CN111926042A - Therapeutic dendritic cell cancer vaccine and application thereof - Google Patents

Abstract

The invention discloses a therapeutic dendritic cell cancer vaccine and application thereof, belonging to the technical field of biology. The invention constructs a shuttle plasmid pGBC-IRES-WSL and recombinant adenovirus which can simultaneously express tumor-associated antigen, granulocyte-megakaryocyte colony stimulating factor (GM-CSF), CD40L and 4-1BBL, and infects human peripheral blood-derived mononuclear cells with the recombinant adenovirus to obtain the therapeutic dendritic cell cancer vaccine. The cancer vaccine can exert an anti-tumor effect by inducing an organism to produce antigen-specific T lymphocytes, thereby being used for treating various blood system tumors and solid tumors.

Description

Therapeutic dendritic cell cancer vaccine and application thereof

Technical Field

The invention belongs to the preparation of a therapeutic cancer vaccine in the field of biotechnology, and particularly relates to a therapeutic dendritic cell cancer vaccine and application thereof.

Background

Cancer is a major health problem of continuing concern worldwide. Immunotherapy has now become the most promising approach for cancer therapy, including the immune checkpoint blockade PD-1/PD-L1, CAR-T technology, oncolytic viruses and therapeutic cancer vaccines, which have been the major and hot areas of immunotherapy development, and to date the us FDA has approved a therapeutic cancer vaccine [ Provenge (Sipuleucel-T) ], six immune checkpoint blockades [ including the CTLA-4 inhibitor ipilimumab (yervoy); PD-1 inhibitors nivolumab (opsivo), pembrolizumab (keytruda); the PD-L1 inhibitors Atezolizumab (Tecntriq), Durvalumab (Imfinzi) and Avelumab (Bavencio), two products of CAR-T technology [ Yescata and Kymriah ], and one product of oncolytic virus [ talimogen laherparepvec (Imygic), T-Vec ] were used clinically with good clinical results. However, the above-mentioned immunotherapy has a significant efficacy only for a small fraction of tumors, while it does not show a significant advantage in most tumors, which is clearly linked to the biological diversity of different tumors and to the complexity of the body's immune system.

Among them, therapeutic cancer vaccines have been one of the focus and hot spots of research in the field of tumor immunotherapy. Therapeutic cancer vaccines are cancer vaccines for therapeutic purposes, and usually achieve an anti-tumor effect by inducing an active immune response specific to a tumor in the body. The purpose can be achieved by various means or strategies, such as various peptide vaccines, various vector vaccines and tumor vaccines based on Dendritic Cells (DCs) and tumor vaccines of various organic combinations of vaccine components, such as peptide-coated DCs, transfection or infection of DCs with various vectors (RNA, DNA and viral vectors), and particularly, neoantigen tumor vaccines, i.e., therapeutic tumor vaccines prepared by using various antigen peptides generated from mutant proteins encoded by mutant genes in tumors, are used for treating tumor patients, and even clinical studies on individual cases or small samples show incredible clinical therapeutic effects. However, to induce an active immune response that is tumor specific, a key step is the presentation of tumor Antigen peptides to T cells for recognition by in vivo Antigen Presenting Cells (APCs). DCs are the strongest APCs in vivo known to date, and therefore therapeutic DC neoplasms are the dominant therapeutic cancer vaccines.

The main functions of DCs are to take up, process and present antigens, and to transmit the information of antigens to naive T cells, to promote T cells to proliferate, differentiate and activate into Cytotoxic T Lymphocytes (CTL) capable of recognizing and killing tumor cells, and it is seen that the most important anti-tumor immune effect is also T cell response, and DCs are the most critical loop for inducing such tumor-specific T lymphocyte response. For example, the first therapeutic DCs tumor vaccine in the world approved by the FDA in the united states in 2010 [ Sipuleucel-t (provenge) ], is a prostate cancer vaccine for the treatment of asymptomatic or microsomal, castration-resistant metastatic advanced prostate cancer, suggesting the advent of the cancer immunotherapy era. Following the approval of Sipuleucel-T, the FDA has successively approved nearly ten tumor immunotherapy-related products for clinical use in the last decade. However, there are also cases of failure, such AS the MAGE-A3 therapeutic tumor vaccine (recombinant MAGE-A3 protein plus AS15 immunostimulant, multiple intramuscular injections) from Kuraranshi Scek (GSK) in the phase III clinical study (MAGRIT study) for the treatment of non-small cell lung cancer, which did not achieve the expected effect and was terminated prematurely. Although the reason for the failure is not clear, the design of a therapeutic tumor vaccine of DCs should be considerable in the hope of obtaining a therapeutic cancer vaccine with better clinical therapeutic effect.

Existing culture systems for preparing DC tumor seedlings are to first obtain Peripheral Blood Mononuclear Cells (PBMCs) from a patient and adhere to the PBMCs via plastic adhesion or CD14⁺After the monocytes are obtained by sorting the immunomagnetic beads, GM-CSF and IL-4 are added, the culture medium is replaced for 2-3 days, new cytokines are added, the monocytes can be differentiated into immature DCs in about 5 days, the target antigens are given for stimulation, and then maturation induction factors (cocktail type cytokines combined with TNF-alpha, IL-1 beta, IL-6 and PGE2) are added for 1-2 days, so that the antigen-stimulated mature DCs can be obtained, the whole preparation time is about 7-9 days, the operation steps are more and complicated, the situations of pollution and the like easily occur, a plurality of cytokines are needed, and the preparation cost is higher. Although it has been exploredA method (FastDC) for rapidly obtaining DCs is obtained, namely, monocytes can be differentiated into DCs in 2-3 days in the presence of GM-CSF and IL-4, and the DCs can also induce generation of tumor specific CTL reaction, so that the culture time is shortened. It still requires multi-step laboratory procedures, addition of antigens, cytokine supplementation, etc., and the stimulation of T cells is not generally recognized.

Providing a composite stimulation signal through an optimized combination of co-stimulatory molecules and/or bioactive molecules with adjuvant function is often a rational design approach to increase the efficacy of therapeutic neoplasms. For example, a PSA-TRICOM vaccine against prostate cancer

The design of the vaccine adopts immune adjuvants TRICOM (B7.1, ICAM-1 and LFA-3) consisting of two virus vectors for expressing Prostate Specific Antigen (PSA) and three co-stimulatory molecules or adhesion molecules respectively, wherein the first virus is recombinant vaccinia virus (vaccinia) and the second virus is recombinant Fowlpox virus (Fowlpox), and the two viruses are combined after expressing the antigen and the co-stimulatory molecules respectively to form a PSA-TRICOM vaccine system. In clinical trial studies, the Prime-Boost strategy was used to immunize advanced prostate cancer by first subcutaneously administering the recombinant vaccinia virus encoding PSA-TRICOM (rV-PSA-TRICOM) Prime twice at 2 weeks intervals, and then subcutaneously administering the recombinant fowlpox virus encoding PSA-TRICOM (rF-psatiricom) Boost four times at 4 weeks intervals, and subcutaneously administering GM-CSF to all sites injected with the vaccine 3 consecutive days after sequential administration. In a multicenter randomized controlled double-blind phase II clinical trial study, 125 castration-resistant metastatic prostate cancer patients were enrolled into the study, randomized into 84 patients in a 2:1 ratio treatment group (rV-PSA-TRICOM Prime twice plus rF-PSATRICOM Boost four times and given GM-CSF for 3 consecutive days) and 41 controls (empty rV twice plus empty rF subcutaneous injections, GM-CSF replaced with saline). The primary endpoint of the study was progression free survival (PSF), although there was no significant difference between the two groups, 30% of the treatment groups remained ill following visit to the third year after studyWhile the patients remained alive, only 17% of the patients survived in the control group, with an overall survival time of 25.1 months in the treatment group, 8.5 months longer than in the control group (16.6 months); and the treatment group has slight toxic and side effects, and patients can tolerate the disease well, so that the treatment effect is good. However, such therapeutic neoplasms require the sequential use of three agents (rV-PSA-TRICOM, rF-PSATRICOM and GM-CSF), are relatively complex in administration mode, are direct applications in vivo of two viruses, and have certain safety risks.

Disclosure of Invention

In view of one or more of the problems of the prior art, an aspect of the present invention provides a shuttle plasmid pGBC-IRES-WSL carrying a plurality of genes of interest, the starting vector of the shuttle plasmid being a pDC511 shuttle plasmid, the plurality of genes of interest including expression genes of tumor-associated antigens, granulocyte-megakaryocyte colony stimulating factor (GM-CSF), CD40L and 4-1BBL, and wherein the expression genes of the granulocyte-megakaryocyte colony stimulating factor (GM-CSF), CD40L and 4-1BBL are connected in series by two 2A peptide sequences to form an open reading frame, and an internal nucleic acid entry site IRES sequence is linked to the 5' end of the expression sequence of the tumor-associated antigens.

The expression gene of the tumor-associated antigen includes, but is not limited to, any one of the following genes: tWT1, Survivin, fusion genes tWT1/Survivin, fusion genes tWT1/Survivin/LAMP-1, MUC1, LMP, HPV E6E 7, EGFRvIII, HER-2/neu, MAGE A3, p53(nonmutant and mutant), NY-ESO-1, PSMA, GD2, CEA, Melana/MART1, Ras mutant, gp100, Proteinase3(PR1), bcr-abl, Tyrosinase, PSA, hTERT, Sarcoma translocation peptides, EphA2, PAP, ML-IAP, AFP, EpGE, ERG (TMPRSS2 ETS fusion gene), NA17, PA46X 3, Androgram, Cyclin B5, Polyalsin B5, Fusalin-8, MAG-24, MAG-1, Rho 639, CYP 598, GM 9, CYP 639, GM1, GM 9, MAGE-A, MAGE-598, and MAG-2; preferably, the expression gene of the tumor associated antigen is tWT1 (the nucleotide sequence of which can be shown in SEQ ID NO: 22), Survivin (the nucleotide sequence of which can be shown in SEQ ID NO: 26), fusion gene tWT1/Survivin (the nucleotide sequence of which can be shown in SEQ ID NO: 34) or fusion gene tWT1/Survivin/LAMP-1 (the nucleotide sequence of which can be shown in SEQ ID NO: 29).

The nucleotide sequence of the GM-CSF expression gene can be shown as SEQ ID NO. 7; the nucleotide sequence of the CD40L expression gene can be shown as SEQ ID NO. 15; the nucleotide sequence of the 4-1BBL expression gene can be shown as SEQ ID NO. 11.

The 2A peptide sequence is selected from any one or two combinations of E2A, F2A, T2A and P2A, and the connection positions of the expression genes of GM-CSF, 4-1BBL and CD40L in the open reading frame formed by two 2A peptide sequences in tandem can be interchanged.

The nucleotide sequence of the shuttle plasmid pGBC-IRES-WSL is shown in SEQ ID NO: 33.

The invention also provides a construction method of the shuttle plasmid pGBC-IRES-WSL, which comprises the following steps:

6.1) inserting the CMVp-pA expression cassette sequence into the multiple cloning site of pDC511 shuttle plasmid to obtain plasmid pDC 523;

6.2) synthesizing an oligonucleotide fragment comprising two 2A peptide sequences and a multiple cloning site; optionally, a fragment of the oligonucleotide fragment with the nucleotide sequence shown as SEQ ID NO. 3 when T2A and P2A are selected;

6.3) inserting the oligonucleotide fragment synthesized in step 6.2) into the CMVp-pA expression cassette multiple cloning site of plasmid pDC523 in 6.1) to obtain a shuttle plasmid named pDC 5232X 2A; optionally, the nucleotide sequence of the pDC 5232X 2A shuttle plasmid inserted into SEQ ID NO. 3 is shown in SEQ ID NO. 4;

6.4) inserting the expressed genes of GM-CSF, 4-1BBL and CD40L into the pDC 5232X 2A shuttle plasmid obtained in step 6.3) to obtain a plasmid containing expressed genes of GM-CSF, 4-1BBL and CD40L, which is named pDC523GM-CSF/4-1BBL/CD40L plasmid, and wherein the expressed genes of GM-CSF, 4-1BBL and CD40L are connected in series by two 2A peptide sequences to form an open reading frame;

6.5) inserting the expression gene of the tumor associated antigen into the pDC523GM-CSF/4-1BBL/CD40L plasmid of step 6.4) to obtain pGBC-IRES-WSL shuttle plasmid, wherein the 5' end of the expression gene of the tumor associated antigen is connected with an internal nucleic acid entry site IRES sequence.

