CN106687592B

CN106687592B - Recombinant plasmid, recombinant plasmodium constructed by recombinant plasmid and application of recombinant plasmid

Info

Publication number: CN106687592B
Application number: CN201680001939.7A
Authority: CN
Inventors: 陈小平; 秦莉; 刘权
Original assignee: Guangzhou Cas Lamvac Biotech Co Ltd
Current assignee: Zhongke Lanhua Guangzhou Biomedical Technology Co ltd
Priority date: 2016-12-26
Filing date: 2016-12-26
Publication date: 2020-03-24
Anticipated expiration: 2036-12-26
Also published as: CN106687592A; WO2018119610A1

Abstract

The invention relates to the field of cell immunotherapy of tumors, in particular to a recombinant plasmid, a recombinant plasmodium constructed by the recombinant plasmid and an application of the recombinant plasmid, wherein the recombinant plasmid is obtained by inserting a liver tumor specific antigen gene into a pL0017 plasmid, and the recombinant plasmodium comprises the recombinant plasmid, compared with plasmid DNA and an RNA vector, the recombinant plasmodium can realize a large amount of in-vivo amplification along with the proliferation of plasmodium, and is favorable for increasing the antigen in vivo.

Description

Recombinant plasmid, recombinant plasmodium constructed by recombinant plasmid and application of recombinant plasmid

Technical Field

The invention relates to the field of tumor cell immunotherapy, in particular to a recombinant plasmid, a constructed recombinant plasmodium and application thereof, and specifically relates to construction of the recombinant plasmodium based on the recombinant plasmid and application thereof in anti-liver tumor therapy.

Background

Hepatocellular carcinoma (HCC), accounts for 85% to 90% of the proportion of primary liver cancer. It is one of the five common cancers in the world, with mortality ranking top three of all tumors. The data show 782,500 new liver cancer patients worldwide each year, and 745,500 patients die of liver cancer each year. Currently, the clinical techniques for liver cancer treatment include surgical resection, orthotopic liver transplantation, hepatic artery chemoembolization, and local radio frequency therapy, which have certain therapeutic effects on patients with early HCC. However, patients often find it too late, and 50% of patients with liver cancer are not eligible to use these traditional methods. At the same time, the one-year survival rate of the patients is lower than 50%. The traditional treatment methods have many defects, including incomplete excision, great toxic and side effects, easy relapse and the like. In addition, the medicines for targeted therapy of liver tumors, including Sorafenib, Bevacizumab and the like, also have the problems of side effects of treatment, short life cycle of patients and the like. Therefore, the development of new drugs for treating liver tumors is highly urgent.

At present, bioimmunotherapy is a widely recognized and promising approach to tumor therapy, most of which primarily kills tumor cells by activating specific CD8+ T lymphocytes in patients. Adoptive therapies including the childrenergen receptor T cells (CAR-T) therapy are widely studied and involved in clinical applications, however, the time and money spent on such individual therapies is enormous and not acceptable to all. At present, the method of expressing tumor-specific antigens by vectors and stimulating specific cellular immunity in vivo to combat tumor growth is considered to be a viable and inexpensive approach, including tumor vaccines. In the aspect of liver tumor vaccine research, both AFP (alpha total protein, alpha fetoprotein) DC (dendritic cell) vaccine and GPC3 polypeptide vaccine complete clinical stage II, the survival rate is obviously improved, and the improvement is obviously related to cell-specific cytotoxic T-lymphocyte (CTL) reaction. More tumor vaccines related to liver cancer have made great progress in DNA vaccines, polypeptide vaccines and DC vaccines. However, these vaccines also have a general problem in that the cellular immunity elicited against the liver tumor antigen is strong but does not have the persistence of specific immune activation, and selecting a good tumor antigen carrier may be a good solution to this problem.

HCC-specific antigens are widely discovered and used in the immunotherapy of tumors. These include glypican 3 protein (GPC3), which Pilia et al found by studies on 1 patient with the hyper-growth syndrome. GPC3 is highly expressed in liver cancer tissue, but is not expressed in normal liver tissue, and is a novel carcinoembryonic antigen of hepatocellular carcinoma. Studies have shown that the GPC3 protein is able to activate cytotoxic T-lymphocytes (CTL) against GPC3, including human and mouse, without eliciting autoimmunity, such CTL often being involved in targeted killing reactions against tumor cells. Antibodies against GPC3, polypeptide vaccines, and CAR-T therapy have entered into intensive research, with GPC3 polypeptide vaccine having completed phase II in the clinic, with an overall survival rate that is significantly correlated with GPC 3-specific immune responses activated in the body.

A good expression vector is often required for tumor immunotherapy based on antigen activation of the immune system in vivo. Vectors of more use include plasmid DNA, RNA, defective viruses and bacteria. They are capable of activating strong immune responses in vivo but are not persistent, which may be associated with immune clearance by the body and their inability to persist in replicating presence in vivo. A good antigen expression vector should include the following two capabilities: firstly, strong specific CTL response aiming at antigen in vivo is activated; secondly, the specific CTL response can exist continuously in vivo and play a role in killing tumors. Numerous studies have shown that some viral or bacterial infections are capable of inhibiting tumor growth well. Meanwhile, protozoa are also widely studied for tumor therapy, including toxoplasma and trypanosoma. At the same time, plasmodium can activate a very strong innate immune response in vivo. Plasmodium, which is a natural adjuvant, has its own components such as gpi (glycosyl phosphatylinositolans), genomic DNA and malaria pigment as pathogen-associated model molecules (PAMPs) that are immediately recognized by the body's immune system and immediately activate macrophages, DCs, Natural Killers (NK) cells, γ δ T cells, Natural Killers T (NKT) cells, CD4+ T and CD8+ T cells in vivo. We conclude that these activated natural and adaptive immunity can be used to inhibit tumor growth. We propose herein that the use of plasmodium as an immunotherapeutic vector for HCC is based on: 1, infection with plasmodium can activate a Th1 response in vivo, which is important for the activation of CTLs against tumor antigens; 2, although erythrocytes lack major histocompatibility complex I (MHC I), which does not affect its generation of CTL responses against tumor antigens, studies have shown that both DCs and macrophages are involved in and activate CTL responses against plasmodium antigens in vivo; 3, the plasmodium itself is parasitic in the erythrocytes of the body, which can be present in the body for a long period of time, which facilitates the long-term expression and release of the antigen, activating the CTL response in the body for a long time.

CN 101838611A discloses a recombinant plasmodium expressing exogenous genes, the exogenous genes in the recombinant plasmodium include antigen genes, therapeutic genes, immunomodulator genes or peptide genes, wherein the plasmid adopted by the invention is pL0015 plasmid, the pL0015 plasmid mainly uses single enzyme digestion (Bam HI) to insert target genes, when the target genes are inserted, reverse insertion of the target genes is easy to occur, construction of plasmid vectors is not facilitated, the recombinant plasmodium in the patent can not express a plurality of exogenous genes at the same time, and the expressed genes can not be secreted out of plasmodium.

Disclosure of Invention

Aiming at the problems at present, the invention provides a recombinant plasmid, a recombinant plasmodium constructed by the recombinant plasmid and application of the recombinant plasmid, wherein the adopted pL0017 plasmid uses a double enzyme digestion method (Bam HI/Xba I) to insert a target gene, complex means such as reverse gene insertion and re-sequencing identification in plasmid construction do not exist, and a new plasmodium burgerii Elongation Factor (EF) -1 α promoter is introduced to simultaneously express two or more exogenous genes.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a recombinant plasmid, wherein a liver tumor specific antigen gene is inserted into a pL0017 plasmid.

In the invention, the pL0017 plasmid is selected to use a double-enzyme digestion method (Bam HI/Xba I) to insert a target gene, so that complicated means such as gene reverse insertion and resequencing identification in plasmid construction do not exist.

According to the invention, the liver tumor specific antigen gene is any one or combination of at least two of GPC3 (phosphatidylinositol proteoglycan 3 expression gene), alpha-embryonic protein AFP expression gene, MAGE expression gene or NY-ESO-1 expression gene, preferably GPC3 antigen gene, the GPC3 protein expressed by GPC3 is a liver tumor antigen with strong immunogenicity, and an antibody, a polypeptide vaccine and CAR-T treatment aiming at GPC3 enter into deep research, wherein the GPC3 polypeptide vaccine has already completed the clinical II phase, and the overall survival rate of the GPC3 polypeptide vaccine is obviously related to the activated GPC3 specific immunoreaction in an organism.

According to the invention, a new independent gene function expression element is inserted by modifying the pL0017 plasmid on the basis of the original pL0017 plasmid, and the new independent gene function expression element comprises a new promoter, a target gene open reading frame insertion site and a terminator, wherein the new promoter in the recombinant plasmid is a promoter pbeef1a α (Plasmodium boidinii Elongation Factor (EF) -1 α promoter) expressed in the whole insect stage of Plasmodium boidinii, the new terminator is 3' UTR, and the target gene open reading frame insertion site of the recombinant plasmid is single enzyme cutting sites Not I and Cla I on the plasmid.

Meanwhile, the reconstructed plasmid contains 2 independent gene expression elements, so that the recombinant insect strain can simultaneously express the capacity of one exogenous gene, two or even more than two exogenous genes, and the exogenous genes can be tumor specific antigens or tumor related antigens.

In the invention, two new single enzyme cutting sites Not I and Cla I are introduced, so that open reading frames of two exogenous genes can be formed.

Preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 1.

In a second aspect, the present invention provides a recombinant plasmodium comprising the recombinant plasmid according to the first aspect.

According to the present invention, the long-term presence of plasmodium in vivo is beneficial to the long-term presence and release of antigens, which is beneficial to the tumor growth inhibition effect and survival of tumor model animals, which many expression vectors currently cannot achieve in HCC immunotherapy.

The invention is beneficial to the distinguishability and the tracer of the positive recombinant plasmodium in vivo and in vitro and the further separation of the positive recombinant plasmodium by combining a flow cytometer through the expression of the fluorescent protein and the luciferase, and achieves the optimized effect in the steps of screening, monoclonal and the like of the positive recombinant plasmodium, and the recombinant plasmodium also comprises a fusion protein gGluc of Green Fluorescent Protein (GFP) and renilla luciferase and/or a secretory protein IBIS 1.

The invention relates to a preparation method of recombinant plasmodium, which adopts the conventional technology in the field for preparation, and is not specially limited, and concretely adopts the steps of expressing a liver tumor specific antigen gene on a vector to obtain a recombinant plasmid, copying the recombinant plasmid in escherichia coli, extracting and purifying, and transfecting the extracted recombinant plasmid into plasmodium to obtain the recombinant plasmodium expressing the liver tumor related antigen.

Preferably, the amino acid sequence of the fusion protein gGluc is SEQ ID NO.2, and the nucleotide sequence is SEQ ID NO. 3.

The amino acid sequence shown in SEQ ID NO.2 is as follows:

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKMTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQ.

the nucleotide sequence shown in SEQ ID NO.3 is as follows:

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGATGACTTCGAAAGTTTATGATCCAGAACAAAGGAAACGGATGATAACTGGTCCGCAGTGGTGGGCCAGATGTAAACAAATGAATGTTCTTGATTCATTTATTAATTATTATGATTCAGAAAAACATGCAGAAAATGCTGTTATTTTTTTACATGGTAACGCGGCCTCTTCTTATTTATGGCGACATGTTGTGCCACATATTGAGCCAGTAGCGCGGTGTATTATACCAGACCTTATTGGTATGGGCAAATCAGGCAAATCTGGTAATGGTTCTTATAGGTTACTTGATCATTACAAATATCTTACTGCATGGTTTGAACTTCTTAATTTACCAAAGAAGATCATTTTTGTCGGCCATGATTGGGGTGCTTGTTTGGCATTTCATTATAGCTATGAGCATCAAGATAAGATCAAAGCAATAGTTCACGCTGAAAGTGTAGTAGATGTGATTGAATCATGGGATGAATGGCCTGATATTGAAGAAGATATTGCGTTGATCAAATCTGAAGAAGGAGAAAAAATGGTTTTGGAGAATAACTTCTTCGTGGAAACCATGTTGCCATCAAAAATCATGAGAAAGTTAGAACCAGAAGAATTTGCAGCATATCTTGAACCATTCAAAGAGAAAGGTGAAGTTCGTCGTCCAACATTATCATGGCCTCGTGAAATCCCGTTAGTAAAAGGTGGTAAACCTGACGTTGTACAAATTGTTAGGAATTATAATGCTTATCTACGTGCAAGTGATGATTTACCAAAAATGTTTATTGAATCGGACCCAGGATTCTTTTCCAATGCTATTGTTGAAGGTGCCAAGAAGTTTCCTAATACTGAATTTGTCAAAGTAAAAGGTCTTCATTTTTCGCAAGAAGATGCACCTGATGAAATGGGAAAATATATCAAATCGTTCGTTGAGCGAGTTCTCAAAAATGAACAATAA.

preferably, the secretory protein IBIS1 has the amino acid sequence of SEQ ID NO.4 and the nucleotide sequence of SEQ ID NO. 5.

