biotech Co., Ltd. First, add 250 µL of cell resuspension buffer P1 containing RNase to fully resuspend the bacterial pellet, and transfer the pellet to a 1.5 mL EP tube. Add 250 µL of alkaline cell lysis buffer P2, and gently invert to mix until the liquid is clear. Add 350 µL of neutralization buffer P3, and invert to mix until flocculent precipitate appears. Centrifuge the suspension at 12000 rpm for 10 min at room temperature. Place the DNA adsorption column into a recovery tube, add 500 µL of equilibration buffer to activate the adsorption membrane, centrifuge at 12000 rpm for 1 min, and discard the liquid. Transfer the supernatant obtained from centrifugation in step 4 to the DNA adsorption column, centrifuge at 12000 rpm for 1 min, and discard the liquid. Add 600 µL of washing buffer to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the liquid, and repeat the washing once. Centrifuge the recovery tube at 12000 rpm for 2 min. Replace the collection tube with a new 1.5 mL EP tube and allow the adsorption column to air dry at room temperature for 5 min. Add 70 µL of preheated elution buffer (65℃) or autoclaved purified water, allow to dissolve the DNA completely at room temperature for 5 min, centrifuge at 12000 rpm for 3 min, and collect the liquid. After discarding the adsorption column, determine the plasmid concentration in the tube and label it with the plasmid name, concentration, and extraction date.

[0072] Example 2: Three-dimensional structure prediction of XCL1-EGFR fusion protein expressed by vaccine plasmid

[0073] XCL1 is a chemokine, and its normal chemotactic function requires the preservation of its complete spatial structure. To ensure that XCL1 can still fold its own spatial structure after fusion with the extracellular region of EGFR, we predicted the tertiary structure of the XCL1-EGFR extracellular fusion protein based on the translated nucleotide sequence using the website http: / / raptorx.uchicago.edu / . The results are as follows: Figure 2 The results show that after XCL1 and EGFR are fused, they still retain their original spatial structures and do not affect the chemotactic function of XCL1.

[0074] Example 3: Detection of the expression effect of XCL1-EGFR fusion vaccine plasmid in mammalian cells

[0075] 24 hours before transfection, seed 1×10⁶ cells into 6-well cell culture plates. 6 HEK293T cells were cultured until the cell density reached 70%–80%, at which point transfection was initiated. The cell culture medium and serum-free Opti-MEM medium were preheated in a 37°C water bath before transfection. During transfection, 5 μg of the EGFR expression vector alone, the XCL1-EGFR wild-type and mutant fusion gene plasmid, and 20 μL of PEI transfection reagent were added sequentially to 200 μL of serum-free Opti-MEM, mixed thoroughly, and incubated at room temperature for 10 minutes. The cells to be transfected were then replaced with fresh medium, and the mixture was gently added to the transfection system and gently shaken. The cells were then returned to a cell culture incubator and cultured for 6 hours before the medium was changed. After 48 hours of transfection, the cells were harvested and Western blot was used to detect the expression effect of the XCL1-EGFR fusion vaccine plasmid in HEK293T cells.

[0076] To facilitate the detection of fusion gene expression, a 21-amino acid flag tag (sequence: DYKDHDGDYKDHDIDYKDDDDK) was attached to the C-terminus of the fusion protein for detection using a flag-tagged antibody. Cells were collected, and 60 μL of 0.5% NP40 lysis buffer containing PMSF or a cocktail protease inhibitor was added. Cells were thoroughly resuspended and lysed at 4°C for 30 minutes by rotary oscillation. The lysis buffer was centrifuged at 12000 rpm at 4°C for 10 minutes, and the supernatant was collected into a new 1.5 mL EP tube, discarding the precipitate. 5×SDS-PAGE protein loading buffer was added according to the actual sample volume, and the mixture was thoroughly mixed. The sample was then heated in a 100°C metal bath for 10 minutes, and Western blot was immediately performed using a flag-tagged antibody (Sigma, F3165). The results are shown below. Figure 3The results showed that both the XCL1-EGFR wild-type and mutant fusion gene plasmids could effectively express EGFR using standalone expression vectors, with no significant difference in expression levels.

[0077] Example 4: Detection of the ability of XCL1-EGFR fusion vaccine plasmid to bind antigen cross-presentation dendritic cells

[0078] This invention first transfects the XCL1-EGFR fusion vaccine plasmid into HEK293T cells and obtains lysate after an appropriate time. Then, it verifies whether the plasmid can effectively bind to antigen-cross-presenting dendritic cells.