In the above method, the expression gene of the tumor-associated antigen in step 6.5) is fusion gene tWT1/Survivin/LAMP-1, and the insertion of the fusion gene tWT1/Survivin/LAMP-1 into the pDC523GM-CSF/4-1BBL/CD40L plasmid in step 6.4) is specifically performed as follows:

6.5.1) inserting the signal peptide and the signal classification sequence of the human LAMP-1 gene into the pmRNA IRES-EGFP plasmid to replace the EGFP sequence, and obtaining the pmRNA IRES-LAMP-1 plasmid;

6.5.2) inserting tWT1 and Survivin expression gene sequence into pmRNA IRES-LAMP-1 plasmid to obtain pmRNA IRES-tWT1/Survivin/LAMP-1 plasmid;

6.5.3) obtaining the tumor associated antigen expression gene with the 5' end connected with the IRES sequence by PCR amplification from the pmRNA IRES-tWT1/Survivin/LAMP-1 plasmid of the step 6.5.2), and the gene is named as IRES-tWT1/Survivin/LAMP-1 sequence, and optionally, the primer pair for amplification is an upstream primer shown as SEQ ID NO. 31 and a downstream primer shown as SEQ ID NO. 32;

6.5.4) inserting the IRES-tWT1/Survivin/LAMP-1 sequence obtained by amplification in the step 6.5.3) into the pDC523GM-CSF/4-1BBL/CD40L plasmid obtained in the step 6.4) to obtain pGBC-IRES-WSL shuttle plasmid.

The invention also provides a recombinant adenovirus which is obtained by cotransfecting HEK293T cells with the pGBC-IRES-WSL shuttle plasmid and the adenovirus skeleton plasmid; optionally, the adenovirus backbone plasmid is a pBGHfrt Δ E1,3Cre or pBGHfrt Δ E1,3Cre (5/F35) backbone plasmid.

The invention also provides a therapeutic dendritic cell cancer vaccine which is obtained by infecting the peripheral blood mononuclear cells or dendritic cells of human beings with the recombinant adenovirus and can simultaneously express tumor-associated antigens, GM-CSF, CD40L and 4-1 BBL; optionally, the infection dose of the recombinant adenovirus is 10-100 pfu/cell, and the infection time is 36-48 hours.

The shuttle plasmid pGBC-IRES-WSL or the application of the recombinant adenovirus or the therapeutic dendritic cell cancer vaccine in preparing the medicine for treating the hematological tumor and the solid tumor also belongs to the content of the invention; wherein the hematological and solid tumors are WT1 and/or Survivin positive hematological and solid tumors; optionally, the WT1 and/or Survivin positive hematological tumors include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, and high risk myelodysplastic syndrome (MDS); the WT1 and/or Survivin positive solid tumors include, but are not limited to, lung cancer, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, renal cancer, bladder cancer, glioblastoma, head and neck malignancies, and various types of malignant sarcomas.

According to the recombinant adenovirus provided based on the technical scheme, expression genes of tumor-associated antigens, granulocyte-megakaryocyte colony stimulating factor (GM-CSF), CD40L and 4-1BBL are inserted into an adenovirus vector, so that the tumor-associated antigens, the granulocyte-megakaryocyte colony stimulating factor (GM-CSF), CD40L and 4-1BBL can be obtained simultaneously during one-time expression, when the recombinant adenovirus is used for infecting monocytes derived from human peripheral blood to obtain a therapeutic Dendritic Cell (DCs) cancer vaccine, the GM-CSF expressed by the recombinant adenovirus vector can be used for inducing the monocytes to differentiate into dendritic cells, and the dendritic cells generated by the differentiation can be processed and treated to express the tumor-associated antigens, and antigen information is presented to T cells; and the expressed CD40L induces the DCs to mature and the expressed 4-1BBL is helpful to stimulate the activation of T cells and realize the optimal activation effect of the T cells. In the whole process, co-stimulation factors do not need to be additionally added or two or more expression systems are combined, so that the preparation of the cancer vaccine is simpler, the time consumption is short, and the use is more convenient. The therapeutic DCs cancer vaccine can play an anti-tumor role by inducing an organism to generate antigen-specific T lymphocytes, and achieves the purpose of treating various blood system tumors and solid tumors, thereby having broad-spectrum anti-cancer activity. Compared with the prior art, the invention has the following beneficial effects:

1) the invention uses 5/F35 mosaic type adenovirus as gene expression vector, which can efficiently infect cells of hematopoietic system source, including monocyte and dendritic cell;

2) the recombinant adenovirus vector constructed by the invention contains a tumor-associated antigen tWT1/Survivin fusion gene, and can effectively express a protein product coded by tWT1/Survivin fusion gene after the monocyte is infected by the recombinant adenovirus vector coded by tWT/Survivin fusion gene, thereby providing the two tumor-associated antigens for processing dendritic cells to present tumor antigen peptides, and stimulating T cells to generate WT1 and Survivin specific CTL reaction;

3) in the recombinant adenovirus vector constructed by the invention, the tWT1/Survivin fusion gene is further fused with a lysosome-associated membrane protein 1 (LAMP-1) classification signal, and after the fusion gene protein is processed into antigen peptide by dendritic cells, the antigen peptide can be extracted through an endoplasmic reticulum path to stimulate CD4⁺Activation of T cells, which contributes to the enhancement of specific CTL function;

4) the recombinant adenovirus vector constructed by the invention can simultaneously express GM-CSF, is a dendritic cell differentiation factor, can differentiate dendritic cell precursor cells (such as monocytes) into dendritic cells, can autocrine GM-CSF after the adenovirus vector coding the GM-CSF infects the monocytes, does not need to add exogenous GM-CSF, can differentiate the monocytes into the dendritic cells, greatly simplifies experimental operation and culture time, and can obtain a therapeutic dendritic cell cancer vaccine within 36-48 hours, so that the time consumption is short, the cost is very low, and the method is suitable for popularization and application;

5) the recombinant adenovirus vector constructed by the invention also encodes CD40L gene, CD40L is a strong inducer for dendritic cell maturation, can promote dendritic cell maturation, up-regulate the expression of costimulatory molecules of dendritic cells, provide a second signal for T cells, and promote and enhance the function of specific CTL;

6. the recombinant adenovirus vector constructed by the invention also encodes 4-1BBL, can express 4-1BBL after infecting monocytes, interacts with 4-1BB of T cells, and promotes the functions of the T cells.

Drawings

FIG. 1 is a map of pDC 5232X 2A plasmid;

FIG. 2 is a schematic diagram showing the structure of an open reading frame formed by the tandem connection of three gene GM-CSF, 4-1BBL and CD40L sequences and a 2A peptide sequence in a pDC523GM-CSF/4-1BBL/CD40L plasmid;

FIG. 3 is a schematic structural diagram of fusion gene tWT 1/Survivin/LAMP-1;

FIG. 4 is a spectrum of pGBC-IRES-WSL plasmid;

FIG. 5 is a diagram showing the gene expression and function identification of Ad5F35GBC/WSL after infection of HEK293T cells, wherein A is an ELISA staining pattern of GM-CSF in supernatant after Ad5F35GBC/WSL infects HEK293T cells for 24 hours; b is a flow cytometer detection peak diagram after HEK293T cells are infected by Ad5F35GBC/WSL for 24 hours;

FIG. 6 is a gel identification chart of tWT1/Survivin/LAMP-1 fusion protein after infection of HEK293T cells by Ad5F35GBC/WSL, wherein A is a gel identification chart detected by WT1 antibody; b is a gel identification picture detected by a Survivin antibody;

FIG. 7 is a graph showing the effect of Ad5F35GBC/WSL on inducing differentiation of human peripheral blood mononuclear cells into dendritic cells after infection with human peripheral blood mononuclear cells, wherein panel A is an ELISA staining pattern of GM-CSF in the supernatant after Ad5F35GBC/WSL infection with human peripheral blood mononuclear cells; b is a microscope observation picture of Ad5F35GBC/WSL infected human peripheral blood mononuclear cells;

FIG. 8 is a graph showing the effect of Ad5F35GBC/WSL on the maturation of dendritic cells induced by the enzyme, wherein A is a peak diagram of gene expression in cells detected by flow cytometry; b is a diagram of cell co-stimulatory molecule expression peak detected by a flow cytometer.

Detailed Description

The invention aims to provide a therapeutic Dendritic Cell (DCs) cancer vaccine in a therapeutic cancer vaccine and a preparation method thereof, and the therapeutic dendritic cell cancer vaccine can induce an organism to generate antigen-specific T lymphocyte reaction after infecting human peripheral blood-derived mononuclear cells so as to play an anti-tumor role, thereby being used for treating various blood system tumors and solid tumors.

To achieve the above objectives, the present inventors fully consider that a therapeutic Dendritic Cell (DCs) cancer vaccine must be able to simultaneously provide three types of signals for T cell activation (tumor antigens, costimulatory molecules to induce DC maturation and activation, cytokines to induce anti-tumor immune responses), and also be able to induceFirstly, a recombinant plasmid capable of simultaneously expressing a plurality of target genes is provided on the basis of high-efficiency anti-tumor immune cell reaction, simple and quick operation and low preparation cost, wherein the recombinant plasmid is based on a shuttle plasmid pDC511 and is derived from pcDNA3.1(-) -myc-His B plasmid (purchased from Invitrogen in USA)^TMCompany), and synthesizing a polypeptide sequence containing 2 peptides of 2A and enzyme cutting sites to replace multiple cloning sites in the hCMVP-pA expression cassette, thereby constructing and obtaining an optimized shuttle plasmid (pDC 5232X 2A). Then inserting a plurality of target genes into the optimized shuttle plasmid, wherein different target genes are connected in series at intervals by 2A peptide sequences, a plurality of target genes are connected in series at intervals by a plurality of 2A peptides to form an open reading frame, a recombinant plasmid containing the target genes is obtained, and a tumor-related antigen gene sequence connected with a nucleic acid entry site (IRES) is further inserted, namely the shuttle plasmid capable of expressing a plurality of target genes and tumor-related antigen genes simultaneously. And then co-transfecting the shuttle plasmid containing the target gene and a skeleton plasmid pBHGfrt (del) E1,3Cre or another skeleton plasmid pBHGfrt (del) E1,3Cre (5/F35) derived from the skeleton plasmid into an HEK293T cell by a calcium phosphate transfection method, wherein the shuttle plasmid containing the target gene and the skeleton plasmid are subjected to homologous recombination mediated by Cre recombinase in the HEK293T cell, and a target gene expression cassette of the shuttle plasmid is recombined into an E1 deletion region of the skeleton plasmid, so that a complete adenovirus vector containing the target gene can be formed, and the HEK293T cell can be pathologically diseased to form a hollow spot (Plague), wherein the process is usually 10-14 days. After the cells form plaques, the cells are selected and further infected with HEK293T cells, the required oncolytic adenovirus (namely recombinant adenovirus) can be selected and amplified after several rounds, and the therapeutic DCs cancer vaccine can be obtained by infecting the recombinant adenovirus with mononuclear cells.

The present invention will be described in detail below with reference to specific examples.

The percentage concentrations stated in the following examples are mass/volume (W/V) percentage concentrations or volume/volume (V/V) percentage concentrations, unless otherwise specified.

The primers, DNA sequence synthesis and DNA sequence determination were all performed by Biotechnology engineering, Inc.

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

The present invention is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are given, which will help understanding of the present invention, but the present invention is not limited to the following examples.