The amino acid sequence shown in SEQ ID NO.4 is as follows:

MARNFECKKINSDDMTSSKKYSKNVGEKFNLISCTKLFALSMLFLICQNYENSPQSTSSHQEYQYNGLVLGNRILSELDQAENHTISYKTNNYEDSSVENPNQQTSDSSSLQTDEDKKKDDSDATSIGETSPTTETTSVEETVTIEDTESVEETESVEETPSTSTEETSSTGKKTYVDRIASILNPLINGEKKSTEKKSSEKKSSEKKSSDEQSSSDEQNSSDDQNSFDDQKLFEDIDNLINGIKSRYQEFSAKIKSPEFQNKCKSYMNTAKEMIEERRNCAMSFISRNLNALGIDKIFEDEFGGYALLGKMMLTKVFIDNMFIPDFLRNSSTIILTIVYFLIMMFIVGSYLDINQDTKTERRNTNESKLFNRTQPPM.

the nucleotide sequence shown in SEQ ID NO.5 is as follows:

ATGGCTCGTAATTTTGAATGCAAAAAGATAAATAGTGATGATATGACATCATCCAAGAAATATTCCAAAAATGTTGGCGAGAAATTTAATTTGATTTCTTGTACAAAATTGTTTGCGCTAAGCATGTTATTTTTGATATGCCAAAATTATGAAAATAGCCCACAAAGCACATCTTCACACCAAGAATACCAATATAATGGTTTGGTTTTAGGAAACAGAATATTATCAGAATTGGATCAAGCTGAAAATCATACTATAAGTTATAAAACAAACAATTATGAAGACTCTTCGGTTGAAAATCCAAATCAGCAAACCTCCGATAGTTCATCATTACAAACTGATGAAGATAAAAAAAAAGATGACAGTGATGCAACATCCATTGGAGAAACATCACCAACTACAGAAACGACATCAGTTGAAGAAACAGTAACAATTGAAGACACAGAATCAGTTGAAGAAACAGAATCAGTTGAAGAAACACCATCAACATCAACTGAAGAAACATCATCAACTGGAAAAAAAACATATGTTGATAGAATAGCTTCCATTTTAAATCCATTAATCAATGGCGAAAAAAAATCTACCGAAAAAAAATCTAGTGAAAAAAAATCTAGTGAAAAAAAATCTTCTGATGAACAAAGCTCTTCTGATGAACAAAATTCTTCTGATGACCAAAATTCTTTTGATGACCAAAAACTATTTGAAGATATTGATAATCTAATAAATGGAATCAAATCACGTTATCAAGAGTTCAGCGCTAAAATAAAATCACCAGAATTCCAAAACAAATGTAAAAGCTATATGAATACTGCAAAAGAAATGATTGAAGAACGTAGAAACTGTGCTATGAGCTTTATATCTAGAAATTTAAATGCCTTAGGTATTGATAAGATATTCGAAGATGAGTTTGGTGGTTATGCACTCCTTGGAAAAATGATGTTAACAAAAGTCTTTATTGACAATATGTTCATTCCTGACTTCTTAAGAAATAGCTCAACAATAATTTTAACTATAGTTTATTTCTTAATAATGATGTTCATCGTAGGAAGCTATCTTGATATCAATCAAGATACTAAAACTGAAAGAAGAAATACAAATGAATCTAAATTGTTCAATAGAACACAACCACCTATGTAA.

in the present invention, the expression of tumor-specific antigens in plasmodium can be divided into two cases, i.e., secretory expression and non-secretory expression. Studies have shown that the non-secreted GPC3 protein is able to significantly activate CTLs in vivo against GPC3 protein and significantly inhibit tumor growth, while the secreted GPC3 protein is not able to significantly activate CTLs in vivo against GPC3 protein and inhibit tumor growth. It is demonstrated that in biological immunotherapy using plasmodium as a vector, the expression of tumor specific antigens should be preferentially considered non-secretory expression rather than secretory expression. However, if gene therapy is carried out using Plasmodium, such as expression of P53 or toxic proteins, a secreted protein expression format is contemplated.

In a third aspect, the present invention provides a vaccine comprising a recombinant plasmid according to the first aspect and/or a recombinant plasmodium according to the second aspect.

In the invention, biological immunotherapy of liver tumor always has the problem that activated tumor antigen specific cytotoxic T-lymphocytes (CTL) have strong but not continuous reaction, which greatly reduces the effect of immunotherapy, thus the treatment effect is not ideal. First, a prerequisite for a good increase in the CTL response in vivo is a priority for the evaluation of the expression vector. An important aspect of tumor immunotherapy is the activation of the CD8+ T cell response in vivo specific for tumors. It is not sufficient to use plasmodium as a vector to express only the tumor antigen, and it is also necessary to test whether the plasmodium vector provides good preconditions for activating the CD8+ T cell response in vivo. Herein, the present invention provides good conditions for tumor-specific antigen-activated CTLs and inhibition of tumor growth thereof, mainly by detecting Th1 response (T helper type 1 response) and the predilection differentiation of DCs in the early and middle stages of infection with Plasmodium vaccines.

In a fourth aspect, the present invention provides a recombinant plasmid according to the first aspect, a recombinant plasmodium according to the second aspect or a vaccine according to the third aspect for use in the treatment of liver tumors.

According to the invention, the treatment of liver tumors comprises in particular: combining the recombinant plasmodium as described in the second aspect with the erythrocytic stage of plasmodium to treat liver tumors; or immunizing the body with a vaccine according to the third aspect.

In a fifth aspect, the present invention provides a medicament for liver tumour therapy, the medicament comprising a recombinant plasmid according to the first aspect and/or a recombinant plasmodium according to the second aspect.

Compared with the prior art, the invention has the following beneficial effects:

(1) the pL0017 plasmid adopted by the invention uses a double enzyme digestion method (Bam HI/Xba I) to insert a target gene, complex means such as reverse gene insertion and re-sequencing identification in plasmid construction do not exist, and a new Plasmodium bereudii Elongation Factor (EF) -1 α promoter is introduced to simultaneously express two or even more than two exogenous genes;

(2) the invention provides a method for secretory expression and non-secretory expression of exogenous genes in plasmodium, and relates to application of IBIS1 protein. Different protein expression modes are flexibly selected according to the functions of the foreign proteins and the purpose of immunotherapy;

(3) compared with plasmid DNA and RNA vectors, the recombinant plasmodium provided by the invention can realize a large amount of amplification in vivo, is beneficial to the increase of antigens in vivo, has longer survival time in erythrocytes of a body compared with defective viruses and bacterial vectors, cannot be eliminated by an immune system of the body in a short time, can effectively express exogenous tumor antigens for a long time, and is beneficial to the long-term existence and immune stimulation of the antigens;

(4) the recombinant plasmodium provided by the invention is not only an expression vector, but also can regulate and activate natural immunity and acquired immunity of an organism, the up-regulated Th1 response and the increased proportion of CD8 α + DC are generated after the immunity is carried out, and the generation of CTL (cytotoxic T lymphocyte) aiming at tumor antigens is facilitated;

(5) the recombinant plasmodium provided by the invention can activate the generation of CTL (cytotoxic T lymphocyte) aiming at the tumor antigen (GPC3) in vivo, so that the anti-tumor effect of the vaccine is favorably improved, and the effects of inhibiting the tumor growth and improving the survival rate of tumor-bearing mice of the plasmodium vaccine expressing the tumor antigen are very obvious.

Drawings

FIG. 1 shows the improvement of pL0017 plasmid and the functional verification of fluorescent protein. Wherein, FIG. 1(A) is a schematic diagram of a method for inserting dual foreign gene expression elements; FIG. 1(B) shows the correct construction of pL 0017-gGluc-X plasmid for enzyme digestion identification, wherein channel 1 is a Bam HI/Xba I double enzyme digestion result, channel 2 is a Bam HI/Cla I double enzyme digestion result, and channel 3 is a Cla I/Not I double enzyme digestion result; FIG. 1(C) shows the results of electrophoretic validation of the insertion of gGluc (gfp-Rluc) and mCherry/gpc3 into the open reading frames ORF1 and ORF2, indicating that mCherry or gpc3 can be successfully inserted into ORF 2. In the identification of the plasmid pL 0017-gGluc-mCherry, a channel 1 is subjected to Bam HI/Xba I double enzyme digestion, a channel 2 is subjected to Bam HI/Cla I double enzyme digestion, a channel 3 is subjected to Cla I/Not I double enzyme digestion, and a channel 4 is subjected to Cla I/Xba I double enzyme digestion. In the identification of the plasmid pL 0017-gLuc-gpc 3, a channel 1 is subjected to Cla I/Xba I double enzyme digestion, a channel 2 is subjected to Not I/Cla I double enzyme digestion, a channel 3 is subjected to Bam HI/Not I double enzyme digestion, and a channel 4 is subjected to Bam HI/Xba I double enzyme digestion; FIG. 1(D) is a diagram of detecting the distribution of recombinant Plasmodium P.y-gGluc-mCherry and P.y-gGluc-ex _ mCherry in C57BL/6 mice under a small animal in-vivo imaging instrument; FIG. 1(E) is the confocal laser microscopy result of the constructed recombinant Plasmodium; FIG. 1(F) is a Western blot result of mCherry expressed by insertion in ORF2 in recombinant Plasmodium;

FIG. 2 shows that mouse GPC3 protein is stably expressed in murine Plasmodium yoelii, including secreted and non-secreted expression. Wherein, FIG. 2(A) is a schematic diagram of the insertion of different xgpc3 genes into the pL0017 plasmid site and the insertion of wild type Plasmodium chromosome into the c-rrna region; FIG. 2(B) is a schematic diagram showing the arrangement of the gpc3 gene after gene recombination and integration in the Plasmodium genome and the design of primers for identifying correct insertion; FIG. 2(C) shows PCR cloning of 5 'int, 3' int, gGluc and gpc3 gene fragments from recombinant Plasmodium genomes; FIG. 2(D) is a Western blot result of the expression of GPC3 protein in recombinant Plasmodium strains; FIG. 2(E) is a confocal laser microscopy showing the distribution of secreted (P.y-eGPC3:2F) and non-secreted (P.y-GPC3:2F) GPC3 protein in two recombinant Plasmodium species and erythrocytes; FIG. 2(F) is the GPC3 antibody labeled laser confocal microscopy observation of GPC3 expression and distribution in P.y-GPC3:2F strain in the different somatic stages of the erythrocytic stage (the ring, trophozoite, schizont and gametophyte stages of Plasmodium);

FIG. 3 shows the effect of plasmodium infection on the differentiation of mouse spleen DC cells, including the cell ratios of CD11c + CD8 α + DC and CD11c + CD8 α -DC and the expression levels of CD80 and CD86 protein molecules, wherein, FIG. 3(A) shows the ratio and statistical difference of P.y-WT to P.y-GPC3: CD8 α + DC in the spleen of 2F immunized and non-immunized mice, and FIG. 3(B) shows the expression ratios of CD80 to CD86 in CD8 α + DC and CD8 α -DC, and the statistical difference is P ≦ 0.05,. times.P ≦ 0.01,. times.P ≦ 0.001;

FIG. 4 shows the concentration measurements of Th 1-related cytokines including IL-2, TNF- α, and IFN- γ at different time points in serum after infection of C57BL/6 mice with Plasmodium falciparum, wherein the statistical differences are ≦ 0.05 ≦ P ≦ 0.01 ≦ P ≦ 0.001;

FIG. 5 IFN-. gamma. -ELISAPOT assay Cytotoxic T Lymphocyte (CTL) responses to GPC3 protein in mice immunized with each recombinant Plasmodium species and the statistical differences were P.ltoreq.0.05, P.ltoreq.0.01, P.ltoreq.0.001;

FIG. 6 shows the inhibition of liver tumors after 17 days of immunization of mice with different Plasmodium species, wherein FIG. 6(A) shows the growth of subcutaneous tumors in mice after 17 days of infection of tumor-bearing mice with P.y-WT, P.y-eGPC3:2F, P.y-GPC3:2F, while normal erythrocyte-immunized tumor-bearing mice are experimental controls, tumor size controls on the left, and volume and statistical difference comparisons of tumor sizes on the right, all statistical methods being T-test (unpaired two-labeled Student's T-tests) with P ≦ 0.05, { P ≦ 0.01 }, { P ≦ 0.001 }; FIG. 6(B) shows the detection of Ki67 expression in subcutaneous tumors of various groups of subcutaneous tumor-bearing mice by immunohistochemistry;

FIG. 7 shows the effect of P.y-GPC3:2F and P.y-WT Plasmodium immunization on tumor growth and survival in mice, control mice injected with normal erythrocytes, and a statistical difference marker on the left in FIG. 7; P.y-GPC3:2F was able to significantly increase survival time and survival rate in mice, the statistical difference in FIG. 7; fig. 7, right, is an infection rate curve with statistical differences: p is less than or equal to 0.05, P is less than or equal to 0.01, and P is less than or equal to 0.001.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.

1) Experimental Material

Plasmid 1.1 pL0017 used in this experiment was given by MR4(Malaria Research and ReferenceReagent Resource Center). the pL0017 plasmid contained the murine gene of Toxoplasma dihydrofolate reductase (tgdhfr/ts), which has pyrimethamine resistance.We cloned and inserted the foreign gene into the Bam H I and Xba I sites, replacing the gfp gene on the original plasmid, and the ORF of the inserted gene was transcribed by the P.burghii transcriptional elongation factor promoter (pbeef1 α a). pEGFP-N1(Clontech, #6085-1) plasmid was used to clone the egfp gene, pRL-SV40vector (Promega, # E2231) was used to clone the rRluc gene, and pmCherry-C1 plasmid (Clontech, #632524) was used for mCryry gene cloning.

1.2 primers. Primers for gfp (green fluorescent protein gene), rRluc (renilla luciferase gene), mCherry (red fluorescent protein gene), ibis1, and gpc3 (glypican 3) were designed using Primer premier 5.0 software, and synthesized by huada gene corporation. The plasmid preserved in the laboratory is used as a template to amplify the ORF of the required gene, and the primers required for the experiment can be searched in the first embodiment.

1.3 cloning of the main enzymes and related reagents. Bam H I (Thermo, FD0054), Not I (Thermo, FD0594), Cla I (Thermo, FD0144), Xba I (Thermo, FD0684), Eco31I (Thermo, ER 0291).

1.4 antibodies. GPC3 antibody (Aviva Systems Biology Corp., San Diego, CA, Rabbit, # ARP37665), Flag antibody (Sigma, monoclonal Anti-Flag)^@M2, mouse, #088K6018), the antibody AlexaFluor @488donkey Anti-mouse IgG (H + L) (Life technologies, #1423052), the Anti-mouse IgG-HRP antibody (Cell Signaling Technology, #7076), the Anti-Rabbit IgG-HRP antibody (Cell Signaling Technology, #7074), the mCherry antibody (Transgen).

1.5 mice, Plasmodium, and Hepa1-6 cells. Pathogen Free (Specific Pathologen Free SPF) grade female C57BL/6 mice, about 5 weeks old. Purchased from Leidel laboratory animals, Inc. of Shanghai (license: SCXK (HU) 2007-. The mice were raised by the laboratory animal center of Guangzhou biomedical and health research institute of Chinese academy of sciences, regularly in light for 12 hours and in darkness for 12 hours. The operation of animal experiments conforms to the relevant regulations of experimental animal use, and all experiments are approved by the laboratory animal use management committee of Guangzhou institute of biological medicine and health of Chinese academy of sciences. Murine non-lethal Plasmodium yoelii 17XNL (MRA-593) was gifted by MR4(the Malaria Research and reference Reagent Resource Center). Hepa1-6 was from the national Academy of Sciences Cell Bank (the Chinese Academy of Sciences, Shanghai, China).

1.6 other main reagents and experimental equipment. AxyPrep plasmid DNA small-scale kit (Axygen, # AP-MN-P-50), AxyPrep gel recovery kit (Axygen, AP-GX-50), peptone, yeast powder, NaCl, agarose, pyrimethamine; PCR apparatus (american society), agarose gel electrophoresis apparatus (six, beijing), ultraviolet spectrophotometer (beckmann), optical microscope (lycra), cryocentrifuge (Eppendorf, germany), biochemical incubator (shanghai-heng instrument), semi-dry membrane transfer apparatus (Bio-Red), electrotransfection apparatus (Amaxa, germany), C6, Arial, Fortessa flow cytometer (BD), laser confocal microscope (lycra).