[0079] (1) Isolation and purification of CD11c+ DC cells

[0080] Mouse whole spleens were used as the source of CD11c+ DC cells. First, mouse spleens were homogenized into single cells using a 70µm pore size cell sieve in 1640 medium. After centrifugation at 200g for 10 min, the supernatant was discarded, and the spleen cells were subjected to erythrocyte lysis buffer (Catalog No. WL2000) from R&D Company. Specifically, lysis buffer A was diluted 10-fold with distilled water to prepare working solution. 2 mL of working solution was added to each spleen for resuspending, and the cells were incubated at room temperature for 10 min. During this period, neutralization buffer B was diluted 10-fold with distilled water to prepare working solution. After 10 min, 10 mL of neutralization buffer B was added to the lysis buffer for neutralization, followed by centrifugation at 200g for 10 min. The cell clusters were washed once with PBS buffer containing 1% inactivated FBS and counted. A total of 1×10⁶ cells was collected. 8 One cell line was resuspended in 400 μL of PBS buffer containing 1% inactivated FBS, and 100 μL of CD11c magnetic beads (Miltenyi Biotec: 130-108-338) was added. The column was incubated at 4°C in the dark for 20 min. During this time, the column was equilibrated with PBS buffer containing 1% inactivated FBS. After 20 min, the cells and magnetic bead mixture were transferred to the magnetic bead column, and the column was placed on a magnetic rack. Once the cells were fully incorporated into the column, the cells were washed three times with 3 mL of PBS buffer containing 1% inactivated FBS each time. The column was then removed from the magnetic rack and placed on top of a 15 mL centrifuge tube. 5 mL of PBS buffer containing 1% inactivated FBS was added, and the cells were rapidly eluted. The cells were counted and centrifuged at 200 g for 10 min. The resulting CD11c+ DCs were resuspended in serum-free 1640 medium to a concentration of 1×10⁶ cells / mL. 6 The sample was prepared at a concentration of 1 mL / well in a 24-well plate, with 1 mL per well.

[0081] (2) Detection of the ability of cross-presenting dendritic cells to bind antigens

[0082] Cells were divided into three groups: a EGFR control group, an XCL1-EGFR wild-type experimental group, and an XCL1-EGFR mutant experimental group. Each group was given an equal volume of cell lysis buffer, mixed thoroughly, and incubated at 37°C in a CO2 incubator for 40 minutes. Cells were then collected, centrifuged at 500g, and washed twice with PBS buffer containing 1% inactivated FBS. Flow cytometry staining was performed: MHC-II APC, Flag-dye light 488, and CD8α-Percp / Cy5.5. Cells were collected at medium speed using a BD LSRII instrument, and the proportion of MHC-II+, Flag-dye light 488+, and CD8α+ cells among the different groups was analyzed. Each group was repeated three times.

[0083] The results showed that, compared with the EGFR-only control group, the lysate of the XCL1-EGFR fusion vaccine plasmid bound to antigen-cross-presenting dendritic cells more strongly in both the XCL1-EGFR wild-type experimental group and the XCL1-EGFR mutant experimental group, with binding abilities approximately 5 times and 6 times that of the EGFR-only control group, respectively. Figure 4 As shown.

[0084] Example 5: Intervention effect of XCL1-EGFR fusion vaccine plasmid on the occurrence of LL2 lung cancer cell allogeneic tumors.

[0085] Given that the XCL1-EGFR fusion vaccine plasmid can be normally expressed in mammalian cells, and its lysate can effectively chemotactically bind to MHC-II+CD11c+CD8a+ antigen-cross-presenting dendritic cells (DCs), we extracted EGFR alone, wild-type XCL1-EGFR, and mutant nucleic acid vaccine plasmids, and administered them to mice via immunization using a Wealtec gene gun (GDS-80). The LL2 lung cancer cell line is a cell line that highly expresses EGFR; therefore, we used LL2 lung cancer cell line for allogeneic transplantation to observe the inhibitory effect of nucleic acid vaccine immunization on lung cancer tumor growth.

[0086] (1) Culture of LL2 lung cancer cells

[0087] Resuscitating LL2 lung cancer cells: Preheat cell culture medium (the cell culture medium was prepared by adding 10% FBS to Corning's DMEM medium). Remove the frozen cells to be resuscitated from liquid nitrogen or a -80°C freezer and quickly thaw them in a 37°C water bath. After complete thawing, centrifuge at 1500 rpm for 3 min, while adding 8 mL of preheated cell culture medium to the culture dish. After centrifugation, discard the supernatant, add 1 mL of preheated cell culture medium, resuspend the cells, and transfer them to prepared culture dishes, labeling them with cell name, date, and passage number. Incubate the cells in a 37°C, 5% CO2 incubator. Passage the cells on the second day, preheating the cell culture medium, trypsin, and PBS buffer. Use a vacuum pump to aspirate the old culture medium from the culture dish. Rinse the cells once with 7 mL of PBS buffer, then aspirate the PBS. Add 1 mL of trypsin and incubate at 37°C for the appropriate digestion time. Add 3 mL of culture medium to stop digestion. Use a power pipette to pipette the cells into a single-cell suspension and transfer to a 15 mL centrifuge tube. Centrifuge at 1500 rpm for 3 min. Meanwhile, prepare new culture dishes and add 8 mL of culture medium to each. After centrifugation, discard the supernatant. Resuspend the cells in an appropriate amount of culture medium according to the required passage ratio, pipette the cells into a single-cell suspension, and add 1 mL of the single-cell suspension to a new culture dish. Return the dish to a 37°C incubator with 5% CO2 for further culture.