Example 1 engineering of shuttle plasmid

This example is a suitable construction of an adenovirus shuttle plasmid encoding multiple genes, AdMAX^TMAnother shuttle plasmid pDC 5232X 2A is constructed on the basis of pDC511 shuttle plasmid (purchased from MicroBix, Canada) in the system, and specifically comprises the following steps:

1.1, synthesizing a pair of primers, wherein an upstream primer is 5'-gcg cta gct agctca ata ttg gcc att agc c-3' (Nhe I site is introduced at the end of SEQ ID NO:1 and 5 '), a downstream primer is 5'-gaa gat cta taa ctt cgt ata atg tat gct ata cga agt tat cga tcg agc cat aga gcc c-3'(BglII site and LoxP sequence are introduced at the end of SEQ ID NO:2 and 5'), and pcDNA3.1(-) -myc-His B plasmid (Invitrogen)^TM) Performing PCR amplification by using high-fidelity Q5 PCR enzyme (NEB) as a template under the conditions of 98 ℃ for 10s, 72 ℃ for 20s and 72 ℃ for 60s for 30 cycles to obtain a 1180bp PCR fragment (namely a CMVp-pA expression cassette) by amplification, carrying out double digestion on the fragment by using Nhe I and BglII, recovering the digested fragment (digested fragment 1), carrying out double digestion on a pDC511 shuttle plasmid by using Xba I and BamH I, and carrying out double digestion on the pDC511 shuttle plasmidAnd recovering a restriction enzyme large fragment (restriction enzyme fragment 2), connecting the restriction enzyme fragment 1 and the restriction enzyme fragment 2 in the presence of ligase, then transforming to DH5 alpha competence, selecting, cloning, performing restriction enzyme digestion, identifying a PCR fragment, inserting the PCR fragment into a pDC511 plasmid, then sequencing, confirming that the CMVp expression cassette obtained by PCR has a correct sequence, and naming the obtained plasmid as pDC 523.

1.2, synthesizing an oligonucleotide fragment comprising two 2A peptide sequences (wherein the 2A peptide sequence can be any one or a combination of two of E2A, F2A, T2A and P2A, the example uses T2A and P2A peptide sequences) and a multiple cloning site (the nucleotide sequence of the oligonucleotide fragment is shown as SEQ ID NO:3 in the sequence table), double-digesting the oligonucleotide fragment with Nhe I and Hind III, then recovering a small fragment (digested fragment 3), double-digesting the pDC523 plasmid obtained in the step 1.1 using Nhe I and Hind III, then recovering a large fragment (digested fragment 4), ligating the digested fragment 3 and the digested fragment 4 in the presence of a ligase, then transforming into DH5 alpha, picking, digesting, identifying the shuttle, inserting the oligonucleotide fragment into the pDC523, sequencing, naming the plasmid with correct sequence as pDC 5232X 2A plasmid, the nucleotide sequence of which is shown as SEQ ID NO:4, as shown in fig. 1, a spectrum of the pDC5232 × 2A plasmid used in the following example 2 for inserting a gene of interest is shown.

Example 2 cloning of WT1, Survivin, GM-CSF, CD40L, and 4-1BBL genes

This example is to insert the desired genes WT1, Survivin, GM-CSF, CD40L, and 4-1BBL into the pDC 5232X 2A plasmid obtained in the above example 1, and specifically includes the following steps:

2.1, extracting 5ml of peripheral blood of a healthy person, separating Peripheral Blood Mononuclear Cells (PBMCs) by using lymphocyte separating medium, stimulating the peripheral blood mononuclear cells for 12 hours by using LPS (10ng/ml), extracting total RNA of cells by using an RNA extraction kit (Qiagen), and synthesizing cDNA (named as PBMCs cDNA) by using a reverse transcription kit (Takara) by using the RNA as a template; extraction of 1X 10 with RNA extraction kit (Qiagen)⁶Total RNA of human K562 leukemia cells was used as a template to synthesize cDNA using a reverse transcription kit (Takara), which was designated as K562 cDNA.

2.2, 6 pairs of primers are designed in the step for amplifying the target gene and constructing related plasmids, and the method specifically comprises the following steps:

2.2.1, designing primers for amplifying GM-CSF gene, wherein an upstream primer is 5'-cta gct agc atg tgg ctg cag agc ctg ctg-3' (SEQ ID NO:5, Nhe I site is introduced at the 5 'end), a downstream primer is 5'-gga att cct cct gga ctg gct ccc agc ag-3'(EcoR I site is introduced at the 5' end), the PBMCs cDNA obtained in the step 2.1 is used as a template for PCR amplification, high-fidelity Q5 PCR enzyme (NEB) is used for amplification, the amplification conditions are 98 ℃ for 10s, 72 ℃ for 20s and 72 ℃ for 20s, 30 cycles, a PCR fragment with the size of 450bp is obtained by amplification, the restriction enzyme fragments are recovered by using Nhe I and EcoR I double restriction enzymes, the large fragment is recovered by using Nhe I and EcoR I double restriction enzymes for pDC 5232X 2A plasmid, the PCR fragment and the pDC 5232X 2A shuttle plasmid are connected, transforming to DH5 alpha competence, selecting clone enzyme digestion to identify PCR fragment and inserting into pDC 5232X 2A plasmid for sequencing, confirming that the fragment obtained by PCR is completely consistent with human GM-CSF sequence (GenBank: NM-000758, nucleotide sequence is shown as SEQ ID NO:7 in sequence table, corresponding amino acid sequence is shown as SEQ ID NO: 8), and naming the obtained plasmid as pDC523 GM-CSF;

2.2.2, designing primers for amplifying the 4-1BBL gene, wherein an upstream primer is 5'-gcg cga aga tct atg gaa tac gcc tct gac gc-3' (a BglII site is introduced at the 5 'end of SEQ ID NO: 9), a downstream primer is 5'-cgg gat cct tcc gac ctc ggt gaa gg-3'(a BamH I site is introduced at the 5' end of SEQ ID NO: 10), the PBMCs cDNA in the step 2.1 is used as a template for PCR amplification, high-fidelity Q5 PCR enzyme (NEB) is used for amplification, amplification conditions are that 98 ℃, 10 and 72 ℃ for 20s and 72 ℃, 30 cycles are carried out, a PCR fragment with the size of 770bp is obtained by amplification, the fragment is subjected to double enzyme digestion by BglII and BamH I, then an enzyme digestion plasmid (enzyme digestion fragment 7) is recovered, pDC523GM-CSF obtained in the step 2.2.1 is subjected to enzyme digestion by a BamH I site and dephosphorylation treatment, then an enzyme digestion fragment (enzyme digestion fragment 8) is recovered, the digested fragment 7 and the digested fragment 8 are connected in the presence of ligase, then are transformed to DH5 alpha competence, the PCR fragment is identified by picking cloning and enzyme digestion and is inserted into a pDC523GM-CSF plasmid for sequencing, the fragment obtained by PCR is confirmed to be completely consistent with a human 4-1BBL sequence (GenBank: NM-003811, the nucleotide sequence of the fragment is shown as SEQ ID NO:11, and the corresponding amino acid sequence is shown as SEQ ID NO: 12), and the obtained plasmid is named as pDC523GM-CSF/4-1 BBL.

2.2.3, designing primers for amplifying the CD40L gene, wherein an upstream primer is 5'-cgc gga tcc atg atc gaa aca tac aac c-3' (SEQ ID NO:13, a BamH I site is introduced at the 5 'end), a downstream primer is 5'-ccg ctc gag tca gag ttt gag taa gcc-3'(an Xho I site is introduced at the SEQ ID NO:14, the 5' end), the PBMCs cDNA in the step 2.1 is used as a template for PCR amplification, high-fidelity Q5 PCR enzyme (NEB) is used for amplification, the amplification conditions are 98 ℃ for 10s, 72 ℃ for 20s and 72 ℃ for 30s, 30 cycles are used for amplification, a PCR fragment with the size of 790bp is obtained, and the fragment is subjected to double enzyme digestion by BamH I and Xho I and then gel recovery of an enzyme digestion fragment (enzyme digestion fragment 9); the pDC523GM-CSF/4-1BBL plasmid obtained in the above step 2.2.2 was double-digested with Bgl II and Xho I to recover the large fragment (digested fragment 10), the digested fragment 9 and the digested fragment 10 were ligated in the presence of ligase, and then transformed into DH5 alpha competence, the PCR fragment was identified by picking, cloning, and digestion, and inserted into pDC523GM-CSF/4-1BBL plasmid, and then sequenced, confirming that the fragment obtained by PCR was completely identical to human CD40L sequence (GenBank: NM-000074, whose nucleotide sequence is shown in SEQ ID NO:15, and the corresponding amino acid sequence is shown in SEQ ID NO: 16), and the plasmid obtained was named pDC523GM-CSF/4-1BBL/CD40L, and the sequences of the three genes GM-CSF, 4-1BBL and CD40L were respectively connected in series by two 2A peptide sequences to form an open reading frame, wherein the three genes GM-CSF, 4-1BBL and CD 40B, The positions of 4-1BBL and CD40L can be interchanged (the sequence of the ligation used in this example is GM-CSF/T2A/4-1BBL/P2A/CD40L, the nucleotide sequence of which is shown in SEQ ID NO:17, and the corresponding amino acid sequence of which is shown in SEQ ID NO: 18), as shown in FIG. 2, a schematic diagram of the structure of the open reading frame is shown.

2.2.4, synthesizing an oligonucleotide fragment (the nucleotide sequence of which is shown in SEQ ID NO: 19), the sequence of which comprises the signal peptide sequence of the human LAMP-1 gene and the lysosome targeting sequence, cleaving the oligonucleotide fragment with Nco I and Hind III and then collecting a small fragment (cleaved fragment 11), cleaving the pmRNA IRES-EGFP plasmid (Tan X, Wan Y. enhanced protein expression by internal ribosome entry site-drive mRNA translation as a novel approach for in vitro cloning of recombinant cells with human immunoglobulin 2008 Jan; 69(1):32-40.) and also cleaving with Nco I and Hind III and then collecting a large fragment (cleaved fragment 12), ligating the cleaved fragment 11 with the cleaved fragment 12, and identifying the correct one of the RNA-LAMP-1-PMIRES.

2.2.5, designing primers for amplifying tWT1 gene, wherein an upstream primer is 5'-acg cgt cga cgg ctc cga cgt gcg gga cc-3' (SEQ ID NO:20, Sal I site is introduced into 5 'end), a downstream primer is 5'-gcg cgc gcg cgc tct aga ctg aat gcc tct gaa gac acc g-3'(SEQ ID NO:21, Xba I site is introduced into 5' end), the K562 cDNA obtained in the step 2.1 is used as a template for PCR amplification, high fidelity Q5 PCR enzyme (NEB) is used for amplification, amplification conditions are 98 ℃ for 10s, 72 ℃ for 20s and 72 ℃ for 30s, 30 cycles are carried out, a PCR fragment with the size of 900bp is obtained by amplification, the fragment is subjected to double digestion with Sal I and Xba I, then an enzyme digestion fragment (enzyme digestion fragment 13) is recovered, pmRNA IRES-LAMP-1 plasmid obtained in the 2.2.4 is also subjected to double digestion with Sal I and Xba I, then a large fragment (enzyme digestion fragment 14) is recovered, the restriction fragment 13 and the restriction fragment 14 are connected, then the obtained product is transformed to DH5 alpha competence, the PCR fragment is picked, cloned, restricted and identified to be inserted into pmRNA IRES-LAMP-1 plasmid and then sequenced, the fragment obtained by PCR is proved to be completely consistent with the amino acid sequence 70-366 (GenBank: NM-024426, the nucleotide sequence is shown as SEQ ID NO:22, the corresponding amino acid sequence is shown as SEQ ID NO: 23) of the WT1, the fragment is a truncated WT1 gene (tWT1), the potential oncogenic site sequence in the WT1 gene is deleted, and the obtained plasmid is named pmRNA IRES-tWT 1/LAMP-1.