EXAMPLE 1 preparation of recombinant plasmid pL 0017-gGluc-X

First, the basic molecular cloning technique used in this experiment:

1) and (4) extracting plasmids.

This was done with reference to the AxyPrep plasmid DNA minikit instructions from Axygen (Axygen, # AP-MN-P-50).

2) Extracting gel DNA fragments.

Reference is made to AxyPrep DNA gel recovery kit instructions from Axygen.

3) And (3) carrying out enzyme digestion on DNA and identifying a vector.

Preparing a proper reaction system for plasmid DNA by using the corresponding restriction enzyme and a buffer solution matched with the restriction enzyme, carrying out water bath at 37 ℃ for 3-4 h, and identifying the enzyme digestion result.

The reaction system for recovering DNA by enzyme digestion is as follows: mu.l of 10 XBuffer, 20. mu.l of digested vector DNA, 0.5. mu.l each of the enzymes E1/E2 (restriction enzymes selected according to the purpose of the experiment), 6. mu.l of ddH₂O, total volume 30. mu.l.

4) And connecting the target fragment with a vector and transforming.

Referring to the TaKaRa T Vector instructions, the Vector and the amount of the DNA of interest are proportioned to be about 1: 4, adding Solution I (2X) containing ligase or rapid T4 ligase and buffer, ligating at 37 ℃ for 15-30min, and performing heat shock transformation, wherein Escherichia coli is competent to be purchased from Transgen.

The specific transformation steps are as follows: (1) taking two tubes of frozen competent cells, adding 1 mul of plasmid DNA to be transformed or 10 mul of DNA ligation product into one tube, mixing uniformly, using the other tube as negative control, adding no DNA, and treating the other tubes in the same way;

(2) ice-cooling for 30min, heat-shocking at 42 deg.C for 90sec, and immediately ice-cooling for 5 min;

(3) adding 500 mul of new nonresistant LB37 ℃ for shake culture for 1 hour, and coating LB on an LB solid culture medium plate containing corresponding antibiotics;

(4) inverting the plate, placing the plate in a constant-temperature incubator at 37 ℃ for culturing for 10-16 h, and observing the growth condition of bacterial colonies;

(5) selecting single colony number according to requirement, amplifying in LB corresponding to antibiotic, taking part in 15% glycerol, preserving bacteria, storing in-80 deg.C refrigerator, and extracting plasmid according to requirement.

5) And (4) determining the DNA sequence.

And (3) sending the DNA fragment, the vector or the bacterial liquid to Huada gene company for DNA sequence determination, detecting the sequence by software, and cloning correctly.

Primers required for cloning:

egfp-Bam H I-F(SEQ ID NO.6):5’-GGATCCATGGTGAGCAAGGGCGAGGAGCTGT-3’

egfp-R(SEQ ID NO.7):5’-CTGGATCATAAACTTTCGAAGTCATCTTGTACAGCTCGTCCATGCCGAGA-3’

Rluc-F(SEQ ID NO.8):5’-TCTCGGCATGGACGAGCTGTACAAGATGACTTCGAAAGTTTATGATCCAG-3’

Rluc-Not I–R(SEQ ID NO.9):5’-GCGGCCGCTTATTGTTCATTTTTGAGAGAACTCGC-3’

gpc3-Cla I–F(SEQ ID NO.10):5’-ATATCGATATGGCCGGGACCGTGCGCACCGCGT-3’

gpc3-Xba I–R(SEQ ID NO.11):5’-GTCTAGAGGTCAGTGCACCAGGAAAAAAAAGCAC-3’

3’UTR-pbeef1aa(F)-Not I(SEQ ID NO.12):5’-GCGGCCGCGATCCCGTTTTTCTTACTTATATAT-3’

3’UTR-pbeef1aa(R)-Cla I(SEQ ID NO.13):5’-ATATCGATCCCTATGTTTTATAAAATTTTTTAT-3’

gpc3-Bam H I-F(SEQ ID NO.14):5’-AGGATCCATGGCCGGGACCGTGCGCACCGCGT-3’

gpc3-Eco31I-Xba I-2Flag-R(SEQ ID NO.15):5’-GGTCTAGAGAGACCTTACTTATCGTCGTCATCCTTGTAATCCTTATCGTCGTCATCCTTGTAATCGTGCACCAGGAAAAAAAAGCACGCC-3’

ibis1-Bam H I–F(SEQ ID NO.16):5’-AGGATCCATGGCTCGTAATTTTGAATGCAAAA-3’

ibis1-linker-R(SEQ ID NO.17):5’-CCGCCAGATCCACCTCCACCACTTCCGCCACCTCCCATAGGTGGTTGTGTTCTATTGAA-3’

gpc3-F-linker(SEQ ID NO.18):5'-GGAGGTGGCGGAAGTGGTGGAGGTGGATCTGGCGGTGGAGGAAGCATGGCCGGGACCGTGCGCACCGCGT-3'

gpc3-Xba I-2Flag-R(SEQ ID NO.19):5-GGTCTAGAGTTACTTATCGTCGTCATCCTTGTAATCCTTATCGTCGTCATCCTTGTAATCGGTGATCTCGTTGTCCTTCTGATTT-3’

mCherry-Cla I-F(SEQ ID NO.20):5'-AATCGATATGGTGAGCAAGGGCGAGGAGGATA-3'

mCherry-XbaI-R(SEQ ID NO.21):5'-AGTCTAGATTACTTGTACAGCTCGTCCATGCCGCCG-3'

PyL739(SEQ ID NO.22):5'-ATGTAATATTTGGATATTTC-3’

PyL740(SEQ ID NO.23):5'-TCACCTACGGAAACCTTGTTAC-3’

L665(SEQ ID NO.24):5'-GTTGAAAAATTAAAAAAAAAC-3’

L635(SEQ ID NO.25):5'-TTTCCCAGTCAGTCACGACGTTG-3’。

secondly, Trizol extracts the total mRNA of Hepa1-6 cells and P.y17XNL-WT plasmodium, and clones the gpc3 and ibis1 genes.

(1) Preparing a reagent: chloroform, isopropanol, 75% ethanol (aqueous DEPC), RNAase-free water or 0.5% SDS solution (water to RNase-free glass bottle, DEPC to a final concentration of 0.01% (V/V), overnight and high pressure, SDS was also prepared with treated DEPC water);

(2) adherently growing cells Hepa 1-6: 3.5cm diameter plate plus 1ml Trizol, blow repeatedly, (1ml Trizol for 10 cm)²The area of the complex is small, DNA pollution can be caused due to insufficient Trizol), and the complex is placed for 5min at room temperature to ensure complete dissociation of the nucleoprotein complex; P.y17XNL-WT plasmodium blood with infection rate more than 30%, naked plasmodium body obtained from erythrocyte lysate, and Trizol dissolution;

(3) adding 0.2mL chloroform to each 1mL Trizol in the prepared Hepa1-6 sample and naked plasmodium body, covering the tube cover, mixing by inverting for 15s, standing at room temperature for 2-3min, centrifuging at 4 deg.C for 15min, and not more than 12000 g;

(4) taking the upper water phase into a new tube, if DNA or protein is separated, the organic phase can be retained, 0.5ml isopropanol is added into each 1ml Trizol, and the incubation is carried out for 10min at room temperature;

(5)12000g 15min, 4 ℃. Removing supernatant, adding at least 1ml 75% ethanol per 1ml Trizol, mixing with vortex, 7500g × 5min, removing supernatant at 4 deg.C, short-time air drying RNA precipitate, dissolving with RNase-free water or 0.5% SDS to obtain Hepa1-6 cell and P.y17XNL-WT plasmodium total mRNA;

(6) blowing and beating for several times, incubating at 55-60 deg.C for 10min, and storing at-80 deg.C;

(7) obtaining total cDNA of a Hepa1-6 cell and plasmodium by RT-PCR;

a) adding 2 mul of extracted total RNA, 2 mul L M-MLV reverse transcription buffer,2 mul of dNTP and 1 mul of RNase into a PCR tube without RNase, adding 9 mul of ultra-pure water without RNase until the total volume is 16 mul, and fully and uniformly mixing;

b) taking two new PCR tubes without RNase, respectively marking RT + and RT-, and equally dividing the 16 mu l mixed solution into the two tubes;

c) mu.l of gpc3 or ibis1 Reverse primer was added to each tube, 1. mu.l of M-MLV Reverse Transcriptase was added to the RT + tube, 1. mu.l of RNase-free ultrapure water was added to the RT-tube, and the mixture was mixed well.

d) Reverse transcription is carried out for 1 hour in a water area at 42 ℃;

e) inactivating reverse transcriptase at 93 deg.C for 3 min;

f) taking the reverse transcribed cDNA as a template, carrying out PCR and electrophoresis detection, and cloning genes of gpc3 and ibis 1;

and thirdly, cloning genes of mCherry (Cla I/Xba I), ibis 1-mChery (Cla I/Xba I), gpc3(Cla I/Xba I), 3' UTR-pbeef1aa (Not I/Cla I), gGluc (BamH I/Not I), gpc3:2F (BamH I/Xba I) and ibis1-gpc3:2F (BamH I/Xba I).

According to the above-mentioned molecular cloning technique and the synthesized gene cloning primer (the primer sequence is shown in primer portion of experimental material), making it pass through reverse transcription kit (

II 1st strand cDNA Synthesis kit, Takara) to obtain total cDNA of Hepa1-6 cells and P.y17XNL-WT plasmodium, using the total cDNA as a template to clone a gpc3(ClaI/Xba I) gene, connecting the gene to a pMD18T vector (TAKARA), carrying out heat shock transformation into Escherichia coli to obtain a single clone strain, and carrying out sequencing and identification.

Fourthly, a new plasmid pL 0017-gGluc-X plasmid containing two independent expression frame elements is constructed, and a pL 0017-gGluc-ibisi-mChery plasmid, a pL 0017-gGluc-gpc 3 plasmid, a pL0017-gpc3 plasmid and a pL0017-ibis1-gpc3 plasmid are constructed on the basis.

The successfully cloned gene fragments mCherry, ibis1-mCherry, gLuc (fusion gene of gfp and Rluc), gpc3:2F, ibis1-gpc3:2F, and 3 ' UTR-pbeef1 α a were inserted between BamHI/XbaI cleavage sites of pL0017 plasmid as shown in FIG. 1(A), wherein the first open reading frame ORF1 was inserted into gLuc gene, and in order to allow the second open reading frame ORF2 to successfully express the target gene, we added a new terminator (3 ' UTR) before ORF2, which was used to terminate normal transcription of ORF1 gene, and after 3 ' UTR we inserted a new promoter pbeef 8a to initiate gene expression of ORF HI 2, and constructed a new plasmid 0017-gLuc-X (FIG. 1(B), which contains no foreign gene insert into the plasmid BamHI-GCIc 2, insert a plasmid containing double restriction endonuclease sequences of BamHI-GCIc-GCIh, and plasmid No restriction endonuclease map of BamHI-GCI7-GCIc 467-GCIc plasmid (FIG. 5-GCIc 467-GCIc), and map 1-GCIc plasmid containing double restriction enzyme cleavage sites of BamHI-GCIp 3-5-GCIp, ORF 5-GCIp, and plasmid (FIG. 5-GCIp 3-5-GCIp III), and plasmid containing no foreign gene insert plasmid III, and plasmid III).

Example 2 construction of recombinant Plasmodium

Firstly, extracting pL 0017-gGluc-mChery plasmid, pL 0017-gGluc-ibisi-mChery plasmid, pL 0017-gGluc-gpc 3 plasmid, pL0017-gpc3:2F plasmid and pL0017-ibis1-gpc3:2F plasmid in large dose.

1) Plasmid major grape

(1) Carrying out streak activation on strains containing a correctly constructed recombinant pL0017 plasmid, namely pL 0017-gGluc-mChery plasmid, pL 0017-gGluc-ibisi-mChery plasmid, pL 0017-gGluc-gpc 3 plasmid, pL0017-gpc3:2F plasmid and pL0017-ibis1-gpc3:2F plasmid, and culturing for 16h in an incubator;

(2) randomly picking 2 monoclonals, and transferring the monoclonals to a 15ml centrifugal tube for culture for 8-12 h;

(3) the culture solution is inoculated into a1 liter triangular flask containing 200ml of ampicillin-resistant LB culture medium in an amplification mode according to the inoculation amount of 1:500, shaking culture is carried out for 12 hours at 37 ℃, bacterium liquid is collected, plasmids are extracted by a QIAGEN large quality plasmid kit (QIfilterplasmid Maxi kit), the operation is carried out according to the instruction, the concentration and the purity of the plasmids are measured by an extracted ultraviolet spectrophotometer, and the plasmids are stored at-80 ℃ for later use.

Secondly, activation of plasmodium P.yoelii 17XNL, preparation of electrotransformation plasmodium and acquisition of recombinant plasmodium, wherein the plasmodium comprises P.y-gGluc-mCherry, P.y-gGluc-ex _ mCherry, P.y-GPC3, P.y-GPC3:2F and P.y-eGPC3: 2F.