[0088] (2) The preventive effect of XCL1-EGFR fusion vaccine plasmid immunization on the occurrence of LL2 lung cancer cell allogeneic tumors

[0089] We follow Figure 5 The immunization strategy, time-annotated in section A, involved gene gun plasmid injection into mice. Six- to eight-week-old male C57B6 mice (purchased from Beijing Vital River Laboratory Animal Co., Ltd.) were divided into three groups of five mice each, receiving either a single EGFR vaccine plasmid, a wild-type XCL1-EGFR vaccine plasmid, or a mutant XCL1-EGFR vaccine plasmid. Hair was removed from the right side of the mice near the inguinal lymph nodes using depilatory cream. Following this, 50 μg of the vaccine plasmid was injected into the hair-removed area using a gene gun, once a week for a total of five injections. Two days after the third injection, an appropriate amount of LL2 lung cancer cells was inoculated to induce tumor formation. Tumor formation time was observed, and the long axis (a) and short axis (b) of the tumor were measured every two days. Tumor volume was calculated as a*b*b / 2, and a tumor growth curve was plotted. Results are as follows: Figure 5 B and Figure 5 As shown in C, immunization with XCL1-EGFR wild-type and XCL1-EGFR mutant vaccine plasmids can effectively delay tumor growth, indicating that the therapeutic effect of this fusion nucleic acid vaccine is significant.