2.2.6, designing primers for amplifying Survivin genes, wherein an upstream primer is 5'-gga cta gtg gtg ccc cga cgt tgc ccc-3' (SpeI site is introduced at the 5 'end of SEQ ID NO: 24), a downstream primer is 5'-gcg cga aga tct atc cat ggc agc cag ctg ctc g-3'(BglII site is introduced at the 5' end of SEQ ID NO: 25), performing PCR amplification by taking the K562 cDNA obtained in the step 2.1 as a template, amplifying by using high-fidelity Q5 PCR enzyme (NEB) under conditions of 98 ℃ 10s, 72 ℃ 20s and 72 ℃ 20s for 30 cycles to obtain a PCR fragment with the size of 440bp, performing double digestion on the fragment by using SpeI and BglII, recovering a digested fragment (digested fragment 15), performing double digestion on the pmRNA IRES-tWT 1/351 plasmid obtained in the step 2.2.5 by using Xba I and BglII, and recovering a large fragment (digested fragment 16), connecting the enzyme-cleaved fragment 15 and the enzyme-cleaved fragment 16, then transforming to DH5 alpha competence, selecting clone, enzyme-cleaved and identifying the PCR fragment, inserting into pmRNA IRES-tWT/LAMP-1 plasmid, sequencing, confirming that the fragment obtained by PCR is completely consistent with the full-length amino acid sequence of human Survivin (GenBank: NM-001168, the nucleotide sequence of which is shown as SEQ ID NO:26, and the corresponding amino acid sequence is shown as SEQ ID NO: 27), naming the obtained plasmid as pmRNA IRES-tWT1/Survivin/LAMP-1, which contains internal nucleic acid entry site IRES sequence (the nucleotide sequence of which is shown as SEQ ID NO:28, and is a guide sequence for translation of mRNA into protein) and fusion gene tWT1/Survivin/LAMP-1 sequence of targeting lysosome pathway (the nucleotide sequence is shown as SEQ ID NO:29, and the corresponding amino acid sequence is shown as SEQ ID NO: 30), the introduction of IRES sequence is helpful for the recombinant adenovirus to co-express GM-CSF, 4-1BBL and CD40L and simultaneously effectively express tumor associated antigen, thereby fully expressing four target genes encoded by the recombinant adenovirus. The structural diagram of the fusion gene tWT1/Survivin/LAMP-1 sequence is shown in FIG. 3.

2.2.7 designed to amplify IRES-tWT1/Survivin/LAMP-1 sequence, with an upstream primer of 5'-ccg ctc gag att ccg ccc ccc ccc ccc-3' (SEQ ID NO:31, XhoI site introduced at 5 'end), a downstream primer of 5'-gcg cgc gcc cgg ggt taa ctt aga tag tct ggt agc ctg cg-3'(SEQ ID NO:32, Hpa I site introduced at 5' end), the pmRNA IRES-tWT1/Survivin/LAMP-1 obtained in the above step 2.2.6 as a template, carrying out PCR amplification with high fidelity Q5 PCR enzyme (NEB) under conditions of 98 ℃ 10s, 72 ℃ 20s, 72 ℃ 60s, 30 cycles, obtaining a PCR fragment of 2130bp size, carrying out double enzyme digestion of the fragment with XhoI and Hpa I, recovering the enzyme digested fragment (enzyme digested fragment 17), the pDC523GM-CSF/4-1BBL/CD40L plasmid obtained in the step 2.2.3 is subjected to double enzyme digestion by using Xho I and Hpa I to recover a large fragment (an enzyme digestion fragment 18), the enzyme digestion fragment 17 and the enzyme digestion fragment 18 are connected and then transformed to DH5 alpha competence, the PCR fragment is selected, cloned, enzyme digestion and identified to be inserted into the pDC523GM-CSF/4-1BBL/CD40L plasmid and then sequenced, the recombinant plasmid with correct sequencing is named as shuttle plasmid pDC523GM-CSF/4-1BBL/CD 40L-IRES-tWT 1/Survivin/1, the shuttle plasmid pGBC-IRES-WSL is abbreviated as shuttle plasmid pGBC, the nucleotide sequence is SEQ ID NO:33, for the construction of recombinant adenoviruses and the preparation of therapeutic DCs cancer vaccines in the following examples, as shown in FIG. 4, the spectrum of the recombinant plasmid pGBC-IRES-WSL is shown.

The shuttle plasmid pGBC-IRES-WSL constructed in this example is inserted with tWT1/Survivin/LAMP-1 fusion gene as tumor-associated antigen, and it will be understood by those skilled in the art that the expression gene of the tumor-associated antigen inserted in the shuttle plasmid pGBC-IRES-WSL constructed in this example may also be any of tWT1, Survivin, fusion gene tWT1/Survivin (the nucleotide sequence of which is shown in SEQ ID NO:34, and the corresponding amino acid sequence of which is shown in SEQ ID NO: 35), MUC1, LMP, HPV E6E 7, EGFRvIII, HER-2/neu, MAGE A3, p53(nonmutant and mutant), NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras, gp100, Proteinase3(PR1), bcr-abl, Sargasl, introversion, PSA 2, hTAT-E, hTAT, hAATE, and hTAT, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, android receiver, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe (a), CYP1B1, PLAC1 and GM 3.

Example 3 construction of recombinant adenovirus vectors capable of simultaneously expressing multiple genes

This embodiment is as AdMax^TMThe Cre recombinant adenovirus system instructions perform the construction of recombinant adenovirus expressing the encoded polygenes. The shuttle Plasmid pGBC-IRES-WSL obtained in example 2 was extracted with a Plasmid extraction Kit (Endofree Plasmid Midi Kit, QIAGEN), the ratio of 260/280 was measured to be about 1.9, and the Plasmid concentration was adjusted to 1. mu.g/. mu.l; pBGHfrt delta E1,3Cre (for constructing adenovirus vector type 5) (purchased from Microbix Canada) and pBGHfrt delta E1,3Cre (5/F35) (for constructing adenovirus vector type 5/F35, purchased from Beijing Benyuan Zhengyang Gene Co., Ltd.) adenovirus backbone plasmids were extracted with Plasmid Large extract Kit (EndoFree Plasmid Maxi Kit, QIAGEN), the ratio of 260/280 was determined to be about 1.9, and the Plasmid concentration was adjusted to 1. mu.g/. mu.l. Passage of low passage (less than 10) HEK293T cells (from Microbix, Canada) to 60mm³In the culture dish of (1), the culture dish is,the shuttle plasmid pGBC-IRES-WSL and the backbone plasmid pBGHfrt delta E1,3Cre or pBGHfrt delta E1,3Cre (5/F35) were precipitated by calcium phosphate precipitation (CalPhos) until the cells fused to 60-70%^TMMammalian Transfection Kit, Clonech). The transfection system is shown in table 1 below, and after the transfection system is prepared, the transfection system is added into HEK293T cells after incubation for 10 minutes.

Table 1: transfection system

After transfection, a culture solution containing agaropectin was prepared according to the system shown in table 2 below.

Table 2: culture solution system containing agaropectin

After the solution A in the table 2 is boiled and dissolved by a microwave oven, the solution A is put into a water bath at 44 ℃, and 30ml of the solution A is mixed with the solution B placed at 44 ℃ after the temperature is reduced to 44 ℃; after the transfection of the plasmid, the culture solution in each dish was aspirated, and 15ml of the A/B mixture was added to each dish.

Each dish was placed at 37 ℃ in 5% CO₂Culturing in incubator, forming solid culture medium containing agar, observing cell condition in culture dish periodically, forming plaques (plagues) about 10-14 days, picking out plaques, further amplifying in HEK293T cells, extracting virus DNA from partial cells, performing PCR identification, correctly cloning and naming Ad5GBC/WSL (type 5) or Ad5F35GBC/WSL (type 5/F35), amplifying for 2 or 3 rounds, collecting HEK293T cells, freeze-thawing, and using adenovirus purification kit (Virabind)^TMTwo types of recombinant Adenovirus [ i.e., Ad5GBC/WSL (type 5) or Ad5F35GBC/WSL (type 5/F35) were obtained after Adenovirus Purification by Adenoviral Purification Kit, Cell Biolabs, Inc]The recombinant adenovirus is used to infect peripheral blood mononuclear cell to obtain the cancer vaccine of DCs. The following examples are for both typesThe recombinant adenovirus is expressed and functionally identified.

Example 4 Ad5F35GBC/WSL infection of HEK293T cells GM-CSF, 4-1BBL and CD40L expression identification

This example utilizes the purified adenovirus Ad5F35GBC/WSL obtained in the above example 3 to infect HEK293T cells for expression and functional identification, and specifically includes the following steps:

4.1 transfer HEK293T cells to 6-well plates (two duplicate wells) at a cell density of 1X 10⁶3 ml/well, after the cells were attached, Ad5F35GBC/WSL was added at a virus titer of 100pfu/cell, after further culturing for 24 hours, the supernatant was collected and assayed for GM-CSF expression by ELISA, and the cells were collected and assayed for 4-1BBL and CD40L by flow cytometry.

4.2, the supernatant collected in the step 4.1 is detected by ELISA (Shenzhendake) after being diluted by 10 times and 100 times according to the stock solution, and the result is shown in A in figure 5, and as can be seen, after the HEK293T cell is infected by Ad5F35GBC/WSL for 24 hours, the HEK293T cell can secrete a large amount of GM-CSF, and the secretion can reach 10-15 ng/1 × 10⁶cells/ml/24h。

4.3, digesting the cells collected in the step 4.1 by 0.25% pancreatin + 0.01% EDTA to form a single cell suspension, washing the single cell suspension twice by PBS, staining the single cell suspension by human 4-1BBL-PE (BD) and CD40L-PE (BD), and detecting the single cell suspension by a flow cytometer, wherein the result is shown in a panel B in figure 5, and the result shows that the human 4-1BBL and the CD40L can be effectively expressed after HEK293T cells are infected by Ad5F35GBC/WSL for 24 hours.

The expression of GM-CSF, 4-1BBL and CD40L was identified following infection of HEK293T cells with the purified Ad5GBC/WSL obtained in example 3 above, according to the above procedure, with results similar to those obtained with the Ad5F35GBC/WSL infected HEK293T cells described above.

Example 5 detection of expression of tWT1/Survivin/LAMP-1 fusion protein following infection of HEK293T cells with Ad5F35GBC/WSL

In this example, the purified adenovirus Ad5F35GBC/WSL obtained in the above example 3 was used to infect HEK293T cells, and then Western blot was used to detect the expression of tWT1/Survivin/LAMP-1 fusion protein, specifically including the following steps:

5.1, 1X 10⁶The HEK293T cells are transferred to a 6-well plate, Ad5F35GBC/WSL is added to the HEK293T cells according to the virus titer of 100pfu/cell, and the HEK293T cells are collected after culturing for 48 hours to carry out Western blot detection on the expression of tWT1/Survivin/LAMP-1 fusion protein, wherein the detection is carried out by using WT1 and Survivin antibodies respectively.

5.2, WT1 antibody was obtained using R & D rabbit polyclonal antibody (AF5729), and as shown in panel A of FIG. 6, it was found that HEK293T cells infected with Ad5F35GBC/WSL (lane B) showed a band of about 55kDa, which was consistent with the predicted molecular weight of tWT1/Survivin/LAMP-1 fusion protein (55kDa), while HEK293T cells not infected with virus showed no band (lane A). To demonstrate the reliability of the WT1 antibody, we used K562 cells as a positive control, K562 cells expressing wild-type WT1, and Western Blot detected a specific band (43kDa) with a smaller molecular weight than the tWT1/Survivin/LAMP-1 fusion protein (lane C).

5.3, Survivin antibody was R & D rabbit polyclonal antibody (AF886), and as shown in panel B of FIG. 6, HEK293T cells infected with Ad5F35GBC/WSL (lane B) showed two bands, one of which was wild-type Survivin protein (molecular weight about 19kDa) and the other of which was 55kDa, which was consistent with the predicted molecular weight of tWT1/Survivin/LAMP-1 fusion protein, while HEK293T cells not infected with virus (lane A) also showed two bands, one of which was wild-type Survivin protein and the other of which was unknown at about 51kDa (the pictures shown in the antibody specification also show this band).

The expression of the tWT1/Survivin/LAMP-1 fusion protein was identified after infection of HEK293T cells with purified Ad5GBC/WSL obtained in example 3 above, according to the above method, and results similar to those obtained when HEK293T cells were infected with Ad5F35GBC/WSL as described above were obtained.

Example 6 Ad5F35GBC/WSL Induction of differentiation of human peripheral blood mononuclear cells into dendritic cells after infection

In this example, the purified adenovirus Ad5F35GBC/WSL obtained in example 3 was used to infect human peripheral blood mononuclear cells, and the differentiation of the human peripheral blood mononuclear cells was observed, which specifically includes the following steps:

6.1 collecting peripheral blood of healthy human 10ml, separating mononuclear cells (PBMCs) with lymphocyte separation medium at 1X 10⁶The cells at/ml were inoculated into a culture dish, Ad5F35GBC/WS was added to monocytes at a viral titer of 100pfu/cell, culture supernatants were collected at 36 hours and 72 hours of culture, respectively, and the content of GM-CSF was measured by ELISA, and as a result, as shown in FIG. 7, panel A, it was observed that a large amount of GM-CSF was still secreted from 36 hours to 72 hours after infection of monocytes by Ad5F35 GBC/WS.