(1) Taking the cryopreserved plasmodium yoelii 17XNL strain P.yoelii from the liquid nitrogen tank, and thawing the strain in a water bath at 37 ℃;

(2) injecting C57BL/6 mouse into abdominal cavity with 1ml syringe, injecting two mice, each insect blood is 0.5ml, diluting to 1% infection rate;

(3) and (3) starting the third day of inoculation, taking blood smears from the tail tips every day, observing the infection rate, dyeing the smears for 30min by using diluted giemsa dye liquor after the smears are fixed and air-dried by methanol, slowly washing the stains off by using clear water, counting the infection rate under a microscope oil microscope, and counting the average infection rate of each piece under 10 visual fields, namely the protozoan infection rate of the mouse. When the infection rate reaches more than 10%, the eyeball is picked to take blood and preserve the seeds, and 3.8% trisodium citrate solution or heparin sodium solution is anticoagulated;

(4) after blood collection is finished, uniformly mixing with equal volume of plasmodium cryopreservation liquid or 10% glycerol PBS solution, repeatedly reversing and uniformly mixing, subpackaging in cell cryopreservation tubes, and storing in a liquid nitrogen tank;

(5) collecting 20-50 μ L blood from mouse tail with infection rate of about 10%, dissolving in 0.6ml PBS, and performing intraperitoneal injection to 3C 57BL/6 mice, 0.2ml each as Day 0;

(6) on Day 4, starting to smear blood and observing the infection rate of each mouse, generally about Day 7-Day 10, and continuing to the next step when the infection rate is 10% -20%, and waiting until the infection rate is reached if the infection rate is not reached;

(7) this step was generally carried out at night in RPMI 1640 complete medium, 200ml, equipped with 10% FBS. Wherein 25mM HEPES (5.96g), 0.85g NaHCO3, 50mg neomycin sulfate are added into each 100ml of culture medium, and the mixture is filtered and sterilized at 0.22 mu M;

(8) 5ml of complete culture medium containing heparin sodium anticoagulation is added into a 50ml centrifuge tube. Performing heart sterile blood collection on the 3C 57BL/6 mice, adding the blood into the 50ml centrifuge tube, and centrifuging for 8min at 450 g/min;

(9) the blood pellet was resuspended in 50ml complete medium and 50 of this volume was addedThe ml is divided equally into two 250ml cell culture bottles, each bottle is supplemented with 25ml of complete culture medium, and the culture bottles are filled with medium containing 5% CO through a 0.22 mu m filter₂,5％O₂,90％N₂After the cover is tightly covered, the culture plate is placed in an incubator at 37 ℃, and is slowly shaken for culture overnight;

(10) opening a culture bottle, taking 0.5ml of culture solution into a 1.5ml EP tube, centrifuging for 5s at the maximum speed, removing the supernatant, taking a sediment smear for dyeing, if the majority of the sediment smear is mature schizont under the microscope visual field, then, waiting for 1-2 hours before coating for observation, and finishing in-vitro culture generally 12-16 hours after culture;

(11) evenly dividing 100ml of cultured plasmodium culture into 3 centrifugal tubes of 50ml, evenly mixing 30ml of Nycodenz and 20ml of PBS by using a sterile Bass tube to prepare 60% Nycodenz liquid, carefully adding the Nycodenz liquid into a centrifugal tube filled with the plasmodium culture from the bottom, evenly mixing, centrifuging at the room temperature of 450g/min for 20min by using a horizontal rotary head, wherein the centrifugal starting acceleration is 3, and the centrifugal braking acceleration is 0.

(12) Carefully collecting the middle layer after centrifugation by using a Pasteur tube, collecting 20-25ml by using 3 tubes, adding fresh complete worm culture medium to the total volume of 40ml, centrifuging for 8min by using 450g, discarding supernatant, and carefully re-suspending the worms in 1ml of electrotransformation solution;

(13) subpackaging into 10 sterile 1.5mL EP tubes, 10 uL 10 ug-30 uG linearized plasmids including pL 0017-gGluc-mCherry plasmid, pL 0017-gGluc-ibisi-mChery plasmid, pL 0017-gGluc-gpc 3 plasmid, pL0017-gpc3:2F plasmid and pL0017-ibis1-sgpc3:2F, Sac II or Apa I enzyme for linearization;

(14) mixing, transferring into an electric cup, incubating plasmid and Plasmodium for 5min, and performing electric conversion with a nucleotide electric converter of Amaxa company with the procedure of T-16;

(15) after electrotransformation, the electroporated protozoa were carefully aspirated into a 1.5ml EP tube using a mini-pasteur tube, 50. mu.l of complete medium was added, a total of 150. mu.l was added, and 150. mu.l of the mixture was injected into a C57BL/6 mouse via the caudal vein using an insulin syringe;

(16) after 24h, mice were injected intraperitoneally with 200. mu.l pyrimethamine (pH 4.0, dissolved in DMSO and in PBS at a dose of 1mg/kg or less) daily for selection of recombinant insect strains. Cutting a tail tip blood smear every day after four days to observe whether plasmodium exists or not;

(17) when the infection rate reaches 10%, transferring a new mouse, taking out blood eyeballs of the mouse, collecting blood by heparin anticoagulation, mixing with equal volume of plasmodium cryopreservation liquid, mixing uniformly, subpackaging in a cell cryopreservation tube, and storing in a liquid nitrogen tank.

Extraction and identification of recombinant plasmodium yoelii genome DNA

(1) In this experiment we used the genomic DNA of wild type P.y17XNL-WT as a negative control. When the infection rate of P.y-GPC3, P.y-GPC3:2F and P.y-eGPC3:2F insect strains in mice reaches 10%, the eyeballs are picked to take blood, 3.8% trisodium citrate or heparin sodium is used for anticoagulation, the blood is collected in a 1.5ml EP tube, the tube is centrifuged for 8min at 450g/min, supernatant blood is discarded, the tube is resuspended and cleaned by PBS once, and the tube is centrifuged for 8min at 450 g/min;

(2) lysing with erythrocyte lysate, placing on ice for 5min, reversing and uniformly mixing for 2-3 times, centrifuging for 8min at 600g/min, discarding the supernatant, carrying out heavy suspension and cleaning once with PBS (phosphate buffer solution), centrifuging for 8min at 450g/min, discarding the supernatant, precipitating the nude plasmodium released from the erythrocytes, carrying out heavy suspension with 200 mu l of PBS, and extracting plasmodium genomic DNA with QIAamp DNA Blood Kits of QIAGEN company;

(3) after quantification and concentration determination by an ultraviolet spectrophotometer, the solution was stored at-20 ℃.

PCR identification of recombinant P.yoelii P.y-GPC3, P.y-GPC3:2F, and P.y-eGPC3: 2F.

(1) Taking genomes of wild plasmodium and recombined P.y-GPC3, P.y-GPC3:2F and P.y-eGPC3:2F as templates, taking 500ng as a template for PCR reaction, preparing a PCR reaction system, and taking the genome of the wild plasmodium yoelii as a negative control;

(2) the method comprises the step of verifying the cloning of gpc3 (containing ibis1-gpc3), gGluc, 5 'int and 3' int, wherein the cloning of 5 'int and 3' int is successful, and the pL0017 sequence is successfully and stably inserted into a plasmodium genome c-rrna element (figure 2(A) and figure 2 (B)).

The primers used in this identification are as described in example one, with the primers used to identify gpc3 being gpc3-Cla I-F (SEQ ID NO.10) and gpc3-Xba I-R (SEQ ID NO. 11); primers used for identifying ibis1-gpc3 are ibis1-BamH I-F (SEQ ID NO.16) and gpc3-Xba I-R (SEQ ID NO. 11); primers used for identifying gRluc are egfp-Bam HI-F (SEQ ID NO.6) and Rluc-Not I-R (SEQ ID NO. 9); primers used for identifying 5' int are L635(SEQ ID NO.25) and PyL739(SEQ ID NO. 22); primers used to identify 3' int were PyL740(SEQ ID NO.23) and L665(SEQ ID NO. 24).

The PCR amplification reaction system is as follows:

one group of negative controls was water samples.

The reaction conditions were as follows:

(3) after the PCR is finished, 1% agarose gel is prepared, the sample is subjected to electrophoresis and is imaged on a gel imager, and whether the gpc3 gene exists in the plasmodium genome after the electric transformation is identified.

As shown in FIG. 2(C), it can be seen that, compared with the negative control P.y-WT, 5 'int and 3' int were successfully detected in P.y-GPC3:2F, P.y-GPC3 and P.y-eGPC3:2F, indicating that the various engineered pL0017 plasmids were successfully embedded into the genome of Plasmodium.

5) Extraction of recombinant Plasmodium proteins

(1) When the infection rate of the mice newly infected with the insects reaches more than 10%, picking eyeballs and taking blood, anticoagulating heparin, collecting the blood in a 1.5ml EP tube, centrifuging for 8min at 450g/min, discarding supernatant plasma, carrying out heavy suspension cleaning once by PBS, and centrifuging for 8min at 450 g/min;

(2) lysing with erythrocyte lysate, placing on ice for 5min, reversing and mixing uniformly for 2-3 times, centrifuging for 8min at 600g/min, discarding supernatant, resuspending and cleaning with PBS once, centrifuging for 8min at 450g/min, discarding supernatant;

(3) precipitating to obtain naked plasmodium released from erythrocyte, adding 300 μ l RIPA into the plasmodium, rapidly blowing with gun, mixing, placing on ice, cracking for 20min, flicking every 2-3min to open the tube wall, and centrifuging at 4 deg.C 12000g/min for 20 min;

(4) the supernatant was transferred to a new tube, and the concentration of the protein was determined using a protein quantification kit and recorded. To each sample was added 1/4 volumes of 5 × loading buffer, mixed well with a gun, boiled in a boiling water bath for 5min, and centrifuged at 4 ℃ for 10min at maximum speed.

(5) The supernatant was transferred to a new tube and split charged and stored in a refrigerator at-80 ℃.

6) Western blot identification of GPC3 expression GPC3 protein in recombinant plasmodium and mCherry expression in mCherry-expressing plasmodium

(1) Cleaning the glass plate: one hand fastens the glass plate and the other hand dips in washing powder and lightly scrubs. Washing both sides with tap water, washing with distilled water, and air drying;

(2) pouring glue and loading sample: after being aligned, the glass plate is placed into a clamp for clamping, and then the glass plate is vertically clamped on a frame for preparing glue pouring: preparing 10% separating glue according to the formula, adding TEMED, immediately shaking up to fill the glue, sucking 5ml of glue with a 10ml gun and discharging along the glass when filling the glue, and when the glue surface rises to the height of the middle line of the green belt. The gelation was accelerated by liquid-sealing with isopropanol. Standing at room temperature for 30min, pouring off isopropanol when gelation is observed, washing with clear water, and blotting with absorbent paper. 4 percent of concentrated glue is added, and the mixture is immediately shaken up after TEMED is added, so that glue can be filled. The remaining space was filled with the gel concentrate and a comb was then inserted into the gel concentrate. Because the volume of the gel is reduced due to shrinkage during solidification, the sample loading volume of the sample loading hole is reduced, and therefore, the gel is frequently supplemented on two sides in the solidification process of the concentrated gel. After the concentrated gel is solidified, the two hands respectively hold the two sides of the comb and slightly pull the comb out vertically and upwards. Washing the concentrated gel with water, and placing the gel into an electrophoresis tank;

(3) loading the extracted protein sample, loading 30 mu g of the protein sample to each electrophoresis hole (comprising P.y-GPC3, P.y-GPC3:2F, P.y-gGluc-mCherry and P.y-gGluc-ex _ mCherry), starting to prepare the sample after adding enough electrophoresis liquid (the electrophoresis liquid at least needs to spread over a small glass plate for internal measurement), sucking the sample by adhering a microsyringe, sucking the sample out without sucking air bubbles, inserting a sample injector needle into the sample adding hole, slowly adding the sample (the sample can be flushed out of the sample adding hole when the sample is too fast, the sample can overflow if the air bubbles exist, and when the next sample is added, the sample injector needs to be washed in an external groove electrophoresis buffer solution for several times so as to avoid cross contamination);

(4) electrophoresis: the electrophoresis time is generally 4-5 h, the voltage is preferably 40V, or 60V (80V for increasing the speed, and 140V for 120-140V after the gel is concentrated and separated, and the result is good, and the total time is about 2 h) can be used for electrophoresis until the bromophenol blue just runs out, the electrophoresis can be stopped, and the membrane is transferred;

(5) film transfer: wetting a PVDF membrane in methanol before membrane conversion, then soaking in a membrane conversion buffer solution, cutting off concentrated gel of the gel, soaking the gel in the membrane conversion buffer solution, wetting two pieces of thick filter paper by the membrane conversion buffer solution, assembling a membrane conversion sandwich layer, sequentially arranging the filter paper, the PVDF membrane, the gel and the filter paper from bottom to top, connecting a power supply, and performing constant-current 60mA membrane conversion for 1 hour;

(6) and (3) sealing: washing the membrane by PBST, soaking the membrane in a sealing solution of 5% skimmed milk powder prepared by PBST, and sealing overnight at 4 ℃;

(7) primary antibody hybridization: washing the sealed membrane by PBST, soaking the membrane in an antibody (1: 2000) of rabbit GPC3 prepared by PBST or a mouse Flag antibody and a primary antibody hybridization solution of mCherry, and gently shaking the membrane in the temperature for hybridization for 2 hours;

(8) washing the membrane: washing the membrane with primary antibody hybridization with PBST for 6 times, each time for 4min, and shaking on a shaker at high speed;

(9) and (3) hybridization of a second antibody: soaking the washed membrane in a second antibody hybridization solution of horseradish peroxidase labeled anti-rabbit IgG (1: 2000) or anti-mouse IgG (1: 2000) prepared by a confining liquid, and carrying out hybridization for 1 hour by gentle shaking in the chamber;

(10) washing the membrane: washing the membrane hybridized by the second antibody with PBST for 6 times, each time for 4min, and washing with high-speed shaking on a shaking table;

(11) chemiluminescence, development, fixation: mixing the reagent A and the reagent B on the preservative film in equal volume; after 1min, the membrane protein surface is downward and fully contacted with the mixed solution; after 1min, the film was transferred to another plastic wrap, the residual liquid was removed, wrapped and placed in an X-ray film holder. In a dark room, pouring 1 Xdeveloping solution and fixing solution into a plastic tray respectively; taking out the X-ray film under a red light, and cutting the film into a proper size (the size is 1cm larger than the length and the width of the film) by using a paper cutter; opening the X-ray film holder, placing the X-ray film on the film, once the X-ray film is placed on the film, the X-ray film holder cannot be moved, closing the X-ray film holder, and starting timing; properly adjusting the exposure time according to the strength of the signal, generally 10min, and optionally pressing for multiple times at different times to achieve the best effect; after exposure, opening the X-ray film clamp, taking out the X-ray film, quickly immersing the X-ray film in a developing solution for development, and stopping development immediately after an obvious strip appears. The developing time is generally 1-2 min (20-25 ℃), and when the temperature is too low (lower than 16 ℃), the developing time needs to be properly prolonged; after the development is finished, immediately immersing the X-ray film into the fixing solution, wherein the fixing time is generally 5-10 min, and the film is transparent; after washing off the residual fixer with tap water, the plate was dried at room temperature.

The results are shown in FIG. 1(F), and it can be seen that the mCherry protein is successfully expressed in P.y-gGluc-mCherry and P.y-gGluc-ex _ mCherry strains, which indicates that the mCherry protein connected with the IBIS1 protein has partial shearing, part of the mChery protein can exist in the form of independent protein, and the IBIS1 protein mainly functions in transporting the protein outside the insect body. Meanwhile, as shown in FIG. 2(D), it can be seen that both P.y-GPC3 (the insect strain expresses gGluc) with double ORF expressing GPC3 and P.y-GPC3:2F (the insect strain does not express gGluc) with single ORF expressing GPC3 can successfully express GPC3, GPC3 protein is successfully expressed in P.y-GPC3:2F and P.y-GPC3 insect strains, and the control group is protein sample prepared by cracking P.y-WT plasmodium.