[0090] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Beijing Promise Future Technology Co., Ltd. Beijing Baisai Technology Co., Ltd. <120> Tumor nucleic acid vaccines targeting EGFR <130> MP2028641 <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 622 <212> PRT <213> Mouse <400> 1 Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Arg Leu Thr Gln Leu 1 5 10 15 Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Tyr Asn Asn 20 25 30 Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg Asn 35 40 45 Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val 50 55 60 Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu Gln 65 70 75 80 Ile Ile Arg Gly Asn Ala Leu Tyr Glu Asn Thr Tyr Ala Leu Ala Ile 85 90 95 Leu Ser Asn Tyr Gly Thr Asn Arg Thr Gly Leu Arg Glu Leu Pro Met 100 105 110 Arg Asn Leu Gln Glu Ile Leu Ile Gly Ala Val Arg Phe Ser Asn Asn 115 120 125 Pro Ile Leu Cys Asn Met Asp Thr Ile Gln Trp Arg Asp Ile Val Gln 130 135 140 Asn Val Phe Met Ser Asn Met Ser Met Asp Leu Gln Ser His Pro Ser 145 150 155 160 Ser Cys Pro Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp Gly 165 170 175 Gly Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala Gln 180 185 190 Gln Cys Ser His Arg Cys Arg Gly Arg Ser Pro Ser Asp Cys Cys His 195 200 205 Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu 210 215 220 Val Cys Gln Lys Phe Gln Asp Glu Ala Thr Cys Lys Asp Thr Cys Pro 225 230 235 240 Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro 245 250 255 Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro Arg 260 265 270 Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Pro 275 280 285 Asp Tyr Tyr Glu Val Glu Glu Asp Gly Ile Arg Lys Cys Lys Lys Cys 290 295 300 Asp Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe 305 310 315 320 Lys Asp Thr Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Tyr 325 330 335 Cys Thr Ala Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Lys 340 345 350 Gly Asp Ser Phe Thr Arg Thr Pro Pro Leu Asp Pro Arg Glu Leu Glu 355 360 365 Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala 370 375 380 Trp Pro Asp Asn Trp Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile 385 390 395 400 Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val 405 410 415 Gly Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser 420 425 430 Asp Gly Asp Val Ile Ile Ser Gly Asn Arg Asn Leu Cys Tyr Ala Asn 435 440 445 Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Pro Asn Gln Lys Thr Lys 450 455 460 Ile Met Asn Asn Arg Ala Glu Lys Asp Cys Lys Ala Val Asn His Val 465 470 475 480 Cys Asn Pro Leu Cys Ser Ser Glu Gly Cys Trp Gly Pro Glu Pro Arg 485 490 495 Asp Cys Val Ser Cys Gln Asn Val Ser Arg Gly Arg Glu Cys Val Glu 500 505 510 Lys Cys Asn Ile Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser 515 520 525 Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile 530 535 540 Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr 545 550 555 560 Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Ile Met Gly 565 570 575 Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn Asn Val Cys 580 585 590 His Leu Cys His Ala Asn Cys Thr Tyr Gly Cys Ala Gly Pro Gly Leu 595 600 605 Gln Gly Cys Glu Val Trp Pro Ser Gly Pro Lys Ile Pro Ser 610 615 620 <210> 2 <211> 622 <212> PRT <213> Artificial sequence <400> 2 Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Arg Leu Thr Gln Leu 1 5 10 15 Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Tyr Asn Asn 20 25 30 Cys Glu Val Val Leu Gly Asn Leu Glu Ile Leu Tyr Val Gln Arg Asn 35 40 45 Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val 50 55 60 Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Phe Leu Glu Asn Leu Gln 65 70 75 80 Ile Ile Arg Gly Asn Ala Leu Tyr Glu Asn Thr Tyr Ala Leu Ala Ile 85 90 95 Leu Ser Asn Tyr Gly Thr Asn Arg Thr Gly Leu Arg Glu Leu Pro Met 100 105 110 Arg Asn Leu Gln Glu Ile Leu Ile Gly Ala Val Arg Phe Ser Asn Asn 115 120 125 Pro Ile Leu Cys Asn Met Asp Thr Ile Gln Trp Arg Asp Ile Val Gln 130 135 140 Asn Val Phe Met Ser Asn Met Ser Met Asp Leu Gln Ser His Pro Ser 145 150 155 160 Ser Cys Pro Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp Gly 165 170 175 Gly Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala Gln 180 185 190 Gln Cys Ser His Arg Cys Arg Gly Arg Ser Pro Ser Asp Cys Cys His 195 200 205 Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu 210 215 220 Val Cys Gln Lys Phe Gln Asp Glu Ala Thr Cys Lys Asp Thr Cys Pro 225 230 235 240 Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro 245 250 255 Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro Arg 260 265 270 Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Pro 275 280 285 Asp Tyr Tyr Glu Val Glu Glu Asp Gly Ile Arg Lys Cys Lys Lys Cys 290 295 300 Asp Gly Pro Cys Arg Phe Val Cys Asn Gly Ile Gly Ile Gly Glu Phe 305 310 315 320 Lys Asp Thr Met Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Tyr 325 330 335 Cys Thr Ala Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Lys 340 345 350 Gly Asp Ser Phe Thr Arg Thr Pro Pro Leu Asp Pro Arg Glu Leu Glu 355 360 365 Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala 370 375 380 Phe Pro Asp Asn Trp Thr Asp Leu His Met Phe Glu Asn Leu Glu Ile 385 390 395 400 Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Met 405 410 415 Gly Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser 420 425 430 Asp Gly Asp Val Ile Ile Ser Gly Asn Arg Asn Leu Cys Tyr Ala Asn 435 440 445 Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Pro Asn Gln Lys Thr Lys 450 455 460 Ile Met Asn Asn Arg Ala Glu Lys Asp Cys Lys Ala Val Asn His Val 465 470 475 480 Cys Asn Pro Leu Cys Ser Ser Glu Gly Cys Trp Gly Pro Glu Pro Arg 485 490 495 Asp Cys Val Ser Cys Gln Asn Val Ser Arg Gly Arg Glu Cys Val Glu 500 505 510 Lys Cys Asn Ile Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser 515 520 525 Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile 530 535 540 Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr 545 550 555 560 Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Ile Met Gly 565 570 575 Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn Asn Val Cys 580 585 590 His Leu Cys His Ala Asn Cys Thr Tyr Gly Cys Ala Gly Pro Gly Leu 595 600 605 Gln Gly Cys Glu Val Trp Pro Ser Gly Pro Lys Ile Pro Ser 610 615 620 <210> 3 <211> 114 <212> PRT <213> mouse <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 <210> 4 <211> 747 <212> PRT <213> 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 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Glu Glu Lys 115 120 125 Lys Val Cys Gln Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe 130 135 140 Glu Asp His Phe Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val 145 150 155 160 Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu 165 170 175 Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala 180 185 190 Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg 195 200 205 Gly Asn Ala Leu Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn 210 215 220 Tyr Gly Thr Asn Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu 225 230 235 240 Gln Glu Ile Leu Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu 245 250 255 Cys Asn Met Asp Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe 260 265 270 Met Ser Asn Met Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro 275 280 285 Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu 290 295 300 Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser 305 310 315 320 His Arg Cys Arg Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys 325 330 335 Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln 340 345 350 Lys Phe Gln Asp Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met 355 360 365 Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys 370 375 380 Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val 385 390 395 400 Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr 405 410 415 Glu Val Glu Glu Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro 420 425 430 Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr 435 440 445 Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala 450 455 460 Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser 465 470 475 480 Phe Thr Arg Thr Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys 485 490 495 Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp 500 505 510 Asn Trp Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly 515 520 525 Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn 530 535 540 Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp 545 550 555 560 Val Ile Ile Ser Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn 565 570 575 Trp Lys Lys Leu Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn 580 585 590 Asn Arg Ala Glu Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro 595 600 605 Leu Cys Ser Ser Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val 610 615 620 Ser Cys Gln Asn Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn 625 630 635 640 Ile Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile 645 650 655 Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr 660 665 670 Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly 675 680 685 Pro His Cys Val Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn 690 695 700 Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys 705 710 715 720 His Ala Asn Cys Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys 725 730 735 Glu Val Trp Pro Ser Gly Pro Lys Ile Pro Ser 740 745 <210> 5 <211> 747 <212> PRT <213> Artificial Sequence <400> 5 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 Gly Ser Gly Gly Gly Gly Gly Glu Glu Lys 115 120 125 Lys Val Cys Gln Gly Thr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe 130 135 140 Glu Asp His Phe Leu Ser Leu Gln Arg Met Tyr Asn Asn Cys Glu Val 145 150 155 160 Val Leu Gly Asn Leu Glu Ile Leu Tyr Val Gln Arg Asn Tyr Asp Leu 165 170 175 Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala 180 185 190 Leu Asn Thr Val Glu Arg Ile Phe Leu Glu Asn Leu Gln Ile Ile Arg 195 200 205 Gly Asn Ala Leu Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn 210 215 220 Tyr Gly Thr Asn Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu 225 230 235 240 Gln Glu Ile Leu Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu 245 250 255 Cys Asn Met Asp Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe 260 265 270 Met Ser Asn Met Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro 275 280 285 Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu 290 295 300 Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser 305 310 315 320 His Arg Cys Arg Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys 325 330 335 Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Gln 340 345 350 Lys Phe Gln Asp Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met 355 360 365 Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys 370 375 380 Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val 385 390 395 400 Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Pro Asp Tyr Tyr 405 410 415 Glu Val Glu Glu Asp Gly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro 420 425 430 Cys Arg Phe Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr 435 440 445 Met Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala 450 455 460 Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser 465 470 475 480 Phe Thr Arg Thr Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys 485 490 495 Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Phe Pro Asp 500 505 510 Asn Trp Thr Asp Leu His Met Phe Glu Asn Leu Glu Ile Ile Arg Gly 515 520 525 Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Met Gly Leu Asn 530 535 540 Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp 545 550 555 560 Val Ile Ile Ser Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn 565 570 575 Trp Lys Lys Leu Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn 580 585 590 Asn Arg Ala Glu Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro 595 600 605 Leu Cys Ser Ser Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val 610 615 620 Ser Cys Gln Asn Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn 625 630 635 640 Ile Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile 645 650 655 Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr 660 665 670 Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly 675 680 685 Pro His Cys Val Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn 690 695 700 Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys 705 710 715 720 His Ala Asn Cys Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys 725 730 735 Glu Val Trp Pro Ser Gly Pro Lys Ile Pro Ser 740 745 <210> 6 <211> 2241 <212> DNA <213> Artificial Sequence <400> 6 atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtgga 120. ggtgtaggct ctgaggtctc tgataagagg acatgtgtca gcttgactac ccaacgcctc 180. ccgtctcca gattaaaac ctatacaatc acagaaggct cactgagggc cgtaattttc ataaccaaac ggggactgaa agtatgtgcg gacccacaag ccacctgggt gcgagatgtg gtccggagta tggacagga atcaatacg cgcaataca tgatacagac caagccaacg ggcacacagc aatctacaaa taccgcagta acactgactg gaggaggtgg aggaggtagt ggcggaggag gtggtgagga aaagaaagtc tgccaaggca caagtaacag gctcacccaa 480. ctgggcactt ttgaagacca ctttctgagc ctgcagagga tgtacaacaa ctgtgaagtg gtccttggga acttggaat tacctatgtg caaaggaatt acgacctttc cttcttaaag accatccagg aggtggccgg ctatgtcctc attgccctca acaccgtgga gagaatccct ttggagacc tgcagatcat cagggaat gctcttatg aaaacaccta tgccttagcc 660 atcctgtcca actatgggac aacagaact gggcttaggg aactcccat gcggaactta 720 caggaaatcc tgattgtgc tgtgcgattc agcaacacc ccatccctg caatgatgt 780 actatccagt gggaggacat cgtccaaaac gtctttatga gcacatgtc atggactta 840 cagagccatc cgagcagttg cccaatgt gatccaagct gtcccaatgg aagctgctgg 900 ggaggaggag aggagactg ccagaaattg accaaatca tctgtgccca gcaatgttcc 960 catcgctgtc gtggcaggtc ccccagtgac tgctgccaca accaatgtgc tgcggggtgt 1020 acagggcccc gagagagtga ctgtctggtc tgccaaagt tccagatga ggccacatgc 1080 aaagacacct gcccaccact catgctgtac aaccccacca cctatcagat ggatgtcac 1140 cctgaaggga agtacagctt tggtgccacc tgtgtgaaga agtgccccg aaactacgtg 1200 gtgacagatc atggctcatg tgtccgagcc tgtgggcctg actactacga agtggaagaa 1260 gatggcatcc gcaagtgtaa aaatgtgat gggccctgtc gcaagttttg taatggcata 1320 ggcattggtg aatttaaaga cacactctcc aataatgcta caacatcaa acacttcaaa 1440. tactgcactg ccatcagcgg ggaccttcac atcctgccag tggcctttaa gggggattct ttcacgcgca ctcctcctct agacccacga ttctaaaaac cgtaagga ataacaggct ttttgctgat tcaggcttgg cctgataact ggactgacct ccatgctttc 1620. 1620. 1620. 1620. 1620. 1620. 1620. 1620 gttggcctga acatcacatc actggggctg cgttccctca aggagatcag tgatggggat 1680 gtgatcattt ctggaaccg aaatttgtgc tacgcaaaca caataactg gaaaaaaactc ttcgggacac ccaatcaga aaccaaaatc atgaacaaca gagctgaga agactgcaag gccgtgaacc acgtctgcaa tcctttatgc tcctcggaag gctgctgggg ccctgagccc agggactgtg tctcctgcca gaatgtgagc agaggcaggg agtgcgtgga gaaatgcaac atcctggagg gggaaccaag ggagtttgtg gaaaattctg aatgcatcca gtgccatcca gaatgtctgc cccaggccat gaacatcacc tgtacaggca ggggaccaga caactgcatc cagtgtgccc actacattga tggcccacac tgtgtcaaga cctgcccagc tggcatcatg ggagagaaca acactctggt ctggaagtat gcagatgcca ataatgtctg ccacctatgc cacgccaact gtacctatgg atgtgctggg ccaggtcttc aaggatgtga agtgtggcca 2220 tctgggccaa agataccatc t <210> 7 <211> 2241 <212> DNA <213> Artificial Sequence <400> 7 atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtgga 120. ggtgtaggct ctgaggtctc tgataagagg acatgtgtca gcttgactac ccaacgcctc 180. ccgtctcca gattaaaac ctatacaatc acagaaggct cactgagggc cgtaattttc ataaccaaac ggggactgaa agtatgtgcg gacccacaag ccacctgggt gcgagatgtg gtccggagta tggacagga atcaatacg cgcaataca tgatacagac caagccaacg ggcacacagc aatctacaaa taccgcagta acactgactg gaggaggtgg aggaggtagt ggcggaggag gtggtgagga aaagaaagtc tgccaaggca caagtaacag gctcacccaa ctgggcactt tgagacca ctttctgagc ctgcagagga tgtacaacaa ctgtgaagtg 480 gtccttggga acttggaat tctctatgtg caaggaatt acgacctttc cttctttaag 540 accatccagg aggtggccgg ctatgtccctc attgccctca acaccgtgga gagaatcttt 600 ttggagacc tgcagatcat cagggaat gctcttatg aaaacaccta tgccttagcc 660 atcctgtcca actatgggac aacagaact gggcttaggg aactcccat gcggaactta 720 caggaaatcc tgattgtgc tgtgcgattc agcaacacc ccatccctg caatgatgt 780 actatccagt gggaggacat cgtccaaaac gtctttatga gcacatgtc atggactta 840 cagagccatc cgagcagttg cccaatgt gatccaagct gtcccaatgg aagctgctgg 900 ggaggaggag aggagactg ccagaaattg accaaatca tctgtgccca gcaatgttcc 960 catcgctgtc gtggcaggtc ccccagtgac tgctgccaca accaatgtgc tgcggggtgt 1020 acagggcccc gagagagtga ctgtctggtc tgccaaagt tccagatga ggccacatgc 1080 aaagacacct gcccaccact catgctgtac aaccccacca cctatcagat ggatgtcac 1140 cctgaaggga agtacagctt tggtgccacc tgtgtgaaga agtgcccccg aaactacgtg gtgacagatc atggctcatg tgtccgagcc tgtgggcctg actactacga agtggaagaa gatggcatcc gcaagtgtaa aaaatgtgat gggccctgtc gctttgtttg taatggcata ggcattggtg aatttaaaga cacaatgtcc aataatgcta caacatcaa acacttcaaa 1440. tactgcactg ccatcagcgg ggaccttcac atcctgccag tggcctttaa gggggattct ttcacgcgca ctcctcctct agacccacga ttctaaaaac cgtaagga ataacaggct ttttgctgat tcaggctttc cctgataact ggactgacct ccatatgttc 1620. 1620. 1620. 1620. 1620. 1620. 1620. 