6.2 when monocytes were infected with Ad5F35GBC/WS for 36 hours, they were observed under a microscope, as shown in B panel of FIG. 7, where the morphology of monocytes gradually appeared to protrude pseudopodia and dendrites and grew in clusters, indicating the differentiation of monocytes into dendritic cells.

Differentiation of the cells into dendritic cells after infection of human peripheral blood mononuclear cells with the purified Ad5GBC/WSL obtained in example 3 was examined as described above, and differentiation of monocytes into dendritic cells was obtained in a manner similar to the above Ad5F35GBC/WSL infected human peripheral blood mononuclear cells.

Example 7 Ad5F35GBC/WSL Induction of dendritic cell maturation

In this example, the purified adenovirus Ad5F35GBC/WSL obtained in example 3 was used to infect human peripheral blood mononuclear cells, and the maturation of dendritic cells induced by Ad5F35GBC/WSL was observed, which specifically includes the following steps:

7.1 collecting peripheral blood of healthy human 10ml, separating mononuclear cells (PBMCs) with lymphocyte separation medium at 1X 10⁶The cells with the cell density of/ml are inoculated into a culture dish, Ad5F35GBC/WSL is added into the monocytes according to the virus titer of 100pfu/cell, the cells are collected after 36 hours of culture respectively for the identification of cell gene expression, and the result is shown in A frame in figure 8, the monocytes obviously express 4-1BBL and CD40L, and the monocytes still present the CD14 phenotype.

7.2, after 36 hours of infection of monocytes by Ad5F35GBC/WSL, flow cytometric analysis is carried out on the phenotype of the co-stimulatory molecules, and the result is shown in B panel in FIG. 8, CD40, CD80 and CD83 are obviously up-regulated, while CD86 and HLA-DR are highly expressed, and are not obviously changed after infection of Ad5F35GBC/WSL, which indicates that after infection of monocytes by Ad5F35GBC/WSL, in addition to induction of differentiation to dendritic cells, the expression of various co-stimulatory molecules is up-regulated, the maturation of dendritic cells is induced, and the mature dendritic cells can process and process the expressed tumor-associated antigens, and present the antigen information to T cells, so that the initial T cells can be effectively activated, and are in the central link of initiating, regulating and maintaining immune response.

The maturation of dendritic cells induced by infection of human peripheral blood mononuclear cells with the purified Ad5GBC/WSL obtained in example 3 was examined as described above, and the results were similar to those obtained when human peripheral blood mononuclear cells were infected with Ad5F35GBC/WSL, i.e., the expression of various co-stimulatory molecules was upregulated after infection of human peripheral blood mononuclear cells with Ad5GBC/WSL, thereby inducing dendritic cell maturation.

In summary, the present invention provides a recombinant adenovirus capable of simultaneously expressing multiple target genes, wherein tumor-associated antigens, granulocyte-megakaryocyte colony stimulating factor (GM-CSF), CD40L, and 4-1BBL can be simultaneously obtained in a single expression, wherein after the recombinant adenovirus is infected with human peripheral blood mononuclear cells, the GM-CSF expressed by the recombinant adenovirus can be used to induce the differentiation of the monocytes into dendritic cells, and the dendritic cells generated by the induced differentiation can be processed, treated with the expressed tumor-associated antigens, and then presented with antigen information to T cells; and the expressed CD40L induces DCs to mature and the expressed 4-1BBL is helpful to stimulate the activation of T cells to realize the optimal T cell activation effect, thereby obtaining a therapeutic Dendritic Cell (DCs) cancer vaccine which can exert anti-tumor effect by inducing the body to produce antigen-specific T lymphocytes, and further can treat various hematological and solid tumors, such as WT1 and/or Survivin-positive hematological tumors including but not limited to acute myeloid leukemia, chronic myeloid leukemia and high risk myelodysplastic syndrome (MDS), and the like, and including but not limited to lung cancer, liver cancer, cholangiocarcinoma, stomach cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, renal cancer, bladder cancer, malignant tumor, and the likeWT1 and/or Survivin positive solid tumors such as gliomas, head and neck malignancies, and various types of malignant sarcomas. Therefore, the recombinant adenovirus and the therapeutic Dendritic Cell (DCs) cancer vaccine and the like provided by the invention can be used for preparing medicines for treating the hematological tumors and solid tumors. When therapeutic Dendritic Cell (DCs) cancer vaccines are administered, intradermal injection and intravenous injection may be used, wherein subcutaneous injection is 1-3 × 10⁷Resuspending the dendritic cells with 1ml of physiological saline, and injecting the dendritic cells into the skin at multiple points (6-8 points) near the groin and the armpit, wherein each part is injected with about 150 mu l; 1-3 × 10 for intravenous injection⁷The dendritic cells were resuspended in 100ml of normal saline and the infusion was completed in about half an hour.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Shenzhen Hao Shi Biotech limited

<120> a therapeutic dendritic cell cancer vaccine and uses thereof

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ttcatcaata atatacctta ttttggattg aagccaatat gataatgagg gggtggagtt 60

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tgtgcgccgg tgtacacagg aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt 240

aaatttgggc gtaaccgagt aagatttggc cattttcgcg ggaaaactga ataagaggaa 300

gtgaaatctg aataattttg tgttactcat agcgcgtaat atttgtctag ggccgcgggg 360

actttgaccg tttacgtgga gactcgccca ggtgtttttc tcaggtgttt tccgcgttcc 420

gggtcaaagt tggcgtttta ttattatagt cagctctaga tcaatattgg ccattagcca 480

tattattcat tggttatata gcataaatca atattggcta ttggccattg catacgttgt 540

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atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 720

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tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 840

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accaaaatca acgggacttt ccaaaatgtc gtaacaactg cgatcgcccg ccccgttgac 1140

gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa 1200

ccgtcagatc actagaagct ttattgcggt agtttatcac agttaaattg ctaacgcagt 1260

cagtgcttct gacacaacag tctcgaactt aagctgcagt gactctctta aggtagcctt 1320

gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag 1380

gagaccaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac 1440

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ccaatttcag tttgctcaag caggctggtg atgtggagga gaatcccggt ccgagatcta 1740

ctagttaact cgaggtttaa accgctgatc agcctcgact gtgccttcta gttgccagcc 1800

atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 1860

cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 1920

ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 1980

tggggatgcg gtgggctcta tggctcgatc gagatccagt taggggcggg actatggttg 2040

ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc 2100

cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc 2160

ctggggactt tccacaccct aactgacaca cattccagat ccataacttc gtatagcata 2220

cattatacga agttatgatc cgaagttcct attcttacta gagtatagga acttcgacta 2280

tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag 2340

cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt 2400

cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca 2460

ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggccctg cattaatgaa 2520

tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 2580

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 2640

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 2700

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 2760

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 2820

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 2880

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat 2940

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 3000

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 3060

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 3120

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 3180

gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 3240

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 3300

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 3360

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 3420

ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 3480

atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 3540

tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 3600

gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 3660

ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 3720

caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 3780

cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 3840

cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 3900

cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 3960

agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 4020

tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 4080

agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac 4140

atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 4200

ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 4260

cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 4320

caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 4380

attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 4440

agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct 4500

aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcact ctaggcaaaa 4560

tagcaccctc ccgctccaga acaacataca gcgcttcaca gcggcagcct aacagtcagc 4620

cttaccagta aaaaagaaaa cctattaaaa aaacaccact cgacacggca ccagctcaat 4680

cagtcacagt gtaaaaaagg gccaagtgca gagcgagtat atataggact aaaaaatgac 4740

gtaacggtta aagtccacaa aaaacaccca gaaaaccgca cgcgaaccta cgcccagaaa 4800

cgaaagccaa aaaacccaca acttcctcaa atcgtcactt ccgttttccc acgttacgta 4860

acttcccatt ttaagaaaac tacaattccc aacacataca agttactccg ccctaaaacc 4920

tacgtcaccc gccccgttcc cacgccccgc gccacgtcac aaactccacc ccctcattat 4980

catattggct tcaatccaaa ataaggtata 5010

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctagctagca tgtggctgca gagcctgctg 30

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggaattcctc ctggactggc tcccagcag 29

<210> 7

<211> 435

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc 60

cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg 120

cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc 180

tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag 240

cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac 300

tacaagcagc actgccctcc aaccccggaa acttcctgtg caacccagat tatcaccttt 360

gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420

ccagtccagg agtga 435

<210> 8

<211> 144

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile

1 5 10 15

Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His

20 25 30

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

35 40 45

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

50 55 60

Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys

65 70 75 80

Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met

85 90 95

Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser

100 105 110

Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys

115 120 125

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

130 135 140

<210> 9

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gcgcgaagat ctatggaata cgcctctgac gc 32

<210> 10

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cgggatcctt ccgacctcgg tgaagg 26

<210> 11

<211> 765

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atggaatacg cctctgacgc ttcactggac cccgaagccc cgtggcctcc cgcgccccgc 60

gctcgcgcct gccgcgtact gccttgggcc ctggtcgcgg ggctgctgct gctgctgctg 120

ctcgctgccg cctgcgccgt cttcctcgcc tgcccctggg ccgtgtccgg ggctcgcgcc 180

tcgcccggct ccgcggccag cccgagactc cgcgagggtc ccgagctttc gcccgacgat 240

cccgccggcc tcttggacct gcggcagggc atgtttgcgc agctggtggc ccaaaatgtt 300

ctgctgatcg atgggcccct gagctggtac agtgacccag gcctggcagg cgtgtccctg 360

acggggggcc tgagctacaa agaggacacg aaggagctgg tggtggccaa ggctggagtc 420

tactatgtct tctttcaact agagctgcgg cgcgtggtgg ccggcgaggg ctcaggctcc 480

gtttcacttg cgctgcacct gcagccactg cgctctgctg ctggggccgc cgccctggct 540

ttgaccgtgg acctgccacc cgcctcctcc gaggctcgga actcggcctt cggtttccag 600

ggccgcttgc tgcacctgag tgccggccag cgcctgggcg tccatcttca cactgaggcc 660

agggcacgcc atgcctggca gcttacccag ggcgccacag tcttgggact cttccgggtg 720

acccccgaaa tcccagccgg actcccttca ccgaggtcgg aataa 765

<210> 12

<211> 254

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro

1 5 10 15

Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val

20 25 30

Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe

35 40 45

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

50 55 60

Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp

65 70 75 80

Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val

85 90 95

Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp

100 105 110

Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu

115 120 125

Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe

130 135 140

Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser

145 150 155 160

Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala

165 170 175

Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala

180 185 190

Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala

195 200 205

Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His

210 215 220

Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val

225 230 235 240

Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

245 250

<210> 13

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cgcggatcca tgatcgaaac atacaacc 28

<210> 14

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ccgctcgagt cagagtttga gtaagcc 27