7) Laser confocal microscope observation mCherry and GPC3 protein distribution in plasmodium

(1) Activation of P.y-gGluc-mCherry, P.y-gGluc-ex _ mCherry, P.y-WT, P.y-GPC3:2F and P.y-eGPC3:2F in C57BL/6 mice;

(2) blood was drawn from the tail of the mice at different times and plated on Concanavalin a (Sigma) -treated slides. Fixing with PBS containing 4% PFA and 0.0075% glutaraldehyde or 4% PFA at room temperature for 20min, and fixing with ice methanol for 10 s;

(3) 5% fetal bovine serum blocking sample, 1% Triton X-100 permeabilized cell sample for 15 min;

(4) PBS containing 2% FBS was washed 3 times for 5min each. Incubating the sample with anti-GPC 3 antibody or anti-Flag antibody at 4 ℃ overnight;

(5) washing with PBS containing 2% FBS for 3 times, each time for 5min, incubating with fluorescently-labeled secondary antibody at room temperature for 1 hr, and keeping out of the sun;

(6) washing with PBS containing 2% FBS for 3 times, each for 5min, and incubating the sample with DAPI working solution (Biyunyan day) for 15 min;

(7) washing with PBS containing 2% FBS for 3 times, each for 5min, and sealing with anti-fluorescence quenching sealing liquid (Biyuntian);

(8) the expression and distribution of GPC3 protein in Plasmodium are observed by laser confocal fluorescence microscope (Lycra).

The results are shown in FIG. 1(E), and mCherry and GFP are successfully expressed in P.y-gGluc-mCherry, P.y-gGluc-ex _ mCherry, while the positioning of P.y-gGluc-ex _ mChery strain shows that IBIS1 protein successfully guides mChery protein out of plasmodium body and into erythrocyte. 2(E) and 2(F), it can be seen that FIG. 2(E) illustrates the successful transport of the GPC3 protein out of the Plasmodium and secretion into erythrocytes by the IBIS1 protein in the P.y-eGPC3:2F insect strain, which is consistent with the results of FIG. 1 (E); FIG. 2(F) shows the stable expression of GPC3 protein in P.y-GPC3:2F recombinant Plasmodium species in different somatic states during the erythrocytic stage.

8) In vivo imaging system for small animals to observe distribution of plasmodium in mice

(1) Injecting activated plasmodium P.y-WT, P.y-gGluc-mCherry, P.y-gGluc-ex _ mCherry into abdominal cavity of mice, and injecting plasmodium infected erythrocyte 5 x 10 into each mouse⁵One/only.

(2) After the mice were infected with plasmodium 7 days, D-fluorescein potassium salt was intraperitoneally injected at a dose of 15mg/ml, 200 μ l per mouse, and in vivo imaging observation was performed 10 minutes later.

As shown in FIG. 1(D), the Renilla luciferase proteins were successfully expressed in the strains P.y-gGluc-mCherry, P.y-gGluc-ex _ mCherry, and the distribution of these strains in vivo at the red phase was shown.

Example 3 immunoassay of murine Plasmodium yoelii immunized mice

Including CD8 α + DC, Th 1-related cytokines, and CTL response assays against GPC3 protein.

1. Experimental materials C57BL/6 mouse, Hepa1-6 cell, penicillin and streptomycin Bio Basic Inc., pancreatin Amresco anti-CD 11C-FITC anti-CD 8 α -PE anti-CD 86-APC, anti-CD 80-PerCP-Cy5.5 antibody (available from eBioscience, San Diego, CA, USA), BD FACSAria flow cytometer, FlowJo analysis software (Tree Star, Inc.), mouse Th related multifactorial detection Kit (BioLegend), BDM ELISPOT MouseeI-gamma Kit (BD Biosciences, #552569), Fortessa flow analyzer (BD), Legendlex analysis software (LGLegend), 1 Xerythrocyte lysate (BD), 200 mesh cell filter, 1mL syringe, German cryo magnetic bead (CSF), Fortessa flow analyzer (BD), Legendlex culture medium (GM-Biotech), 1 Xerythrocyte lysate (BD), 1 XE culture medium (GM-Biotech), PCR culture medium (GM-PCR medium), PCR culture medium (GM-BCE) 2-CD medium), PCR culture medium (GM-BCE) culture medium), PCR culture medium (GM-CD 2-DME), PCR culture medium (GM-CD culture medium), PCR culture medium (GM-GM culture medium, PCR (BioLenge) and PCR culture medium)

2. The recombinant mouse plasmodium yoelii immune tumor-bearing mouse detects the change condition of CD8 α + DC cells and the expression condition of CD80 and CD86 in the spleen.

1) Taking out frozen Hepa1-6 cells from liquid nitrogen, thawing at 37 ℃, and activating and culturing cells;

2) taking out the cryopreserved P.y-WT and P.y-GPC3:2F plasmodium strains from liquid nitrogen, dissolving and injecting the strains into the abdominal cavity of two mice;

3) after 3 days, observing the infection rate of plasmodium in the mice, and counting the infection rate of the plasmodium and the concentration (per ml) of red blood cells when the infection rate is between 3 and 5 percent;

4) the immunized mice were divided into three groups of 4 mice, the first group was inoculated with P.yoelii P.y-WT in an amount of 5 x 10⁵One/one; the second group was inoculated with empty vector-transfected P.beijerinckii P.y-GPC3:2F, inoculated withThe amount is 5 x 10⁵One/one; the third group was inoculated with the same number of red blood cells of normal mice as a blank control group. The day was designated D0;

5) at the same time, three groups of experimental mice were inoculated subcutaneously in the back with Hepa1-6 cells 5 x 10⁵One/one;

6) on the fourteenth day after the mice were inoculated with plasmodium and tumor cells, spleens were removed and splenocytes were isolated. Filtering spleen cells with a 200-mesh filter screen;

7) adding erythrocyte lysate (BD), lysing on ice for 20min, and centrifuging at 300g for 5 min;

8) washed twice with PBS containing 2% FBS and centrifuged at 300g for 5 min. If the red blood cells are not fully cracked, the red blood cell lysate is cracked once on ice;

9) filtering spleen cells once again by using a 200-mesh filter screen;

10) staining, removing 106 spleen cells, staining the samples with flow antibodies CD11c-FITC, CD8 α -PE, CD86-APC, CD80-PerCP-Cy5.5 (purchased from eBioscience, San Diego, Calif., USA), wherein unstained samples, CD11c-FITC single staining, CD11c-FITC and CD8 α -PE double staining are used as negative controls, and each sample is stained in dark for 10 min;

11) washing twice with PBS containing 2% FBS, and centrifuging at 300g for 5 min;

12) and detecting the cell sample on a flow type computer. The flow meter was BD FACSAria, and the flow analysis software was FlowJo (TreeStar, Inc.);

the results are shown in FIGS. 3(A) to 3(B), in which FIG. 3(A) to 3(B) show that the ratio of CD8 α + DC is increased by infection with Plasmodium, CD8 α + DC expresses CD80 and CD86 both higher than the expression of CD8 α -DC in mice, the ratio of CD8 α + DC in spleen of mice immunized with Plasmodium is significantly increased than that of mice immunized with no Plasmodium, and in each group of mice, CD80 and CD86 in CD8 α + DC in P.y-GPC3:2F immunization group are significantly increased compared with the other two groups, and CD80 and CD86 are both expressed in CD8 α + DC, while the expression ratio of CD80 to CD86, especially CD80 in CD8 α -DC is reduced.

3. The recombinant plasmodium yoelii immunizes tumor-bearing mice, and detects the expression condition of Th1 related cell factors in the serum of the immunized mice.

4) the immunized mice were divided into three groups of 4 mice, the first group was inoculated with P.yoelii P.y-WT in an amount of 5 x 10⁵One/one; the second group was inoculated with empty vector-transfected P.beijerinckii P.y-GPC3:2F in an amount of 5X 10⁵One/one; the third group was inoculated with the same number of red blood cells of normal mice as a blank control group. The day was designated D0;

5) at the same time, three groups of experimental mice were inoculated subcutaneously in the back with Hepa1-6 cells 5 x 10⁵One/only.

On the first, third, seventh and fourteenth days, i.e., D1, D3, D7 and D14, 150 μ l of blood was obtained by ocular aspiration with a capillary glass tube, and the same volume of saline was intraperitoneally injected. Standing the sample blood at normal temperature for 1h, centrifuging at 1000rpm/min for 10min after clotting, taking out supernatant serum, subpackaging 2 tubes, and freezing and storing in a refrigerator at-80 ℃.

The concentration of the cytokine in serum was measured using a BioLegend multifactorial detection kit, which detects the concentration of the Th 1-associated cytokine. The specific kit detection operation refers to product specifications and technical support.

The results are shown in fig. 4, the plasmodium carrier insect strain vaccine can effectively activate Th1 related cell factors including IL-2, IFN-gamma and TNF- α, the detection result shows that the concentrations of the two factors reach the highest value at 7 days of immunization and are significantly different from the control group, and the statistical difference analysis shows that P is less than or equal to 0.05, P is less than or equal to 0.01, P is less than or equal to 0.001.

4. ELISAPOT detection of recombinant P.yoelii immunized mice, CTL detection in vivo against GPC3 protein

1) In vitro culture of mouse BM-DC cells

(1) Get

C57BL/6 mice 4, killing the mice by dislocation of the neck, and soaking the mice in 70% ethanol for 10min for disinfection;

(2) peeling and shearing two hind legs of the mouse off by using sterilization forceps and scissors in a sterile operating platform, putting the mouse in a culture dish with the diameter of 10cm, soaking the mouse in a cell culture medium RPMI 1640, and peeling off muscles on the leg bones by using the scissors;

(3) obtaining a hind leg femur, cutting joints at two ends of the femur by using scissors, injecting an RPMI 1640 complete culture medium into a leg bone cavity by using an injector, and washing for multiple times to obtain cells in the bone cavity;

(4) centrifuging the cell sap obtained by filtering with a 200-mesh sterile filter screen for 8min at 300 g;

(5) taking a 15mL centrifuge tube, adding 2mL of erythrocyte lysate for resuspension and lysis of cells at the bottom of the centrifuge tube, lysing for 8min on ice, adding 5mL of PBS, centrifuging for 8min again at 300g, and washing again with PBS;

(6) the cells were plated on a 10cm culture dish, and RPMI 1640 culture medium containing 10% FBS, GM-CSF (50g/l) and 2-Me (50. mu.M) was added for induction culture;

(7) after culturing for 24-48 h, BM-DC cells will grow adherently and part of suspension cells will be removed. The culture was carried out for 7 days.

2) Obtaining spleen cells and CD8 α + T cells of plasmodium immune mice.

(1) Taking out frozen Hepa1-6 cells from liquid nitrogen, thawing at 37 ℃, and activating and culturing cells;

(2) taking out the frozen P.y-WT, P.y-GPC3:2F and P.y-eGPC3:2F plasmodium strains from liquid nitrogen, dissolving and injecting the strains into the abdominal cavity of two mice;

(3) after 3 days, observing the infection rate of plasmodium in the mice, and counting the infection rate of the plasmodium and the concentration (per ml) of red blood cells when the infection rate is between 3 and 5 percent;

(4) dividing the immunized mice into four groups of 5 mice, inoculating the first group with plasmodium yoelii P.y-WT, and inoculating 5 × 105 mice; the second group was inoculated with Plasmodium P.y-GPC3:2F at 5 x 10⁵One/one; third group of Plasmodium beijerinckii inoculated with empty vectorP.y-eGPC3:2F, inoculum size 5 x 10⁵One/one; the fourth group was inoculated with the same number of red blood cells of normal mice as a blank control group. The day was designated D0;

(5) at the same time, three groups of experimental mice were inoculated subcutaneously in the back with Hepa1-6 cells 5 x 10⁵One/one;

(6) on day 17 of plasmodium infection of mice, the mice were sacrificed and the spleen was removed and isolated to obtain spleen cells;

(7) aseptically placing spleen into a sterile plate containing RPMI 1640 complete medium, washing and carefully removing fascia and adipose tissue;

(8) placing a sterilized 200 mesh steel mesh on a small sterile plate, placing the spleen thereon and dropping 1ml of 1640 medium containing 2% FBS;

(9) 1ml of RPMI 1640 medium containing 2% FBS was extracted with a 2ml syringe and injected from the spleen side to swell the spleen;

(10) cutting spleen into 1-2mm small pieces with curved scissors, grinding with 2ml inner core of syringe, washing with 5-6ml RPMI 1640 medium containing 2% FBS to grind spleen cells, collecting cells into a 50ml centrifuge tube, and placing the tube into ice;

(11) after the spleen to be treated is sequentially operated according to the method, centrifuging at the temperature of 4 ℃ for 300g multiplied by 10min, removing supernatant, and gently shaking up cell sediment;

(12) adding into 10ml erythrocyte lysate, slowly shaking to resuspend the cells, gently moving to avoid forming cell clump, and standing at room temperature for 4-5 min;

(13) adding 25mL of RPMI 1640 medium containing 2% FBS to terminate lysis, centrifuging at 4 ℃ for 200g × 10min, discarding the supernatant, adding 15mL of culture solution, and slowly shaking to resuspend cells;

(14) standing the tube for 2-3min, taking the upper 12ml of cells for re-suspension, and transferring into another new tube;

(15) centrifuging at 4 deg.C for 200g × 10min, discarding supernatant, adding 6ml RPMI 1640 complete culture medium, and resuspending cells;

(16) and D, separating CD8 α + T cells in splenocytes by virtue of the magnetic beads of the America whirlwind mouse CD8 α.

3) CD8 α + T cells were co-cultured with BM-DC and IFN-. gamma.ELISPOT detected IFN-. gamma.secretion from CD8 α + T cells.

(1) CD8 α + T lymphocytes obtained from each mouse were counted and 2X 10 cells were removed⁶CD8 α + T lymphocytes were plated in 24-well plates at 5X 10 cells/well⁵Adding 400 μ l of complete medium containing 30 μ g/ml of mouse GPC3 protein into the pre-cultured BM-DC, and incubating for 7 days in a cell culture box;

(2) an ELISPOT 96-well plate pre-coated with IFN-gamma antibody was prepared, and 2X 10 wells were added per well⁵BM-DC pre-incubated with murine GPC3 protein and 5X 10⁵Adding 100 mu l of complete culture medium into each CD8 α + T cell, and incubating and culturing for 2 days in a cell culture box;

(3) removing the culture medium, washing with deionized water for 2 times, each for 5min (without touching the membrane during washing);

(4) dripping 200 μ l of Wash Buffer into each hole, and cleaning for 3 times, 5min each time;

(5) dropping 100 μ l Antibody detection Solution (depletion Antibody Solution) into each well, covering the cover, and incubating at room temperature for 2 hr;

(6) removing antibody detection solution, dripping 200 μ l Wash Buffer into each hole, and cleaning for 3 times, each time for 2 min;

(7) mu.l of HRP-labeled streptavidin (streptavidin-HRP) was added to each well. Covering the cover, and incubating at room temperature for 1 hour;

(8) removing the HRP-labeled streptomycin avidin, dropwise adding 200 mul of Wash Buffer into each hole, washing for 4 times, each time for 2min, washing for 2 times by PBS, and each hole for 200 mul;

(9) dropwise adding AEC solution (AEC substrate solution) into each hole for dyeing, and determining the color development time according to the color development condition, wherein the color development time is generally 5-60 min;

(10) the reaction was terminated by washing with deionized water. After the last washing, the plate is reversely buckled on clean absorbent paper;

(11) air-drying overnight in dark, removing the outer plastic plate frame, storing in dark, and reading with a plate reader.