1620 atgggcctga acatcacatc actggggctg cgttccctca aggagatcag tgatggggat 1680. gtgatcattt ctggaaccg aaatttgtgc tacgcaaaca caataactg gaaaaaaactc ttcgggacac ccaatcaga aaccaaaatc atgaacaaca gagctgaga agactgcaag gccgtgaacc acgtctgcaa tcctttatgc tcctcggaag gctgctgggg ccctgagccc agggactgtg tctcctgcca gaatgtgagc agaggcaggg agtgcgtgga gaaatgcaac 1920 atcctggagg gggaaccaag ggagtttgtg gaaaattctg aatgcatcca gtgccatcca 1980 gaatgtctgc cccaggccat gaacatcacc tgtacaggca ggggaccaga caactgcatc 2040 cagtgtgccc actacattga tggcccacac tgtgtcaaga cctgcccagc tggcatcatg 2100 ggagagaaca acactctggt ctggaagtat gcagatgcca ataatgtctg ccacctatgc 2160 cacgccaact gtacctatgg atgtgctggg ccaggtcttc aaggatgtga agtgtggcca 2220 tctgggccaa agataccatc t 2241 <210> 8 <211> 1866 <212> DNA <213> Artificial Sequence <400> 8 gaggaaaaga aagtctgcca aggcacaagt aacaggctca cccaactggg cacttttgaa 60 gaccactttc tgagcctgca gaggatgtac aacaactgtg aagtggtcct tgggaacttg 120 gaaattacct atgtgcaaag gaattacgac ctttccttct taaagaccat ccaggaggtg 180 gccggctatg tcctcattgc cctcaacacc gtggagagaa tccctttgga gaacctgcag 240 atcatcaggg gaaatgctct ttatgaaaac acctatgcct tagccatcct gtccaactat 300 gggacaaaca gaactgggct tagggaactg cccatgcgga acttacagga aatcctgatt 360 ggtgctgtgc gattcagcaa caaccccatc ctctgcaata tggatactat ccagtggagg 420 gacatcgtcc aaaacgtctt tatgagcaac atgtcaatgg acttacagag ccatccgagc 480 agttgcccca aatgtgatcc aagctgtccc aatggaagct gctggggagg aggagaggag 540 aactgccaga aattgaccaa aatcatctgt gcccagcaat gttcccatcg ctgtcgtggc 600 aggtccccca gtgactgctg ccacaaccaa tgtgctgcgg ggtgtacagg gccccgagag 660 agtgactgtc tggtctgcca aaagttccaa gatgaggcca catgcaaaga cacctgccca 720 ccactcatgc tgtacaaccc caccacctat cagatggatg tcaaccctga agggaagtac 780 agctttggtg ccacctgtgt gaagaagtgc ccccgaaact acgtggtgac agatcatggc 840 tcatgtgtcc gagcctgtgg gcctgactac tacgaagtgg aagaagatgg catccgcaag 900 tgtaaaaaat gtgatgggcc ctgtcgcaaa gtttgtaatg gcataggcat tggtgaattt 960 aaagacacac tctccataaa tgctacaaac atcaaacact tcaaatactg cactgccatc 1020 agcggggacc ttcacatcct gccagtggcc tttaaggggg attctttcac gcgcactcct 1080 cctctagacc cacgagaact agaaattcta aaaaccgtaa aggaaataac aggctttttg 1140 ctgattcagg cttggcctga taactggact gacctccatg ctttcgagaa cctagaaata 1200 atacgtggca gaacaaagca acatggtcag ttttctttgg cggtcgttgg cctgaacatc 1260 acatcactgg ggctgcgttc cctcaaggag atcagtgatg gggatgtgat catttctgga 1320 aaccgaaatt tgtgctacgc aaacacaata aactggaaaa aactcttcgg gacacccaat 1380 cagaaaacca aaatcatgaa caacagagct gagaaagact gcaaggccgt gaaccacgtc 1440 tgcaatcctt tatgctcctc ggaaggctgc tggggccctg agcccaggga ctgtgtctcc 1500 tgccagaatg tgagcagagg cagggagtgc gtggagaaat gcaacatcct ggagggggaa 1560 ccaagggagt ttgtggaaaa ttctgaatgc atccagtgcc atccagaatg tctgccccag 1620 gccatgaaca tcacctgtac aggcagggga ccagacaact gcatccagtg tgcccactac 1680 attgatggcc cacactgtgt caagacctgc ccagctggca tcatgggaga gaacaacact 1740 ctggtctgga agtatgcaga tgccaataat gtctgccacc tatgccacgc caactgtacc 1800 tatggatgtg ctgggccagg tcttcaagga tgtgaagtgt ggccatctgg gccaaagata 1860 ccatct 1866 <210> 9 <211> 1866 <212> DNA <213> Artificial Sequence <400> 9 gaggaaaaga aagtctgcca aggcacaagt aacaggctca cccaactggg cacttttgaa 60 gaccactttc tgagcctgca gaggatgtac aacaactgtg aagtggtcct tgggaacttg 120 gaaattctct atgtgcaaag gaattacgac ctttccttct taaagaccat ccaggaggtg 180 gccggctatg tcctcattgc cctcaacacc gtggagagaa tctttttgga gaacctgcag 240 atcatcaggg gaaatgctct ttatgaaaac acctatgcct tagccatcct gtccaactat 300 gggacaaaca gaactgggct tagggaactg cccatgcgga acttacagga aatcctgatt 360 ggtgctgtgc gattcagcaa caaccccatc ctctgcaata tggatactat ccagtggagg 420 gacatcgtcc aaaacgtctt tatgagcaac atgtcaatgg acttacagag ccatccgagc 480 agttgcccca aatgtgatcc aagctgtccc aatggaagct gctggggagg aggagaggag 540 aactgccaga aattgaccaa aatcatctgt gcccagcaat gttcccatcg ctgtcgtggc 600 aggtccccca gtgactgctg ccacaaccaa tgtgctgcgg ggtgtacagg gccccgagag 660 agtgactgtc tggtctgcca aaagttccaa gatgaggcca catgcaaaga cacctgccca 720 ccactcatgc tgtacaaccc caccacctat cagatggatg tcaaccctga agggaagtac 780 agctttggtg ccacctgtgt gaagaagtgc ccccgaaact acgtggtgac agatcatggc 840 tcatgtgtcc gagcctgtgg gcctgactac tacgaagtgg aagaagatgg catccgcaag 900 tgtaaaaaat gtgatgggcc ctgtcgcttt gtttgtaatg gcataggcat tggtgaattt 960 aaagacacaa tgtccataaa tgctacaaac atcaaacact tcaaatactg cactgccatc 1020 agcggggacc ttcacatcct gccagtggcc tttaaggggg attctttcac gcgcactcct 1080 cctctagacc cacgagaact agaaattcta aaaaccgtaa aggaaataac aggctttttg 1140 ctgattcagg ctttccctga taactggact gacctccata tgttcgagaa cctagaaata 1200 atacgtggca gaacaaagca acatggtcag ttttctttgg cggtcatggg cctgaacatc 1260 acatcactgg ggctgcgttc cctcaaggag atcagtgatg gggatgtgat catttctgga 1320 aaccgaaatt tgtgctacgc aaacacaata aactggaaaa aactcttcgg gacacccaat 1380 cagaaaacca aaatcatgaa caacagagct gagaaagact gcaaggccgt gaaccacgtc 1440 tgcaatcctt tatgctcctc ggaaggctgc tggggccctg agcccaggga ctgtgtctcc 1500 tgccagaatg tgagcagagg cagggagtgc gtggagaaat gcaacatcct ggagggggaa 1560 ccaagggagt ttgtggaaaa ttctgaatgc atccagtgcc atccagaatg tctgccccag 1620 gccatgaaca tcacctgtac aggcagggga ccagacaact gcatccagtg tgcccactac 1680 attgatggcc cacactgtgt caagacctgc ccagctggca tcatgggaga gaacaacact 1740 ctggtctgga agtatgcaga tgccaataat gtctgccacc tatgccacgc caactgtacc 1800 tatggatgtg ctgggccagg tcttcaagga tgtgaagtgt ggccatctgg gccaaagata 1860 ccatct 1866 <210> 10 <211> 342 <212> DNA <213> Artificial Sequence <400> 10 atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60 ggtgtaggct ctgaggtctc tgataagagg acatgtgtca gcttgactac ccaacgcctc 120 ccagtctcca gaattaaaac ctatacaatc acagaaggct cactgagggc cgtaattttc 180 ataaccaaac ggggactgaa agtatgtgcg gacccacaag ccacctgggt gcgagatgtg 240 gtccggagta tggacaggaa atcaaatacg cgcaataaca tgatacagac caagccaacg 300 ggcacacagc aatctacaaa taccgcagta acactgactg ga 342 <210> 11 <211> 21 <212> PRT <213> Artificial Sequence <400> 11 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> 12 <211> 24 <212> PRT <213> Artificial Sequence <400> 12 Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr 1 5 10 15 Ala Leu Cys Ala Ala Gly Gly Ala 20 <210> 13 <211> 22 <212> PRT <213> Artificial Sequence <400> 13 Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr 1 5 10 15 Lys Asp Asp Asp Asp Lys 20