<210> 15

<211> 786

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgatcgaaa catacaacca aacttctccc cgatctgcgg ccactggact gcccatcagc 60

atgaaaattt ttatgtattt acttactgtt tttcttatca cccagatgat tgggtcagca 120

ctttttgctg tgtatcttca tagaaggttg gacaagatag aagatgaaag gaatcttcat 180

gaagattttg tattcatgaa aacgatacag agatgcaaca caggagaaag atccttatcc 240

ttactgaact gtgaggagat taaaagccag tttgaaggct ttgtgaagga tataatgtta 300

aacaaagagg agacgaagaa agaaaacagc tttgaaatgc aaaaaggtga tcagaatcct 360

caaattgcgg cacatgtcat aagtgaggcc agcagtaaaa caacatctgt gttacagtgg 420

gctgaaaaag gatactacac catgagcaac aacttggtaa ccctggaaaa tgggaaacag 480

ctgaccgtta aaagacaagg actctattat atctatgccc aagtcacctt ctgttccaat 540

cgggaagctt cgagtcaagc tccatttata gccagcctct gcctaaagtc ccccggtaga 600

ttcgagagaa tcttactcag agctgcaaat acccacagtt ccgccaaacc ttgcgggcaa 660

caatccattc acttgggagg agtatttgaa ttgcaaccag gtgcttcggt gtttgtcaat 720

gtgactgatc caagccaagt gagccatggc actggcttca cgtcctttgg cttactcaaa 780

ctctga 786

<210> 16

<211> 261

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly

1 5 10 15

Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu

20 25 30

Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg

35 40 45

Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val

50 55 60

Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser

65 70 75 80

Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys

85 90 95

Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu

100 105 110

Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser

115 120 125

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

130 135 140

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

145 150 155 160

Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr

165 170 175

Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser

180 185 190

Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala

195 200 205

Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His

210 215 220

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

225 230 235 240

Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe

245 250 255

Gly Leu Leu Lys Leu

260

<210> 17

<211> 2163

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc 60

cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg 120

cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc 180

tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag 240

cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac 300

tacaagcagc actgccctcc aaccccggaa acttcctgtg caacccagac tatcaccttt 360

gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420

ccagtccagg aggaattccg agccaaacga agcggctctg gagaaggccg tggatctctg 480

ctgacctgcg gagacgtaga ggagaatcca gggcccggat ctatggaata cgcctctgac 540

gcttcactgg accccgaagc cccgtggcct cccgcgcccc gcgctcgcgc ctgccgcgta 600

ctgccttggg ccctggtcgc ggggctgctg ctgctgctgc tgctcgctgc cgcctgcgcc 660

gtcttcctcg cctgcccctg ggccgtgtcc ggggctcgcg cctcgcccgg ctccgcggcc 720

agcccgagac tccgcgaggg tcccgagctt tcgcccgacg atcccgccgg cctcttggac 780

ctgcggcagg gcatgtttgc gcagctggtg gcccaaaatg ttctgctgat cgatgggccc 840

ctgagctggt acagtgaccc aggcctggca ggcgtgtccc tgacgggggg cctgagctac 900

aaagaggaca cgaaggagct ggtggtggcc aaggctggag tctactatgt cttctttcaa 960

ctagagctgc ggcgcgtggt ggccggcgag ggctcaggct ccgtttcact tgcgctgcac 1020

ctgcagccac tgcgctctgc tgctggggcc gccgccctgg ctttgaccgt ggacctgcca 1080

cccgcctcct ccgaggctcg gaactcggcc ttcggtttcc agggccgctt gctgcacctg 1140

agtgccggcc agcgcctggg cgtccatctt cacactgagg ccagggcacg ccatgcctgg 1200

cagcttaccc agggcgccac agtcttggga ctcttccggg tgacccccga aatcccagcc 1260

ggactccctt caccgaggtc ggaaggatcc agagctaaga ggtcgggttc cggcgccacc 1320

aatttcagtt tgctcaagca ggctggtgat gtggaggaga atcccggtcc gagatccatg 1380

atcgaaacat acaaccaaac ttctccccga tctgcggcca ctggactgcc catcagcatg 1440

aaaattttta tgtatttact tactgttttt cttatcaccc agatgattgg gtcagcactt 1500

tttgctgtgt atcttcatag aaggttggac aagatagaag atgaaaggaa tcttcatgaa 1560

gattttgtat tcatgaaaac gatacagaga tgcaacacag gagaaagatc cttatcctta 1620

ctgaactgtg aggagattaa aagccagttt gaaggctttg tgaaggatat aatgttaaac 1680

aaagaggaga cgaagaaaga aaacagcttt gaaatgcaaa aaggtgatca gaatcctcaa 1740

attgcggcac atgtcataag tgaggccagc agtaaaacaa catctgtgtt acagtgggct 1800

gaaaaaggat actacaccat gagcaacaac ttggtaaccc tggaaaatgg gaaacagctg 1860

accgttaaaa gacaaggact ctattatatc tatgcccaag tcaccttctg ttccaatcgg 1920

gaagcttcga gtcaagctcc atttatagcc agcctctgcc taaagtcccc cggtagattc 1980

gagagaatct tactcagagc tgcaaatacc cacagttccg ccaaaccttg cgggcaacaa 2040

tccattcact tgggaggagt atttgaattg caaccaggtg cttcggtgtt tgtcaatgtg 2100

actgatccaa gccaagtgag ccatggcact ggcttcacgt cctttggctt actcaaactc 2160

tga 2163

<210> 18

<211> 720

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile

1 5 10 15

Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His

20 25 30

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

35 40 45

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

50 55 60

Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys

65 70 75 80

Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met

85 90 95

Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser

100 105 110

Cys Ala Thr Gln Thr Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys

115 120 125

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

130 135 140

Glu Phe Arg Ala Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu

145 150 155 160

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

165 170 175

Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro Pro Ala

180 185 190

Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val Ala Gly

195 200 205

Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe Leu Ala

210 215 220

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

225 230 235 240

Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala

245 250 255

Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln

260 265 270

Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly

275 280 285

Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr

290 295 300

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

305 310 315 320

Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser

325 330 335

Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala

340 345 350

Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn

355 360 365

Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln

370 375 380

Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp

385 390 395 400

Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro

405 410 415

Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Ser Arg Ala

420 425 430

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

435 440 445

Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Ser Met Ile Glu Thr Tyr

450 455 460

Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly Leu Pro Ile Ser Met

465 470 475 480

Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu Ile Thr Gln Met Ile

485 490 495

Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg Arg Leu Asp Lys Ile

500 505 510

Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val Phe Met Lys Thr Ile

515 520 525

Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser Leu Leu Asn Cys Glu

530 535 540

Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys Asp Ile Met Leu Asn

545 550 555 560

Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu Met Gln Lys Gly Asp

565 570 575

Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys

580 585 590

Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser

595 600 605

Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg

610 615 620

Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg

625 630 635 640

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

645 650 655

Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser

660 665 670

Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe

675 680 685

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

690 695 700

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

705 710 715 720

<210> 19

<211> 235

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ccatggcggc ccccggcagc gcccggcgac ccctgctgct gctactgctg ttgctgctgc 60

tcggcctcat gcattgtgcg tcagcagtcg acacgcgctc tagagcggaa gatctctgat 120

ccccatcgct gtgggtggtg ccctggcggg gctggtcctc atcgtcctca tcgcctacct 180

cgtcggcagg aagaggagtc acgcaggcta ccagactatc taagttaaca agctt 235

<210> 20

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

acgcgtcgac ggctccgacg tgcgggacc 29

<210> 21

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gcgcgcgcgc gctctagact gaatgcctct gaagacaccg 40

<210> 22

<211> 891

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ggctccgacg tgcgggacct gaacgcgctg ctgcccgccg tcccctccct gggtggcggc 60

ggcggctgtg ccctgcctgt gagcggcgcg gcgcagtggg cgccggtgct ggactttgcg 120

cccccgggcg cttcggctta cgggtcgttg ggcggccccg cgccgccacc ggctccgccg 180

ccacccccgc cgccgccgcc tcactccttc atcaaacagg agccgagctg gggcggcgcg 240

gagccgcacg aggagcagtg cctgagcgcc ttcactgtcc acttttccgg ccagttcact 300

ggcacagccg gagcctgtcg ctacgggccc ttcggtcctc ctccgcccag ccaggcgtca 360

tccggccagg ccaggatgtt tcctaacgcg ccctacctgc ccagctgcct cgagagccag 420

cccgctattc gcaatcaggg ttacagcacg gtcaccttcg acgggacgcc cagctacggt 480

cacacgccct cgcaccatgc ggcgcagttc cccaaccact cattcaagca tgaggatccc 540

atgggccagc agggctcgct gggtgagcag cagtactcgg tgccgccccc ggtctatggc 600

tgccacaccc ccaccgacag ctgcaccggc agccaggctt tgctgctgag gacgccctac 660

agcagtgaca atttatacca aatgacatcc cagcttgaat gcatgacctg gaatcagatg 720

aacttaggag ccaccttaaa gggagttgct gctgggagct ccagctcagt gaaatggaca 780

gaagggcaga gcaaccacag cacagggtac gagagcgata accacacaac gcccatcctc 840

tgcggagccc aatacagaat acacacgcac ggtgtcttca gaggcattca g 891

<210> 23

<211> 297

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro Ser

1 5 10 15

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

20 25 30

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

35 40 45

Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro

50 55 60

Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly Ala

65 70 75 80

Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe Ser

85 90 95

Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe Gly

100 105 110

Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe Pro

115 120 125

Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile Arg

130 135 140

Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr Gly

145 150 155 160

His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe Lys

165 170 175

His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln Tyr

180 185 190

Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser Cys

195 200 205

Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn

210 215 220

Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln Met

225 230 235 240

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

245 250 255

Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu Ser

260 265 270

Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile His

275 280 285

Thr His Gly Val Phe Arg Gly Ile Gln

290 295

<210> 24

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ggactagtgg tgccccgacg ttgcccc 27

<210> 25

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gcgcgaagat ctatccatgg cagccagctg ctcg 34

<210> 26

<211> 423

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ggtgccccga cgttgccccc tgcctggcag ccctttctca aggaccaccg catctctaca 60

ttcaagaact ggcccttctt ggagggctgc gcctgcaccc cggagcggat ggccgaggct 120

ggcttcatcc actgccccac tgagaacgag ccagacttgg cccagtgttt cttctgcttc 180

aaggagctgg aaggctggga gccagatgac gaccccatag aggaacataa aaagcattcg 240

tccggttgcg ctttcctttc tgtcaagaag cagtttgaag aattaaccct tggtgaattt 300

ttgaaactgg acagagaaag agccaagaac aaaattgcaa aggaaaccaa caataagaag 360

aaagaatttg aggaaactgc ggagaaagtg cgccgtgcca tcgagcagct ggctgccatg 420

gat 423

<210> 27

<211> 141

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp His

1 5 10 15

Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala Cys

20 25 30

Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr Glu

35 40 45

Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu Glu

50 55 60

Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys His Ser

65 70 75 80

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

85 90 95

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

100 105 110

Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala Glu

115 120 125

Lys Val Arg Arg Ala Ile Glu Gln Leu Ala Ala Met Asp

130 135 140

<210> 28

<211> 571

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ccgccccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt 60

tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc 120

tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca 180

aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac 240

gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg 300

ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt 360

gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct 420

gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg 480

ctttacatgt gtttagtcga ggttaaaaaa cgtctaggcc ccccgaacca cggggacgtg 540

gttttccttt gaaaaacacg atgataatac c 571

<210> 29

<211> 1533

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

atggcggccc ccggcagcgc ccggcgaccc ctgctgctgc tactgctgtt gctgctgctc 60

ggcctcatgc attgtgcgtc agcacaattc gtcgacggct ccgacgtgcg ggacctgaac 120

gcgctgctgc ccgccgtccc ctccctgggt ggcggcggcg gctgtgccct gcctgtgagc 180

ggcgcggcgc agtgggcgcc ggtgctggac tttgcgcccc cgggcgcttc ggcttacggg 240

tcgttgggcg gccccgcgcc gccaccggct ccgccgccac ccccgccgcc gccgcctcac 300

tccttcatca aacaggagcc gagctggggc ggcgcggagc cgcacgagga gcagtgcctg 360

agcgccttca ctgtccactt ttccggccag ttcactggca cagccggagc ctgtcgctac 420

gggcccttcg gtcctcctcc gcccagccag gcgtcatccg gccaggccag gatgtttcct 480

aacgcgccct acctgcccag ctgcctcgag agccagcccg ctattcgcaa tcagggttac 540

agcacggtca ccttcgacgg gacgcccagc tacggtcaca cgccctcgca ccatgcggcg 600

cagttcccca accactcatt caagcatgag gatcccatgg gccagcaggg ctcgctgggt 660

gagcagcagt actcggtgcc gcccccggtc tatggctgcc acacccccac cgacagctgc 720

accggcagcc aggctttgct gctgaggacg ccctacagca gtgacaattt ataccaaatg 780

acatcccagc ttgaatgcat gacctggaat cagatgaact taggagccac cttaaaggga 840

gttgctgctg ggagctccag ctcagtgaaa tggacagaag ggcagagcaa ccacagcaca 900

gggtacgaga gcgataacca cacaacgccc atcctctgcg gagcccaata cagaatacac 960

acgcacggtg tcttcagagg cattcagaga tccggtgccc cgacgttgcc ccctgcctgg 1020

cagccctttc tcaaggacca ccgcatctct acattcaaga actggccctt cttggagggc 1080

tgcgcctgca ccccggagcg gatggccgag gctggcttca tccactgccc cactgagaac 1140

gagccagact tggcccagtg tttcttctgc ttcaaggagc tggaaggctg ggagccagat 1200

gacgacccca tagaggaaca taaaaagcat tcgtccggtt gcgctttcct ttctgtcaag 1260

aagcagtttg aagaattaac ccttggtgaa tttttgaaac tggacagaga aagagccaag 1320

aacaaaattg caaaggaaac caacaataag aagaaagaat ttgaggaaac tgcggagaaa 1380

gtgcgccgtg ccatcgagca gctggctgcc atggatagat ctagactgat ccccatcgct 1440

gtgggtggtg ccctggcggg gctggtcctc atcgtcctca tcgcctacct cgtcggcagg 1500

aagaggagtc acgcaggcta ccagactatc taa 1533

<210> 30

<211> 510

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Gln Phe Val Asp

20 25 30

Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro Ser

35 40 45

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

50 55 60

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

65 70 75 80

Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro

85 90 95

Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly Ala

100 105 110

Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe Ser

115 120 125

Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe Gly

130 135 140

Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe Pro

145 150 155 160

Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile Arg

165 170 175

Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr Gly

180 185 190

His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe Lys

195 200 205

His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln Tyr

210 215 220

Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser Cys

225 230 235 240

Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn

245 250 255

Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln Met

260 265 270

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

275 280 285

Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu Ser

290 295 300

Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile His

305 310 315 320

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

325 330 335

Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp His Arg Ile Ser Thr Phe

340 345 350

Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala Cys Thr Pro Glu Arg Met

355 360 365

Ala Glu Ala Gly Phe Ile His Cys Pro Thr Glu Asn Glu Pro Asp Leu

370 375 380

Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu Glu Gly Trp Glu Pro Asp

385 390 395 400

Asp Asp Pro Ile Glu Glu His Lys Lys His Ser Ser Gly Cys Ala Phe

405 410 415

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

420 425 430

Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys Ile Ala Lys Glu Thr Asn

435 440 445

Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala Glu Lys Val Arg Arg Ala

450 455 460

Ile Glu Gln Leu Ala Ala Met Asp Arg Ser Arg Leu Ile Pro Ile Ala

465 470 475 480

Val Gly Gly Ala Leu Ala Gly Leu Val Leu Ile Val Leu Ile Ala Tyr

485 490 495

Leu Val Gly Arg Lys Arg Ser His Ala Gly Tyr Gln Thr Ile

500 505 510

<210> 31

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ccgctcgaga ttccgccccc ccccccc 27

<210> 32

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gcgcgcgccc ggggttaact tagatagtct ggtagcctgc g 41

<210> 33

<211> 9144

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ttcatcaata atatacctta ttttggattg aagccaatat gataatgagg gggtggagtt 60

tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg 120

atgttgcaag tgtggcggaa cacatgtaag cgacggatgt ggcaaaagtg acgtttttgg 180

tgtgcgccgg tgtacacagg aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt 240

aaatttgggc gtaaccgagt aagatttggc cattttcgcg ggaaaactga ataagaggaa 300

gtgaaatctg aataattttg tgttactcat agcgcgtaat atttgtctag ggccgcgggg 360

actttgaccg tttacgtgga gactcgccca ggtgtttttc tcaggtgttt tccgcgttcc 420

gggtcaaagt tggcgtttta ttattatagt cagctctaga tcaatattgg ccattagcca 480

tattattcat tggttatata gcataaatca atattggcta ttggccattg catacgttgt 540

atctatatca taatatgtac atttatattg gctcatgtcc aatatgaccg ccatgttggc 600

attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 660

atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 720

acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 780

tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 840

tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 900

attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag 960

tcatcgctat taccatggtg atgcggtttt ggcagtacac caatgggcgt ggatagcggt 1020

ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 1080

accaaaatca acgggacttt ccaaaatgtc gtaacaactg cgatcgcccg ccccgttgac 1140

gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa 1200

ccgtcagatc actagaagct ttattgcggt agtttatcac agttaaattg ctaacgcagt 1260

cagtgcttct gacacaacag tctcgaactt aagctgcagt gactctctta aggtagcctt 1320

gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag 1380

gagaccaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac 1440

ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac tcccagttca 1500

attacagctc ttaaggctag agtacttaat acgactcact ataggctagc atgtggctgc 1560

agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc cgctcgccca 1620

gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg cgtctcctga 1680

acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc tcagaaatgt 1740

ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag cagggcctgc 1800

ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac tacaagcagc 1860

actgccctcc aaccccggaa acttcctgtg caacccagac tatcaccttt gaaagtttca 1920

aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag ccagtccagg 1980

aggaattccg agccaaacga agcggctctg gagaaggccg tggatctctg ctgacctgcg 2040

gagacgtaga ggagaatcca gggcccggat ctgaatacgc ctctgacgct tcactggacc 2100

ccgaagcccc gtggcctccc gcgccccgcg ctcgcgcctg ccgcgtactg ccttgggccc 2160

tggtcgcggg gctgctgctg ctgctgctgc tcgctgccgc ctgcgccgtc ttcctcgcct 2220

gcccctgggc cgtgtccggg gctcgcgcct cgcccggctc cgcggccagc ccgagactcc 2280

gcgagggtcc cgagctttcg cccgacgatc ccgccggcct cttggacctg cggcagggca 2340

tgtttgcgca gctggtggcc caaaatgttc tgctgatcga tgggcccctg agctggtaca 2400

gtgacccagg cctggcaggc gtgtccctga cggggggcct gagctacaaa gaggacacga 2460

aggagctggt ggtggccaag gctggagtct actatgtctt ctttcaacta gagctgcggc 2520

gcgtggtggc cggcgagggc tcaggctccg tttcacttgc gctgcacctg cagccactgc 2580

gctctgctgc tggggccgcc gccctggctt tgaccgtgga cctgccaccc gcctcctccg 2640

aggctcggaa ctcggccttc ggtttccagg gccgcttgct gcacctgagt gccggccagc 2700

gcctgggcgt ccatcttcac actgaggcca gggcacgcca tgcctggcag cttacccagg 2760

gcgccacagt cttgggactc ttccgggtga cccccgaaat cccagccgga ctcccttcac 2820

cgaggtcgga aggatccaga gctaagaggt cgggttccgg cgccaccaat ttcagtttgc 2880

tcaagcaggc tggtgatgtg gaggagaatc ccggtccgag atccatcgaa acatacaacc 2940

aaacttctcc ccgatctgcg gccactggac tgcccatcag catgaaaatt tttatgtatt 3000

tacttactgt ttttcttatc acccagatga ttgggtcagc actttttgct gtgtatcttc 3060

atagaaggtt ggacaagata gaagatgaaa ggaatcttca tgaagatttt gtattcatga 3120

aaacgataca gagatgcaac acaggagaaa gatccttatc cttactgaac tgtgaggaga 3180

ttaaaagcca gtttgaaggc tttgtgaagg atataatgtt aaacaaagag gagacgaaga 3240

aagaaaacag ctttgaaatg caaaaaggtg atcagaatcc tcaaattgcg gcacatgtca 3300

taagtgaggc cagcagtaaa acaacatctg tgttacagtg ggctgaaaaa ggatactaca 3360

ccatgagcaa caacttggta accctggaaa atgggaaaca gctgaccgtt aaaagacaag 3420

gactctatta tatctatgcc caagtcacct tctgttccaa tcgggaagct tcgagtcaag 3480

ctccatttat agccagcctc tgcctaaagt cccccggtag attcgagaga atcttactca 3540

gagctgcaaa tacccacagt tccgccaaac cttgcgggca acaatccatt cacttgggag 3600

gagtatttga attgcaacca ggtgcttcgg tgtttgtcaa tgtgactgat ccaagccaag 3660

tgagccatgg cactggcttc acgtcctttg gcttactcaa actctgatat ctcgagattc 3720

cgcccccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt 3780

gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct 3840

ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa 3900

ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg 3960

tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc 4020

caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg 4080

agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg 4140

aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc 4200

tttacatgtg tttagtcgag gttaaaaaac gtctaggccc cccgaaccac ggggacgtgg 4260

ttttcctttg aaaaacacga tgataatacc atggcggccc ccggcagcgc ccggcgaccc 4320

ctgctgctgc tactgctgtt gctgctgctc ggcctcatgc attgtgcgtc agcacaattc 4380

gtcgacggct ccgacgtgcg ggacctgaac gcgctgctgc ccgccgtccc ctccctgggt 4440

ggcggcggcg gctgtgccct gcctgtgagc ggcgcggcgc agtgggcgcc ggtgctggac 4500

tttgcgcccc cgggcgcttc ggcttacggg tcgttgggcg gccccgcgcc gccaccggct 4560

ccgccgccac ccccgccgcc gccgcctcac tccttcatca aacaggagcc gagctggggc 4620

ggcgcggagc cgcacgagga gcagtgcctg agcgccttca ctgtccactt ttccggccag 4680

ttcactggca cagccggagc ctgtcgctac gggcccttcg gtcctcctcc gcccagccag 4740

gcgtcatccg gccaggccag gatgtttcct aacgcgccct acctgcccag ctgcctcgag 4800

agccagcccg ctattcgcaa tcagggttac agcacggtca ccttcgacgg gacgcccagc 4860

tacggtcaca cgccctcgca ccatgcggcg cagttcccca accactcatt caagcatgag 4920

gatcccatgg gccagcaggg ctcgctgggt gagcagcagt actcggtgcc gcccccggtc 4980

tatggctgcc acacccccac cgacagctgc accggcagcc aggctttgct gctgaggacg 5040

ccctacagca gtgacaattt ataccaaatg acatcccagc ttgaatgcat gacctggaat 5100

cagatgaact taggagccac cttaaaggga gttgctgctg ggagctccag ctcagtgaaa 5160

tggacagaag ggcagagcaa ccacagcaca gggtacgaga gcgataacca cacaacgccc 5220

atcctctgcg gagcccaata cagaatacac acgcacggtg tcttcagagg cattcagaga 5280

tccggtgccc cgacgttgcc ccctgcctgg cagccctttc tcaaggacca ccgcatctct 5340

acattcaaga actggccctt cttggagggc tgcgcctgca ccccggagcg gatggccgag 5400

gctggcttca tccactgccc cactgagaac gagccagact tggcccagtg tttcttctgc 5460

ttcaaggagc tggaaggctg ggagccagat gacgacccca tagaggaaca taaaaagcat 5520

tcgtccggtt gcgctttcct ttctgtcaag aagcagtttg aagaattaac ccttggtgaa 5580

tttttgaaac tggacagaga aagagccaag aacaaaattg caaaggaaac caacaataag 5640

aagaaagaat ttgaggaaac tgcggagaaa gtgcgccgtg ccatcgagca gctggctgcc 5700

atggatagat ctagactgat ccccatcgct gtgggtggtg ccctggcggg gctggtcctc 5760

atcgtcctca tcgcctacct cgtcggcagg aagaggagtc acgcaggcta ccagactatc 5820

taagttaacc ccgggcagct tcgaccatca tcatcatcat cattgagttt aaacggtctc 5880

cagcttaagt ttaaaccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5940

tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 6000

ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 6060

tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 6120

tgcggtgggc tctatggctc gatcgagatc cagttagggg cgggactatg gttgctgact 6180

aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc 6240

tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg 6300

actttccaca ccctaactga cacacattcc agatccataa cttcgtatag catacattat 6360

acgaagttat gatccgaagt tcctattctt actagagtat aggaacttcg actatcgtcg 6420

ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct 6480

gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg 6540

cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac 6600

gtttcggcga gaagcaggcc attatcgccg gcatggcggc cctgcattaa tgaatcggcc 6660

aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 6720

cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 6780

ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 6840

aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 6900

acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 6960

gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 7020

ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac 7080

gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 7140

cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 7200

taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 7260

atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 7320

cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 7380

cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 7440

ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 7500

ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 7560

tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 7620

aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 7680

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 7740

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 7800

atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 7860

tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 7920

ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 7980

ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 8040

tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 8100

ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 8160

ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 8220

tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca 8280

gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 8340

taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 8400

cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 8460

agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 8520

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 8580

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 8640

ccattattat catgacatta acctataaaa ataggcgtat cactctaggc aaaatagcac 8700

cctcccgctc cagaacaaca tacagcgctt cacagcggca gcctaacagt cagccttacc 8760

agtaaaaaag aaaacctatt aaaaaaacac cactcgacac ggcaccagct caatcagtca 8820

cagtgtaaaa aagggccaag tgcagagcga gtatatatag gactaaaaaa tgacgtaacg 8880

gttaaagtcc acaaaaaaca cccagaaaac cgcacgcgaa cctacgccca gaaacgaaag 8940

ccaaaaaacc cacaacttcc tcaaatcgtc acttccgttt tcccacgtta cgtaacttcc 9000

cattttaaga aaactacaat tcccaacaca tacaagttac tccgccctaa aacctacgtc 9060

acccgccccg ttcccacgcc ccgcgccacg tcacaaactc caccccctca ttatcatatt 9120

ggcttcaatc caaaataagg tata 9144

<210> 34

<211> 1320

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ggctccgacg tgcgggacct gaacgcgctg ctgcccgccg tcccctccct gggtggcggc 60