As shown in FIG. 5, it was found that neither GPC 3-secreting recombinant Plasmodium P.y-eGPC3:2F nor wild-type Plasmodium P.y-WT immunized mice produced a specific CTL response against GPC 3. While P.y-GPC3:2F Plasmodium possessed the ability to activate a specific CTL response in the mouse against GPC3 and had significant differences (P.ltoreq.0.001. x.), it was found that P.y-GPC3:2F immunized mice were able to detect strong CTLs against GPC3, whereas no CTLs against GPC3 were detected in both secreted GPC 3-expressing insect strain P.y-eGPC3:2F and the control group.

Example 4 inhibitory Effect of recombinant Plasmodium yoelii on subcutaneous liver tumors in tumor-bearing mice

1. Test materials. Plasmodium yoelii, Hepa1-6 cells, vernier caliper, incubator, 4% paraformaldehyde, xylene, ethanol, sodium citrate, PBS, 3% H₂O₂Deionized water, hematoxylin, immunohistochemical detection kit (BD). Ki67 antibody (abcam, # ab 16667).

2. Inhibition effect of recombinant murine plasmodium yoelii immunization on subcutaneous tumor of tumor-bearing mice on day 14

2) taking out the frozen P.y-WT, P.y-GPC3:2F and P.y-eGPC3:2F plasmodium strains from liquid nitrogen, dissolving and injecting the strains into the abdominal cavity of two mice;

4) the immunized mice were divided into four groups of 5 mice each, and the first group was inoculated with Plasmodium yoelii P.y-WT in an amount of 5X 10⁵One/one; the second group was inoculated with Plasmodium P.y-GPC3:2F at 5X 10⁵One/one; the third group was inoculated with empty vector-transfected P.beijerinckii P.y-eGPC3:2F at an inoculum size of 5X 10⁵One/one; the fourth group was inoculated with the same number of red blood cells of normal mice as a blank control group. The day was designated D0;

5) at the same time, each mouse was subcutaneously inoculated with 5X 10 Hepa1-6 cells per group⁵One/one;

6) measuring the size of subcutaneous tumor, insect blood infection rate and survival condition of the mice from day 7;

7) on day 14, the mouse tumors were removed and photographed with a ruler.

The results are shown in fig. 6, and the tumor volume calculation formula is: v ═ ab²) And/2, wherein a is the length of the tumor and b is the width of the tumor. Immunohistochemistry measured Ki67 expression in each group of tumors. The results showed that in mice immunized with Plasmodium, P.y-eGPC3:2F or P.y-WT immunization had some tumor-inhibiting effect (P.ltoreq.0.05X) compared to the control group not immunized with Plasmodium, while Hepa1-6-P.y-GPC3:2F had the most significant tumor-inhibiting effect (P.ltoreq.0.001X) compared to the control group, and it also had a significant tumor-inhibiting effect (P.ltoreq.0.01X) compared to the P.y-eGPC3:2F or P.y-WT treated groups. Immunohistochemistry results also indicated that Ki67 expression was effectively inhibited in the plasmodium-treated group tumors.

3. Subcutaneous tumor of tumor-bearing mice immunized by plasmodium, distribution of Ki67 in tumor was detected

1) The stripped subcutaneous tumor of the mouse is fixed with 4 percent paraformaldehyde, and the distribution condition of Ki67 in the tumor is detected by immunohistochemistry;

2) and (6) washing. After the material is fixed, the fixing solution in the tissue must be washed clean, because the fixing solution remained in the tissue is not beneficial to dyeing in some cases, and precipitation or crystallization is generated in some cases to influence observation;

4) and (4) dehydrating. Dehydrating with 30%, 50%, 70%, 80%, 90% ethanol solution for 40min, and placing into 95% and 100% ethanol solution twice each for 20min (after fixing and washing various materials, the tissue contains a large amount of water, and water and paraffin are insoluble, so water in the tissue must be removed);

5) and (4) transparent. The mixture of 100% alcohol and xylene is mixed for 15min, and xylene is replaced once for 0.5h (or until it is transparent). (because ethanol is not soluble in paraffin, and xylene can be soluble in both ethanol and paraffin, so the transition is carried out through xylene after dehydration, when the tissue is totally occupied by xylene, light can penetrate through the tissue, and the tissue presents a transparent state with different degrees);

6) and (4) wax penetration. And adding a mixed solution of xylene and paraffin for 15min, and adding paraffin I and paraffin II for waxing for 20-30 min respectively. (the purpose of wax penetration is to remove clearing agents such as xylene and the like in tissues, so that paraffin penetrates into the tissues to reach a saturation degree for embedding, the wax penetration time is longer according to the wax penetration time of the tissues, about 1-2day is needed, the wax penetration is carried out in an incubator, the temperature in the incubator is kept to be about 55-60 ℃, the temperature is not required to be too high so as to avoid the tissues from being crisp, and the incubator is placed for 0.5 h);

7) and (4) embedding. Pouring the waxed tissue together with the melted paraffin into a container, and then immediately putting into cold water to solidify into a wax block immediately. The melting point of the paraffin for embedding is between 50 and 60 ℃, and the paraffin with different melting points is selected according to factors such as tissue materials, slice thickness, weather conditions and the like during embedding. The melting point of paraffin commonly used for animal materials is 52-56 ℃;

8) slicing;

9) and (3) pasting.

The SP (streptavidin-peroxidase) method is adopted, and the main steps are as follows:

1) baking the tissue slices in a constant temperature oven at 60 ℃ for 20 min;

2) soaking the tissue slices in xylene (I) for 10min, and soaking for 10min after the tissue slices are replaced by xylene (II);

3) soaking in anhydrous ethanol (I) for 5 min;

4) soaking in anhydrous ethanol (II) for 2 min;

5) soaking in 95% ethanol for 2 min;

6) soaking in 70% ethanol for 2 min;

7) soaking in single distilled water for 2 min;

8) antigen retrieval: repairing with 0.01M sodium citrate by microwave method;

9) standing for 20min to restore the slice to room temperature, and soaking and washing in triple distilled water;

10) washing and soaking for 5min with PBS;

11)3％H₂O₂standing with deionized water at room temperature for 10min, and washing with TBST for 5min for 2-3 times;

12) adding dropwise normal goat serum confining liquid, and keeping room temperature for 15 min;

13) excess liquid was spun off and rabbit anti-CD 8a antibody (1: 200)4 ℃ overnight; 4 ℃ overnight; washing with TBST for 3 times for 3 min;

14) dropwise adding 40-50 μ l of biotinylated secondary antibody, and standing at room temperature for 30 min; washing with TBST for 3 times for 3 min;

15) dripping 40-50 μ l of antibiotic labeled horse radish peroxidase, standing at room temperature for 30min, washing with TBST for 3 times, each for 3 min;

16) DAB color development, and mastering the dyeing degree under a microscope;

17) washing with PBS or tap water for 10 min;

18) counterstaining with hematoxylin for about 1min, and differentiating with 1% hydrochloric acid alcohol;

19) washing with tap water for 10-15 min;

20) and (3) dehydrating: twice with 95% alcohol for 10 seconds, twice with 100% alcohol for 10 seconds, and twice with xylene for 10 seconds;

21) and (6) baking and sealing the sheet.

4. Long-term inhibition effect and survival effect of recombinant plasmodium yoelii immunization on subcutaneous tumor of tumor-bearing mouse

4) the immunized mice were divided into three groups, the first group was inoculated with Plasmodium yoelii P.y-WT in an amount of 5X 10⁵One/only (n ═ 8); the second group was inoculated with Plasmodium P.y-GPC3:2F at 5X 10⁵One/only (n ═ 8); the third group was inoculated with the same number of red blood cells of normal mice as a blank control group (n-9);

7) depending on animal welfare and ethics, the tumor of a mouse cannot exceed any diameter of 20mm, or when two tumors grow, the maximum diameters of the two tumors together cannot exceed 20 mm. If the above regulation is exceeded, the mouse is regarded as dead, and the mouse is subjected to euthanasia operation;

8) the measurement of the tumor size is finished about 32 days, and the survival condition recording is finished until all the experimental mice die;

9) when the sizes of the tumors of the mice in each group are different, the number of the mice in each group is ensured to be more than or equal to 3. When the number of the tumor cells is less than 3, tumor growth difference analysis among groups is not compared;

results as shown in figure 7, tumor lengths exceeding 20mm will be sacrificed and considered dead mice according to animal welfare and ethical regulations, and tumor sizes are stopped when mice per group survive less than 3. The statistics shown on the left in FIG. 7 show that the tumors were significantly inhibited by day 25 after immunization with Plasmodium (P.ltoreq.0.001. star), while P.y-GPC3:2F was more effective in inhibiting tumor growth (P.ltoreq.0.05. star) than P.y-WT; figure 7, right, shows that GPC3 expression in plasmodium did not affect normal growth of plasmodium in the erythrocytic stage. Most importantly, as shown in FIG. 7, mice immunized with Plasmodium P.y-GPC3:2F were not only effective in inhibiting tumor growth but also significantly extended the survival cycle of the mice.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Guangzhou Zhongke Lanhua Biotech Co., Ltd

<120> recombinant plasmid, recombinant plasmodium constructed by the same and application thereof