Claims

1. A fusion protein, the amino acid sequence of which is shown in SEQ ID NO:

5.

2. The nucleic acid encoding the fusion protein of claim 1.

3. The nucleic acid according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

7.

4. A recombinant vector comprising a backbone vector and the nucleic acid as described in claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that, The backbone vector is selected from pcDNA3.1(-) or pcDNA3.1(+).

6. Transformation or transfection of the recombinant host of the recombinant vector of claim 4 or 5.

7. The recombinant host according to claim 6, characterized in that, Its host cells are bacteria or mammalian cells.

8. The recombinant host according to claim 7, characterized in that, The bacteria are Escherichia coli; the mammalian cells are HEK293T cells.

9. The method for preparing the fusion protein according to claim 1, characterized in that, Cultivate the recombinant host according to any one of claims 6 to 8 to obtain a culture containing the fusion protein.

10. The use of the fusion protein of claim 1, the nucleic acid of claim 2 or 3, the recombinant vector of claim 4 or 5, the recombinant host of any one of claims 6 to 8, and / or the fusion protein prepared by the preparation method of claim 9 in the preparation of drugs for the prevention and treatment of lung cancer.

11. A drug for the prevention and treatment of lung cancer, characterized in that, It includes: The fusion protein of claim 1, the nucleic acid of claim 2 or 3, the recombinant vector of claim 4 or 5, the recombinant host of any one of claims 6 to 8, and / or the fusion protein prepared by the preparation method of claim 9.

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