ggcggctgtg ccctgcctgt gagcggcgcg gcgcagtggg cgccggtgct ggactttgcg 120

cccccgggcg cttcggctta cgggtcgttg ggcggccccg cgccgccacc ggctccgccg 180

ccacccccgc cgccgccgcc tcactccttc atcaaacagg agccgagctg gggcggcgcg 240

gagccgcacg aggagcagtg cctgagcgcc ttcactgtcc acttttccgg ccagttcact 300

ggcacagccg gagcctgtcg ctacgggccc ttcggtcctc ctccgcccag ccaggcgtca 360

tccggccagg ccaggatgtt tcctaacgcg ccctacctgc ccagctgcct cgagagccag 420

cccgctattc gcaatcaggg ttacagcacg gtcaccttcg acgggacgcc cagctacggt 480

cacacgccct cgcaccatgc ggcgcagttc cccaaccact cattcaagca tgaggatccc 540

atgggccagc agggctcgct gggtgagcag cagtactcgg tgccgccccc ggtctatggc 600

tgccacaccc ccaccgacag ctgcaccggc agccaggctt tgctgctgag gacgccctac 660

agcagtgaca atttatacca aatgacatcc cagcttgaat gcatgacctg gaatcagatg 720

aacttaggag ccaccttaaa gggagttgct gctgggagct ccagctcagt gaaatggaca 780

gaagggcaga gcaaccacag cacagggtac gagagcgata accacacaac gcccatcctc 840

tgcggagccc aatacagaat acacacgcac ggtgtcttca gaggcattca gagatccggt 900

gccccgacgt tgccccctgc ctggcagccc tttctcaagg accaccgcat ctctacattc 960

aagaactggc ccttcttgga gggctgcgcc tgcaccccgg agcggatggc cgaggctggc 1020

ttcatccact gccccactga gaacgagcca gacttggccc agtgtttctt ctgcttcaag 1080

gagctggaag gctgggagcc agatgacgac cccatagagg aacataaaaa gcattcgtcc 1140

ggttgcgctt tcctttctgt caagaagcag tttgaagaat taacccttgg tgaatttttg 1200

aaactggaca gagaaagagc caagaacaaa attgcaaagg aaaccaacaa taagaagaaa 1260

gaatttgagg aaactgcgga gaaagtgcgc cgtgccatcg agcagctggc tgccatggat 1320

<210> 35

<211> 440

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro Ser

1 5 10 15

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

20 25 30

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

35 40 45

Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro

50 55 60

Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly Ala

65 70 75 80

Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe Ser

85 90 95

Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe Gly

100 105 110

Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe Pro

115 120 125

Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile Arg

130 135 140

Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr Gly

145 150 155 160

His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe Lys

165 170 175

His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln Tyr

180 185 190

Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser Cys

195 200 205

Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn

210 215 220

Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln Met

225 230 235 240

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

245 250 255

Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu Ser

260 265 270

Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile His

275 280 285

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

290 295 300

Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp His Arg Ile Ser Thr Phe

305 310 315 320

Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala Cys Thr Pro Glu Arg Met

325 330 335

Ala Glu Ala Gly Phe Ile His Cys Pro Thr Glu Asn Glu Pro Asp Leu

340 345 350

Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu Glu Gly Trp Glu Pro Asp

355 360 365

Asp Asp Pro Ile Glu Glu His Lys Lys His Ser Ser Gly Cys Ala Phe

370 375 380

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

385 390 395 400

Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys Ile Ala Lys Glu Thr Asn

405 410 415

Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala Glu Lys Val Arg Arg Ala

420 425 430

Ile Glu Gln Leu Ala Ala Met Asp

435 440

Claims (10)

1. A shuttle plasmid pGBC-IRES-WSL carrying a plurality of target genes, wherein the shuttle plasmid is named as pGBC-IRES-WSL, the starting vector of the shuttle plasmid is pDC511 shuttle plasmid, the plurality of target genes comprise tumor-associated antigens, expression genes of granulocyte-megakaryocyte colony stimulating factor (GM-CSF), CD40L and 4-1BBL, and the expression genes of the granulocyte-megakaryocyte colony stimulating factor (GM-CSF), CD40L and 4-1BBL are connected in series through two 2A peptide sequences to form an open reading frame, and an internal nucleic acid entry site IRES sequence is connected to the 5' end of the expression sequence of the tumor-associated antigens.

2. The shuttle plasmid pGBC-IRES-WSL according to claim 1, wherein the expression genes of the tumor associated antigens include, but are not limited to, any of the following genes: tWT1, Survivin, fusion genes tWT1/Survivin, fusion genes tWT1/Survivin/LAMP-1, MUC1, LMP, HPV E6E 7, EGFRvIII, HER-2/neu, MAGE A3, p53(nonmutant and mutant), NY-ESO-1, PSMA, GD2, CEA, Melana/MART1, Ras mutant, gp100, Proteinase3(PR1), bcr-abl, Tyrosinase, PSA, hTERT, Sarcoma translocation peptides, EphA2, PAP, ML-IAP, AFP, EpGE, ERG (TMPRSS2 ETS fusion gene), NA17, PA46X 3, Androgram, Cyclin B5, Polyalsin B5, Fusalin-8, MAG-24, MAG-1, Rho 639, CYP 598, GM 9, CYP 639, GM1, GM 9, MAGE-A, MAGE-598, and MAG-2;

preferably, the expression gene of the tumor associated antigen is tWT1 (the nucleotide sequence of which can be shown in SEQ ID NO: 22), Survivin (the nucleotide sequence of which can be shown in SEQ ID NO: 26), fusion gene tWT1/Survivin (the nucleotide sequence of which can be shown in SEQ ID NO: 34) or fusion gene tWT1/Survivin/LAMP-1 (the nucleotide sequence of which can be shown in SEQ ID NO: 29).

3. The shuttle plasmid pGBC-IRES-WSL according to claim 1 or 2, wherein the nucleotide sequence of the GM-CSF expressible gene is as shown in SEQ ID NO 7; the nucleotide sequence of the CD40L expression gene can be shown as SEQ ID NO. 15; the nucleotide sequence of the 4-1BBL expression gene can be shown as SEQ ID NO. 11.

4. A shuttle plasmid pGBC-IRES-WSL according to any one of claims 1 to 3, wherein the 2A peptide sequences are selected from any one or a combination of two of E2A, F2A, T2A and P2A, and the positions of the junctions of the expressed genes of GM-CSF, 4-1BBL and CD40L in the open reading frame formed by the tandem connection of the two 2A peptide sequences can be interchanged.

5. The shuttle plasmid pGBC-IRES-WSL according to any one of claims 1 to 4, wherein the nucleotide sequence of the shuttle plasmid pGBC-IRES-WSL is set forth in SEQ ID NO. 33.

6. The method for constructing the shuttle plasmid pGBC-IRES-WSL according to any one of claims 1 to 5, comprising the steps of:

6.1) inserting the CMVp-pA expression cassette sequence into the multiple cloning site of pDC511 shuttle plasmid to obtain plasmid pDC 523;

6.2) synthesizing an oligonucleotide fragment comprising two 2A peptide sequences and a multiple cloning site; optionally, a fragment of the oligonucleotide fragment with the nucleotide sequence shown as SEQ ID NO. 3 when T2A and P2A are selected;

6.3) inserting the oligonucleotide fragment synthesized in step 6.2) into the CMVp-pA expression cassette multiple cloning site of plasmid pDC523 in 6.1) to obtain a shuttle plasmid named pDC 5232X 2A; optionally, the nucleotide sequence of the pDC 5232X 2A shuttle plasmid inserted into SEQ ID NO. 3 is shown in SEQ ID NO. 4;

6.4) inserting the expressed genes of GM-CSF, 4-1BBL and CD40L into the pDC 5232X 2A shuttle plasmid obtained in step 6.3) to obtain a plasmid containing expressed genes of GM-CSF, 4-1BBL and CD40L, which is named pDC523GM-CSF/4-1BBL/CD40L plasmid, and wherein the expressed genes of GM-CSF, 4-1BBL and CD40L are connected in series by two 2A peptide sequences to form an open reading frame;

6.5) inserting the expression gene of the tumor associated antigen into the pDC523GM-CSF/4-1BBL/CD40L plasmid of step 6.4) to obtain pGBC-IRES-WSL shuttle plasmid, wherein the 5' end of the expression gene of the tumor associated antigen is connected with an internal nucleic acid entry site IRES sequence.

7. The method according to claim 6, wherein the expression gene of the tumor-associated antigen in step 6.5) is fusion gene tWT1/Survivin/LAMP-1, and the insertion of the fusion gene tWT1/Survivin/LAMP-1 into the pDC523GM-CSF/4-1BBL/CD40L plasmid of step 6.4) is specifically performed as follows:

6.5.1) inserting the signal peptide and the signal classification sequence of the human LAMP-1 gene into the pmRNA IRES-EGFP plasmid to replace the EGFP sequence, and obtaining the pmRNA IRES-LAMP-1 plasmid;

6.5.2) inserting tWT1 and Survivin expression gene sequence into pmRNA IRES-LAMP-1 plasmid to obtain pmRNA IRES-tWT1/Survivin/LAMP-1 plasmid;

6.5.3) obtaining the tumor associated antigen expression gene with the 5' end connected with the IRES sequence by PCR amplification from the pmRNA IRES-tWT1/Survivin/LAMP-1 plasmid of the step 6.5.2), and the gene is named as IRES-tWT1/Survivin/LAMP-1 sequence, and optionally, the primer pair for amplification is an upstream primer shown as SEQ ID NO. 31 and a downstream primer shown as SEQ ID NO. 32;

6.5.4) inserting the IRES-tWT1/Survivin/LAMP-1 sequence obtained by amplification in the step 6.5.3) into the pDC523GM-CSF/4-1BBL/CD40L plasmid obtained in the step 6.4) to obtain pGBC-IRES-WSL shuttle plasmid.

8. A recombinant adenovirus obtained by co-transfecting HEK293T cells with the pGBC-IRES-WSL shuttle plasmid according to any one of claims 1 to 5 or the obtained in step 6.5) of claim 6 and an adenovirus backbone plasmid;

optionally, the adenovirus backbone plasmid is a pBGHfrt Δ E1,3Cre or pBGHfrt Δ E1,3Cre (5/F35) backbone plasmid.

9. A therapeutic dendritic cell cancer vaccine obtained by infecting the recombinant adenovirus of claim 8 to human peripheral-derived blood mononuclear cells or dendritic cells, which can simultaneously express tumor-associated antigens, GM-CSF, CD40L, and 4-1 BBL;

optionally, the infection dose of the recombinant adenovirus is 10-100 pfu/cell, and the infection time is 36-48 hours.

10. Use of the shuttle plasmid pGBC-IRES-WSL of any one of claims 1 to 5 or the recombinant adenovirus of claim 8 or the therapeutic dendritic cell cancer vaccine of claim 9 for the manufacture of a medicament for the treatment of hematologic and solid tumors; wherein the hematological and solid tumors are WT1 and/or Survivin positive hematological and solid tumors;

optionally, the WT1 and/or Survivin positive hematological tumors include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, and high risk myelodysplastic syndrome (MDS); the WT1 and/or Survivin positive solid tumors include, but are not limited to, lung cancer, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, prostate cancer, renal cancer, bladder cancer, glioblastoma, head and neck malignancies, and various types of malignant sarcomas.

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