<130>2016

<160>25

<170>PatentIn version 3.3

<210>1

<211>14873

<212>DNA

<213> artificially synthesized sequence

<400>1

tatgcttgtc tcaaagatta agccatgcaa gtgaaagtat atgcacattt attgcagaaa 60

ctgcgaacgg ctcattaaaa cagttataat ctacttgaca ttttattata aggataacta 120

cggaaaagct gtagctaata cttgttaagt acttttactc cccggagtaa ttgtatgtat 180

ttgttaagac ccctaagaaa aaatgatatt aaaggaatta taacaaagaa gcaacacata 240

atataattat tcagtgtgta tcaatcgagt ttctgaccta tcagcttttg atgttagggt 300

attgacctaa catggctttg acgggtaacg gggaattaga gttcgattcc ggagagggag 360

cctgagaaat agctaccaca tctaaggaag gcagcaggcg cgtaaattac ccaattctaa 420

ataagagagg tagtgacaag aaataacaat ataaggccaa attttggttt tataattgga 480

atgatgggaa tttaaaacct tcccaaaaat caattggagg gcaagtctgg tgccagcagc 540

cgcggtaatt ccagctccaa tagcgtatat taaaattgtt gcagttaaaa cgctcgtagt 600

tgaacttcaa gggtataatt attttaagca actcacttgg aaagaatcat gacttctgtc 660

actgctttta tccttgttgc agttctttta atacagggcc ctttgagagc ccattaattt 720

atgactgggt ttctcgttac tttgagtaaa ttagagtgtt taaagcaaac agataaagcg 780

tattttactg tgtttgaata ctatagcatg gaataacaac attgaatagg tcaaaagttt 840

ttgaaaaatt tttcttattt tggcttagat acagttaata ggagtagctt gggggcattt 900

gtattcagat gtcagaggtg aaattcttag attttctgga gacaaacaac tgcgaaagca 960

tttgcctaaa atacttccat taatcaagaa cgaaagttaa gggagtgaag acgatcagat 1020

accgtcgtaa tcttaaccat aaactatgcc gactaagtgt tggatgaaaa tttataaata 1080

aaactatctt ctttaaagga gtagtttttt agatgcttcc ttcagtacct tatgagaaat 1140

caaagtcttt gggttctggg gcgagtattc gcgcaagcga gaaagttaaa agaattgacg 1200

gaagggcacc accaggcgtg gagcttgcgg cttaatttga ctcaacacgg ggaaactcac 1260

tagtttaaga caagagtagg attgacagat taatagctct ttcttgattt cttggatggt 1320

gatgcatggc cgtttttagt tcgtgaatat gatttgtctg gttaattccg ataacgaacg 1380

agatcttaac ctgctaatta gcggtggata tgtgatattc ttcgaaggtg gactaactat 1440

agcgttttcg aaggtatgtt gcataatcaa attggtttac cctttgtttt tttgtagcat 1500

attcttttat ttcgttgggt tttttcccta gtaaggatgt atctgcttta tttaatgctt 1560

cttagaggaa cgatgtgtgt ctaacacaag gaagtttaag gcaacaacag gtctgtgatg 1620

tccttagata tactaggctg cacgcgtgct acactgatat gtaaaacgag tatttaaaat 1680

tatatctgta tggtagataa tttaatttct acgtattatc agcatatact tttcctacac 1740

tgaaatagtg aaggtaatct ttatcaatac atatcgtgat ggggatagat tattgcaatt 1800

attaatcttg aacgaggaat gcctagtaag catgattcat cagattgtgc tgactacgtc 1860

cctgcccttt gtacacaccg cccgtcgctc ctaccgattg aaagatatga tgaattgttt 1920

ggacaagaaa atagaaattt tatttttatt ttttttggaa ggaccgtaaa tcctatcttt 1980

taaaggaagg agaagtcgta acaaggtttc cgtaggtgaa ttcactggcc gtcgttttac 2040

aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 2100

ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 2160

gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 2220

tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 2280

agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 2340

ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 2400

catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 2460

tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 2520

cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 2580

cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 2640

tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 2700

tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 2760

atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 2820

gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 2880

aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 2940

aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga3000

gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 3060

cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 3120

atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 3180

tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 3240

ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 3300

ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 3360

ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 3420

tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 3480

tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 3540

aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 3600

tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 3660

tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 3720

gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 3780

agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 3840

tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 3900

ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 3960

cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 4020

tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg 4080

acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 4140

gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 4200

ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 4260

tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 4320

attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 4380

cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 4440

ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 4500

aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg 4560

ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc 4620

acacaggaaa cagctatgac catgattacg ccaagcttgc atgcctgcag gtcaacaata 4680

aataataaat aaatattgtg gaaataaaat aacatataat tatttttaat acattgattt 4740

cccttttatt tttttaaatt tcattgatat aaaaatatat aataataaca tatatgattt 4800

caaattaatc ttttcaaaaa tggtgtttat ttttgtatgt ttgtgtatga attaatcaca 4860

taacacatct attaaattga gttggtaata tagacacaaa taaatatata tatttttata 4920

gcttaaaagt gtgttatgaa tattttaagc atattttctt tttctttgga ttgtgtaaaa 4980

tgaactcata taatgcgttt ttttgttttt gttattttgt cattttgtta ttttgctatt 5040

ttatggatta atttttgttt ataaaatggg aaataatttt aacatattta aataaatgga 5100

gaaaaaatat aaaataatta taaaaaaaag ttaatacaca ttttttcctg ttatagacct 5160

tatatttatt tatccatata tatatatata tatatatata tatatatata cataccaagt 5220

gaattaagag gaaagctaat ttattattca gaataatata tgaactatat ataattttta 5280

ttattttggt gtatattaat ctgtctatat gcatacatgc aataatttat cgacttatat 5340

atcaaataac ataaaataga agtgttttaa attatggata tatgctcaat attcattttt 5400

tttaataagt tagctatatt taaattatac attttatata tggtctcttt tttttttaaa 5460

tattatttaa gtgatcatga aaatataaat aatttttttt tatttaatat ccttttgctt 5520

gcatgtggta aatggaaatt tggatgtgtt ttgaargttc ggatatagtt gtatggacat 5580

ataatatatt ttgtgaaaaa ttggttttat gtttatactt atgccaatac tttttgagta 5640

aaacaaagca agtgcttata aataattaaa gccaatttta taatatatat ttttttattt 5700

aatttgaatt tagtagtata attttttatg gtaagtgctc aaagagagtt gcttataaag 5760

tatggtttgt ttctttttcg ccattttgaa ttacacatta aaaatatata gatacatata 5820

ttataatatg aaatcattaa taatttaggg aaattctaca aatttaaaaa cgaataaaat 5880

aattgttttt catcatgcca taacacaata ttgatatata catgtacaaa catttttttt 5940

atttggaaaa tataaattat ataaaaaaaa atgtatagta tacaaaatga gcatattcac 6000

acggggtgga cgttcatttt ttcatttttc ccctgttttt tatgagtata tgataaaatt 6060

ttatgaacat ttacacaaaa tgaaaatgga tatataggaa aaatggagcg gtatttcatt 6120

tatctttgat tgtcatttgg atattatatt accytgggta ggcaattaaa aatgttaaat 6180

aacaatttaa ggaaattata ttttatatat taaaattaac actgtattat atgattcgct 6240

tataaaagcc actctttccc catgcaaagc tgtttaatat caattttaac aaattacaca 6300

catgttaata tatttatata tataatttat atatttataa tttatatatt tatattttta 6360

ttatttatat atttattatt tattgtgtgt gtcaattcgg gtaggatata cctctttttt 6420

attgtttaaa gcgatttgta ttctaaaata taaagrattt gaaaaagaga aagatagaat 6480

atgatcccat catatatagc cctataattt ttatttagca gcgaattaat ttttctatta 6540

agtttatgtg taattaaaat aacggaatat atataataca ataaaaaagt gcataaatta 6600

aaattttttc aattaaattt ttttttttaa ggggttatat aatattaaat atataaaata 6660

cgattatata tttttgctac aattttttat attaagatat aaatagtaaa taaatggtat 6720

tatatggcat gtaatatata aattttttcc aatttttatt ttatatacac ttttcctttt 6780

tttgtcataa aacttaaaca atttacacat tcattttaaa aattgactat ttgtttcaac 6840

attttttgag tttccgtttt ataatagtat tttcatttgt atattgctta tatatataaa 6900

tacacaccta aatgttacaa aggatcaatg cataaaccgg tgtgtctggt cgtcgcgatg 6960

acccccaaga ggggcatcgg catcaacaac ggcctcccgt ggccccactt gaccacagat 7020

ttcaaacact tttctcgtgt gacaaaaacg acgcccgaag aagccagtcg cctgaacggg 7080

tggcttccca ggaaatttgc aaagacgggc gactctggac ttccctctcc atcagtcggc 7140

aagagattca acgccgttgt catgggacgg aaaacctggg aaagcatgcc tcgaaagttt 7200

agacccctcg tggacagatt gaacatcgtc gtttcctctt ccctcaaaga agaagacatt 7260

gcggcggaga agcctcaagc tgaaggccag cagcgcgtcc gagtctgtgc ttcactccca 7320

gcagctctca gccttctgga ggaagagtac aaggattctg tcgaccagat ttttgtcgtg 7380

ggaggagcgg gactgtacga ggcagcgctg tctctgggcg ttgcctctca cctgtacatc 7440

acgcgtgtag cccgcgagtt tccgtgcgac gttttcttcc ctgcgttccc cggagatgac 7500

attctttcaa acaaatcaac tgctgcgcag gctgcagctc ctgccgagtc tgtgttcgtt 7560

cccttttgtc cggagctcgg aagagagaag gacaatgaag cgacgtatcg acccatcttc 7620

atttccaaga ccttctcaga caacggggtt ccctacgact ttgtggttct cgagaagaga 7680

aggaagactg acgacgcagc cactgcggaa ccgagcaacg caatgagctc cttgacgtcc 7740

acgagggaga caactcccgt gcacgggttg caggctcctt cttcggccgc agccattgcc 7800

ccggtgttgg cgtggatgga cgaagaagac cggaaaaaac gcgagcaaaa ggaactgatt 7860

cgggccgttc cgcatgttca ctttagaggc catgaagagt tccagtacct tgatctcatt 7920

gccgacatta ttaacaatgg aaggacaatg gatgaccgaa cgggcgttgg tgtcatctcc 7980

aaattcggct gcactatgcg ctactcgctg gatcaggcct ttccacttct caccacaaag 8040

cgtgtgttct ggaaaggggt cctcgaagag ttgctgtggt tcattcgcgg cgacacgaac 8100

gcaaaccatc tttctgagaa gggcgtgaag atctgggaca agaatgtgac acgcgagttc 8160

ctcgattcgc gcaatctccc ccaccgagag gtcggagaca tcggcccggg ctacggcttc 8220

cagtggagac acttcggcgc ggcatacaaa gacatgcaca cagactacac agggcagggc 8280

gtcgaccagc tgaagaatgt gatccagatg ctgagaacga atccaacaga tcgtcgcatg 8340

ctcatgactg cctggaatcc tgcagcgctg gacgaaatgg cgctgccgcc ttgtcacttg 8400

ttgtgccagt tctacgtgaacgaccagaag gagctgtcgt gcatcatgta tcagcggtcg 8460

tgcgatgtcg gcctcggcgt ccccttcaac atcgcttcct attcgctttt gacgctcatg 8520

gttgcacacg tctgcaacct aaaacctaag gagttcattc acttcatggg gaacacgcat 8580

gtctacacga accatgtcga ggctttaaaa gagcagctgc ggagagaacc gagaccgttc 8640

cccattgtga acatcctcaa caaggaacgc atcaaggaaa tcgacgattt caccgccgag 8700

gattttgagg tcgtgggcta cgtcccgcac ggacgaatcc agatggagat ggctgtctag 8760

cggaaataca gaagctagct ttgatcccgt ttttcttact tatatattta taccaattga 8820

ttgtatttat aactgtaaaa atgtgtatgt tgtgtgcata tttttttttg tgcatgcaca 8880

tgcatgtaaa tagctaaaat tatgaacatt ttattttttg ttcagaaaaa aaaaacttta 8940

cacacataaa atggctagta tgaatagcca tattttatat aaattaaatc ctatgaattt 9000

atgaccatat taaaaattta gatatttatg gaacataata tgtttgaaac aataagacaa 9060

aattattatt attattatta tttttactgt tataattatg ttgtctcttc aatgattcat 9120

aaatagttgg acttgatttt taaaatgttt ataatatgat tagcatagtt aaataaaaaa 9180

agttgaaaaa ttaaaaaaaa acatataaac acaaatgatg ttttttcctt caatttcgat 9240

atcgaattcc tgcagcccag cttaattctt ttcgagctct ttatgcttaa gtttacaatt 9300

taatattcat actttaagta ttttttgtag tatcctagat attgtgcttt aaatgctcac 9360

ccctcaaagc accagtaata ttttcatcca ctgaaatacc attaaatttt caaaaaaata 9420

ctatgcatat aatgttatac atataaacat aaaacgccat gtaaatcaaa aaatatataa 9480

aaatatgtat aaaaataaat atgcactaaa tataagctaa ttatgcataa aaattaaagt 9540

gccctttatt aactagtcgt aattatttat atttctatgt tataaaaaaa tcctcatata 9600

ataatataat taatatatgt aatgtttttt ttattttata attttaatat aaaataatat 9660

gtaaattaat tcaaaaaata aatataattg ttgtgaaaca aaaaacgtaa ttttttcatt 9720

tgccttcaaa atttaaattt attttaatat ttcctaaaat atatatactt tgtgtataaa 9780

tatataaaaa tatatatttg cttataaata aataaaaaat tttataaaac atagggggat 9840

ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 9900

acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 9960

acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 10020

ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 10080

agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 10140

tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 10200

tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 10260

acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 10320

acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 10380

ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 10440

actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 10500

tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaaga 10560

tgacttcgaa agtttatgat ccagaacaaa ggaaacggat gataactggt ccgcagtggt 10620

gggccagatg taaacaaatg aatgttcttg attcatttat taattattat gattcagaaa 10680

aacatgcaga aaatgctgtt atttttttac atggtaacgc ggcctcttct tatttatggc 10740

gacatgttgt gccacatatt gagccagtag cgcggtgtat tataccagac cttattggta 10800

tgggcaaatc aggcaaatct ggtaatggtt cttataggtt acttgatcat tacaaatatc 10860

ttactgcatg gtttgaactt cttaatttac caaagaagat catttttgtc ggccatgatt 10920

ggggtgcttg tttggcattt cattatagct atgagcatca agataagatc aaagcaatag 10980

ttcacgctga aagtgtagta gatgtgattg aatcatggga tgaatggcct gatattgaag 11040

aagatattgc gttgatcaaa tctgaagaag gagaaaaaat ggttttggag aataacttct 11100

tcgtggaaac catgttgcca tcaaaaatca tgagaaagtt agaaccagaa gaatttgcag 11160

catatcttga accattcaaa gagaaaggtg aagttcgtcg tccaacatta tcatggcctc 11220

gtgaaatccc gttagtaaaa ggtggtaaac ctgacgttgt acaaattgtt aggaattata 11280

atgcttatct acgtgcaagt gatgatttac caaaaatgtt tattgaatcg gacccaggat 11340

tcttttccaa tgctattgtt gaaggtgcca agaagtttcc taatactgaa tttgtcaaag 11400

taaaaggtct tcatttttcg caagaagatg cacctgatga aatgggaaaa tatatcaaat 11460

cgttcgttga gcgagttctc aaaaatgaac aataagcggc cgcgatcccg tttttcttac 11520

ttatatattt ataccaattg attgtattta taactgtaaa aatgtgtatg ttgtgtgcat 11580

attttttttt gtgcatgcac atgcatgtaa atagctaaaa ttatgaacat tttatttttt 11640

gttcagaaaa aaaaaacttt acacacataa aatggctagt atgaatagcc atattttata 11700

taaattaaat cctatgaatt tatgaccata ttaaaaattt agatatttat ggaacataat 11760

atgtttgaaa caataagaca aaattattat tattattatt atttttactg ttataattat 11820

gttgtctctt caatgattca taaatagttg gacttgattt ttaaaatgtt tataatatga 11880

ttagcatagt taaataaaaa aagttgaaaa attaaaaaaa aacatataaa cacaaatgat 11940

gttttttcct tcaatttcga tatcgaattc ctgcagccca gcttaattct tttcgagctc 12000

tttatgctta agtttacaat ttaatattca tactttaagt attttttgta gtatcctaga 12060

tattgtgctt taaatgctca cccctcaaag caccagtaat attttcatcc actgaaatac 12120

cattaaattt tcaaaaaaat actatgcata taatgttata catataaaca taaaacgcca 12180

tgtaaatcaa aaaatatata aaaatatgta taaaaataaa tatgcactaa atataagcta 12240

attatgcata aaaattaaag tgccctttat taactagtcg taattattta tatttctatg 12300

ttataaaaaa atcctcatat aataatataa ttaatatatg taatgttttt tttattttat 12360

aattttaata taaaataata tgtaaattaa ttcaaaaaat aaatataatt gttgtgaaac 12420

aaaaaacgta attttttcat ttgccttcaa aatttaaatt tattttaata tttcctaaaa 12480

tatatatact ttgtgtataa atatataaaa atatatattt gcttataaat aaataaaaaa 12540

ttttataaaa catagggatc gatatgtata tcaattatta tatatactat ataaaatatt 12600

tatatatatt cttaaattta tgctcaatgg ccgggaccgt gcgcaccgcg tgcttgctgg 12660

tggcgatgct gctaggcttg ggctgcctgg gacaggcgca gcccccgccg cctccagacg 12720

ccacctgtca ccaggtccgt tctttcttccagagactgca gcccggactc aaatgggttc 12780

cagaaacccc tgtaccagga tcagatttgc aagtatgtct ccccaagggc ccaacatgct 12840

gctcaagaaa gatggaagaa aaataccaac taacagcacg gctgaacatg gaacaactgc 12900

tccagtctgc gagtatggaa ctcaagttct taattattca gaatgctgcg gttttccaag 12960

aggcctttga aattgttgtt cgccatgcca agaactacac caacgccatg ttcaagaata 13020

actaccccag cctgactcca caagcttttg agtttgtcgg tgaatttttc acagatgtgt 13080

ctctctacat cttgggttct gatatcaacg tggatgatat ggtcaatgaa ttgttcgaca 13140

gcctctttcc agtcatctac acccagatga tgaacccagg cctgcctgag tcagtcttag 13200

acatcaacga gtgcctccga ggagcaagac gtgacctgaa agtatttggc agtttcccca 13260

agcttattat gacccaggtt tccaagtcac tgcaagtcac tcgaatcttc cttcaagccc 13320

tgaatctcgg aattgaagtc atcaacacta ccgaccacct caagtttagt aaggactgtg 13380

gccgtatgct cacccgaatg tggtattgct cttactgcca gggactgatg atggttaagc 13440

cttgcggtgg ttattgcaat gtggtcatgc aaggctgtat ggctggtgtg gtggagatcg 13500

acaagtactg gagagaatac attctgtctc ttgaagagct cgtgaatggc atgtacagaa 13560

tctacgacat ggagaatgtg ctgctcggcc tcttttctac catccatgat tccatccagt 13620

atgtgcagaa gaacggaggc aagctgacca ccaccattgg caagttgtgt gcccactccc 13680

agcaacgcca atatagatct gcttattacc ctgaagatct gtttattgac aagaagatat 13740

taaaagtcgc tcatgtcgaa catgaagaaa ccttatccag ccgaagaagg gaactgattc 13800

agaaactgaa gtctttcatc aacttctata gcgctttgcc gggctacatc tgcagccata 13860

gccccgtggc cgaaaatgat accctgtgct ggaacggaca agaacttgtg gagagataca 13920

gccagaaggc ggcaaggaac gggatgaaga atcagtttaa cctccatgag ctgaaaatga 13980

agggccctga gccggtggtt agccagatca ttgacaaact gaagcacatt aaccagctcc 14040

tgagaaccat gtctgtgccc aagggtaaag ttctggataa aagcctggat gaagaaggac 14100

ttgaaagtgg agactgcggt gatgatgaag atgaatgcat tggaagctct ggtgacggga 14160

tggtgaaagt gaagaatcaa ctgcgcttcc ttgcagaact ggcctatgat ctggatgtgg 14220

acgatgctcc ggggaacaag cagcatggaa atcagaagga caacgagatc accacctctc 14280

acagcgtggg gaacatgccg tccccactga agatcctcat cagtgtggcc atctatgtgg 14340

cgtgcttttt tttcctggtg cactgatcta gaagatcccg tttttcttac ttatatattt 14400

ataccaattg attgtattta taactgtaaa aatgtgtatg ttgtgtgcat attttttttt 14460

gtgcatgcac atgcatgtaa atagctaaaa ttatgaacat tttatttttt gttcagaaaa 14520

aaaaaacttt acacacataa aatggctagt atgaatagcc atattttata taaattaaat 14580

cctatgaatt tatgaccata ttaaaaattt agatatttat ggaacataat atgtttgaaa 14640

caataagaca aaattattat tattattatt atttttactg ttataattat gttgtctctt 14700

caatgattca taaatagttg gacttgattt ttaaaatgtt tataatatga ttagcatagt 14760

taaataaaaa aagttgaaaa attaaaaaaa aacatataaa cacaaatgat gttttttcct 14820

tcaatttcgg gtaccgagct cgaattcaac ctggttgatc ttgccagtag tca 14873

<210>2

<211>550

<212>PRT

<213> artificially synthesized sequence

<400>2

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

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

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

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

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Met

225 230 235 240

Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr Gly

245 250 255

Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser Phe

260 265 270

Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile Phe

275 280 285

Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro

290 295 300

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

305 310 315 320

Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His

325 330 335

Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys

340 345 350

Ile Ile Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr

355 360 365

Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu Ser

370 375 380

Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu Glu

385 390 395 400

Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu

405 410 415

Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys Ile Met Arg Lys

420 425 430

Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys

435 440 445

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

450 455 460

Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr Asn

465 470 475 480

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

485 490 495

Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys Phe

500 505 510

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

515 520 525

Asp Ala Pro Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu Arg

530 535 540

Val Leu Lys Asn Glu Gln

545 550

<210>3

<211>1653

<212>DNA

<213> artificially synthesized sequence

<400>3

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagatg 720

acttcgaaag tttatgatcc agaacaaagg aaacggatga taactggtcc gcagtggtgg 780

gccagatgta aacaaatgaa tgttcttgat tcatttatta attattatga ttcagaaaaa 840

catgcagaaa atgctgttat ttttttacat ggtaacgcgg cctcttctta tttatggcga 900

catgttgtgc cacatattga gccagtagcg cggtgtatta taccagacct tattggtatg 960

ggcaaatcag gcaaatctgg taatggttct tataggttac ttgatcatta caaatatctt 1020

actgcatggt ttgaacttct taatttacca aagaagatca tttttgtcgg ccatgattgg 1080

ggtgcttgtt tggcatttca ttatagctat gagcatcaag ataagatcaa agcaatagtt 1140

cacgctgaaa gtgtagtaga tgtgattgaa tcatgggatg aatggcctga tattgaagaa 1200

gatattgcgt tgatcaaatc tgaagaagga gaaaaaatgg ttttggagaa taacttcttc 1260

gtggaaacca tgttgccatc aaaaatcatg agaaagttag aaccagaaga atttgcagca 1320

tatcttgaac cattcaaaga gaaaggtgaa gttcgtcgtc caacattatc atggcctcgt 1380

gaaatcccgt tagtaaaagg tggtaaacctgacgttgtac aaattgttag gaattataat 1440

gcttatctac gtgcaagtga tgatttacca aaaatgttta ttgaatcgga cccaggattc 1500

ttttccaatg ctattgttga aggtgccaag aagtttccta atactgaatt tgtcaaagta 1560

aaaggtcttc atttttcgca agaagatgca cctgatgaaa tgggaaaata tatcaaatcg 1620

ttcgttgagc gagttctcaa aaatgaacaa taa 1653

<210>4

<211>378

<212>PRT

<213> artificially synthesized sequence

<400>4

Met Ala Arg Asn Phe Glu Cys Lys Lys Ile Asn Ser Asp Asp Met Thr

1 5 10 15

Ser Ser Lys Lys Tyr Ser Lys Asn Val Gly Glu Lys Phe Asn Leu Ile

20 25 30

Ser Cys Thr Lys Leu Phe Ala Leu Ser Met Leu Phe Leu Ile Cys Gln

35 40 45

Asn Tyr Glu Asn Ser Pro Gln Ser Thr Ser Ser His Gln Glu Tyr Gln

50 55 60

Tyr Asn Gly Leu Val Leu Gly Asn Arg Ile Leu Ser Glu Leu Asp Gln

65 70 75 80

Ala Glu Asn His Thr Ile Ser Tyr Lys Thr Asn Asn Tyr Glu Asp Ser

85 90 95

Ser Val Glu Asn Pro Asn Gln Gln Thr Ser Asp Ser Ser Ser Leu Gln

100 105 110

Thr Asp Glu Asp Lys Lys Lys Asp Asp Ser Asp Ala Thr Ser Ile Gly

115 120 125

Glu Thr Ser Pro Thr Thr Glu Thr Thr Ser Val Glu Glu Thr Val Thr

130 135 140

Ile Glu Asp Thr Glu Ser Val Glu Glu Thr Glu Ser Val Glu Glu Thr

145 150 155 160

Pro Ser Thr Ser Thr Glu Glu Thr Ser Ser Thr Gly Lys Lys Thr Tyr

165 170 175

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

180 185 190

Lys Ser Thr Glu Lys Lys Ser Ser Glu Lys Lys Ser Ser Glu Lys Lys

195 200 205

Ser Ser Asp Glu Gln Ser Ser Ser Asp Glu Gln Asn Ser Ser Asp Asp

210 215 220

Gln Asn Ser Phe Asp Asp Gln Lys Leu Phe Glu Asp Ile Asp Asn Leu

225 230 235 240

Ile Asn Gly Ile Lys Ser Arg Tyr Gln Glu Phe Ser Ala Lys Ile Lys

245 250 255

Ser Pro Glu Phe Gln Asn Lys Cys Lys Ser Tyr Met Asn Thr Ala Lys

260 265 270

Glu Met Ile Glu Glu Arg Arg Asn Cys Ala Met Ser Phe Ile Ser Arg

275 280 285

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

290 295 300

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

305 310 315 320

Asn Met Phe Ile Pro Asp Phe Leu Arg Asn Ser Ser Thr Ile Ile Leu

325 330 335

Thr Ile Val Tyr Phe Leu Ile Met Met Phe Ile Val Gly Ser Tyr Leu

340 345 350

Asp Ile Asn Gln Asp Thr Lys Thr Glu Arg Arg Asn Thr Asn Glu Ser

355 360 365

Lys Leu Phe Asn Arg Thr Gln Pro Pro Met

370 375

<210>5

<211>1137

<212>DNA

<213> artificially synthesized sequence

<400>5

atggctcgta attttgaatg caaaaagata aatagtgatg atatgacatc atccaagaaa 60

tattccaaaa atgttggcga gaaatttaat ttgatttctt gtacaaaatt gtttgcgcta 120

agcatgttat ttttgatatg ccaaaattat gaaaatagcc cacaaagcac atcttcacac 180

caagaatacc aatataatgg tttggtttta ggaaacagaa tattatcaga attggatcaa 240

gctgaaaatc atactataag ttataaaaca aacaattatg aagactcttc ggttgaaaat 300

ccaaatcagc aaacctccga tagttcatca ttacaaactg atgaagataa aaaaaaagat 360

gacagtgatg caacatccat tggagaaaca tcaccaacta cagaaacgac atcagttgaa 420

gaaacagtaa caattgaaga cacagaatca gttgaagaaa cagaatcagt tgaagaaaca 480

ccatcaacat caactgaaga aacatcatca actggaaaaa aaacatatgt tgatagaata 540

gcttccattt taaatccatt aatcaatggc gaaaaaaaat ctaccgaaaa aaaatctagt 600

gaaaaaaaat ctagtgaaaa aaaatcttct gatgaacaaa gctcttctga tgaacaaaat 660

tcttctgatg accaaaattc ttttgatgac caaaaactat ttgaagatat tgataatcta 720

ataaatggaa tcaaatcacg ttatcaagag ttcagcgcta aaataaaatc accagaattc 780

caaaacaaat gtaaaagcta tatgaatact gcaaaagaaa tgattgaaga acgtagaaac 840

tgtgctatga gctttatatc tagaaattta aatgccttag gtattgataa gatattcgaa 900

gatgagtttg gtggttatgc actccttgga aaaatgatgt taacaaaagt ctttattgac 960

aatatgttca ttcctgactt cttaagaaat agctcaacaa taattttaac tatagtttat 1020

ttcttaataa tgatgttcat cgtaggaagc tatcttgata tcaatcaaga tactaaaact 1080

gaaagaagaa atacaaatga atctaaattg ttcaatagaa cacaaccacc tatgtaa 1137

<210>6

<211>31

<212>DNA

<213> artificially synthesized sequence

<400>6

ggatccatgg tgagcaaggg cgaggagctg t 31

<210>7

<211>50

<212>DNA

<213> artificially synthesized sequence

<400>7

ctggatcata aactttcgaa gtcatcttgt acagctcgtc catgccgaga 50

<210>8

<211>50

<212>DNA

<213> artificially synthesized sequence

<400>8

tctcggcatg gacgagctgt acaagatgac ttcgaaagtt tatgatccag 50

<210>9

<211>35

<212>DNA

<213> artificially synthesized sequence

<400>9

gcggccgctt attgttcatt tttgagagaa ctcgc 35

<210>10

<211>33

<212>DNA

<213> artificially synthesized sequence

<400>10

atatcgatat ggccgggacc gtgcgcaccg cgt 33

<210>11

<211>34

<212>DNA

<213> artificially synthesized sequence

<400>11

gtctagaggt cagtgcacca ggaaaaaaaa gcac 34

<210>12

<211>33

<212>DNA

<213> artificially synthesized sequence

<400>12

gcggccgcga tcccgttttt cttacttata tat 33

<210>13

<211>33

<212>DNA

<213> artificially synthesized sequence

<400>13

atatcgatcc ctatgtttta taaaattttt tat 33

<210>14

<211>32

<212>DNA

<213> artificially synthesized sequence

<400>14

aggatccatg gccgggaccg tgcgcaccgc gt 32

<210>15

<211>90

<212>DNA

<213> artificially synthesized sequence

<400>15

ggtctagaga gaccttactt atcgtcgtca tccttgtaat ccttatcgtc gtcatccttg 60

taatcgtgca ccaggaaaaa aaagcacgcc 90

<210>16

<211>32

<212>DNA

<213> artificially synthesized sequence

<400>16

aggatccatg gctcgtaatt ttgaatgcaa aa 32

<210>17

<211>59

<212>DNA

<213> artificially synthesized sequence

<400>17

ccgccagatc cacctccacc acttccgcca cctcccatag gtggttgtgt tctattgaa 59

<210>18

<211>70

<212>DNA

<213> artificially synthesized sequence

<400>18

ggaggtggcg gaagtggtgg aggtggatct ggcggtggag gaagcatggc cgggaccgtg 60

cgcaccgcgt 70

<210>19

<211>85

<212>DNA

<213> artificially synthesized sequence

<400>19

ggtctagagt tacttatcgt cgtcatcctt gtaatcctta tcgtcgtcat ccttgtaatc 60

ggtgatctcg ttgtccttct gattt 85

<210>20

<211>32

<212>DNA

<213> artificially synthesized sequence

<400>20

aatcgatatg gtgagcaagg gcgaggagga ta 32

<210>21

<211>36

<212>DNA

<213> artificially synthesized sequence

<400>21

agtctagatt acttgtacag ctcgtccatg ccgccg 36

<210>22

<211>20

<212>DNA

<213> artificially synthesized sequence

<400>22

atgtaatatt tggatatttc 20

<210>23

<211>22

<212>DNA

<213> artificially synthesized sequence

<400>23

tcacctacgg aaaccttgtt ac 22

<210>24

<211>21

<212>DNA

<213> artificially synthesized sequence

<400>24

gttgaaaaat taaaaaaaaa c 21

<210>25

<211>23

<212>DNA

<213> artificially synthesized sequence

<400>25

tttcccagtc agtcacgacg ttg 23

Claims

1. A recombinant plasmid is characterized in that a liver tumor specific antigen gene is inserted into pL0017 plasmid;

the liver tumor specific antigen gene is phosphatidylinositolglycan 3 expression gene gpc 3;

the recombinant plasmid further comprises a whole worm stage expression promoter pbeef1a α and a terminator 3' UTR of Plasmodium bereund;

the recombinant plasmid also includes restriction enzyme sites Not I and Cla I.

2. The recombinant plasmid of claim 1, wherein the nucleotide sequence of the recombinant plasmid is shown in SEQ ID No. 1.

3. The recombinant plasmid of claim 1, wherein the recombinant plasmid expresses 2 or more foreign genes simultaneously.

4. A recombinant plasmodium comprising the recombinant plasmid according to any one of claims 1-3.

5. The recombinant Plasmodium falciparum according to claim 4, further comprising a fusion protein gGluc of green fluorescent protein GFP with Renilla luciferase and/or the secretory protein IBIS 1.

6. The recombinant plasmodium according to claim 5, wherein the amino acid sequence of the fusion protein gRluc is SEQ ID No.2, and the nucleotide sequence is SEQ ID No. 3.

7. The recombinant plasmodium according to claim 5, wherein the amino acid sequence of the secretory protein IBIS1 is SEQ ID No.4 and the nucleotide sequence is SEQ ID No. 5.

8. A vaccine comprising the recombinant plasmid of any one of claims 1-3 and/or the recombinant plasmodium of any one of claims 4-7.

9. Use of the recombinant plasmid according to any one of claims 1-3, the recombinant plasmodium according to any one of claims 4-7 or the vaccine according to claim 8 for the preparation of a medicament for the treatment of liver tumors.

10. A medicament for liver tumour therapy, characterised in that it comprises a recombinant plasmid according to any one of claims 1 to 3 and/or a recombinant plasmodium according to any one of claims 4 to 7.