CN106687592A

CN106687592A - Recombinant plasmid, recombinant malaria parasite and its application

Info

Publication number: CN106687592A
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: 2017-05-17
Anticipated expiration: 2036-12-26
Also published as: CN106687592B; WO2018119610A1

Abstract

The present invention relates to the field of cellular immunotherapy for tumors and, more particularly, to a recombinant plasmid for the construction of recombinant malaria parasites and their use, wherein the recombinant plasmid is a tumorigenin-specific antigen gene inserted into the pL0017 plasmid, Recombinant malaria parasite, comprising the recombinant plasmid. Compared with plasmid DNA and RNA vector, the recombinant malaria parasite can be multiplied by the proliferation of Plasmodium, which is beneficial to the increase of the antigen in vivo. Compared with the defective virus and bacterial carrier, it survives in the red blood cells of the body longer, not short-term by the body's immune system to clear, long-term effective expression of exogenous tumor antigen, is conducive to long-term antigen and immune stimulation. The recombinant malaria parasite is capable of activating the high expression of Th1-related cytokines in vivo and increasing the proportion of CD8a + DCs in total CD11c + DCs and further activating specific cytotoxic T lymphocyte responses against tumor antigens, which is beneficial to the antitumor effect of the vaccine.

Description

A kind of recombinant plasmid, its recombinant plasmodium for building and its application

Technical field

The present invention relates to the cellular immunotherapy field of tumour, more particularly to a kind of recombinant plasmid, its restructuring malaria for building Protozoon and its application, the structure of recombinant plasmodium specially based on recombinant plasmid and its in the treatment of anti-liver neoplasm Using.

Background technology

Hepatocellular carcinoma (Hepatocellular carcinoma, HCC), account for primary carcinoma of liver ratio 85% is arrived 90%.It is one of five common big cancers of the world, fatal rate in all tumours ranking first three.The data display whole world is annual new Increase liver cancer patient 782,500, while there are 745,500 patients to die from liver cancer every year.At present, it is clinically used for the skill of liver cancer treatment Including surgery excision, orthotopic liver transplantation, TACE and local RF therapy etc., these methods are to early stage HCC for art Patient has certain curative effect.But patient often has found too late, 50% liver cancer patient is not appropriate for using these conventional methods.Together When, one term of patient survival rate is less than 50%.The defect of traditional treatment means is more, including cuts off incomplete, toxic and side effect greatly, easily Recurrence etc..In addition, the medicine of targeted therapy liver neoplasm, including Sorafenib and Bevacizumab etc., there is also treatment secondary The problems such as effect and patient survival are short.Therefore, the medicine for researching and developing new treatment liver neoplasm is very active demand.

At present, biological immune treatment is that one kind is widely recognized as and promising oncotherapy means, most of means master Tumour cell is killed by activating in patient's body specific C D8+T lymphocytes.Adoptive treatment includes chimeric Antigen receptor T cells (CAR-T) treatments are widely studied and participate in clinical practice, but this for individual Therapeutic modality spend time and money it is all very huge, not all people is receptible.At present, by vector expression Tumour specific antigen, and it is considered as one feasible honest and clean to activate internal specific cellular immunity come the method to neoplasm growth The approach of valency, which includes tumor vaccine.In terms of liver neoplasm vaccine research, by AFP (alpha fetal protein, Alpha-fetoprotein) DC (dendritic cell, BMDC) vaccines and GPC3 polypeptide vaccines be completed the clinical II phases, it is raw Rate is deposited all to be significantly increased, it is this raising and cell-specific toxic T lymphocyte (cytotoxic T-lymphocyte, CTL) reaction has significantly association.More tumor vaccines on liver cancer are all obtained very on DNA vaccination, polypeptide vaccine and DC vaccines Much progress.However, these vaccines there is also a universal problem, the exactly caused cell for liver tumor antigens is exempted from Epidemic disease is very strong but does not possess the continuation of specific immune activation, and selects a kind of good tumour antigen carrier perhaps to solve well Certainly this problem.

HCC specific antigens are widely found and for the immunization therapy of tumour.Including phosphatidylinositols albumen The albumen of glycan 3 (glypican 3, GPC3), it is that Pilia etc. has found by 1 research of undue growth syndrome patient 's.GPC3 expression high in liver cancer tissue, and do not expressed in normal liver tissue, it is a kind of carcinomebryonic antigen of new hepatocellular carcinoma. Research shows that GPC3 albumen can be activated including the cytotoxic T lymphocyte for GPC3 in human body and Mice Body (cytotoxic T-lymphocyte, CTL), without causing autoimmunity, this CTL often participates in the target for tumour cell Reacted to killing.For the antibody of GPC3, polypeptide vaccine and CAR-T treatments have come into in-depth study, wherein GPC3 Polypeptide vaccine has been completed the clinical II phases, and its overall survival has significantly pass with the GPC3 specific immune responses of machine vivo activation Connection.

One immunotherapy of tumors based on antigenic activation vivo immuning system, generally requires a good expression and carries Body.The wherein more carriers of application include DNA, RNA, defective virus and bacterium.They can activate strong in vivo exempting from Epidemic disease react but not persistently, this may with immunity of organism remove and they can not continue to replicate in vivo exist it is relevant.One good Good Antigen Expression Vectors should include following two abilities：One is to swash the intravital strong specific CTL for antigen Reaction；Two is the presence that this specific CTL reaction can be lasting in vivo and performance tumor-killing effect.Numerous studies table Bright, some viruses or bacterium infection can well suppress tumour growth.Meanwhile, it is also wide that protozoan is used for oncotherapy General research, including Infection of Toxoplasma Gondii and taper worm.Meanwhile, plasmodium can activate very strong internal innate immune reaction.Plasmodium Can be used as a kind of natural adjuvant, some compositions such as GPI (the glycosyl phosphatidylinositol of itself Anchors), genomic DNA and malarial pigment can be used as pathogen associative mode molecule (pathogen-associated Molecular patterns, PAMPs) recognized immediately by body immune system, and activate immediately internal macrophage, DCs, Natural killer (NK) cell, gamma delta T cells, natural killer T (NKT) cell, CD4+T and CD8+T cells.And It is concluded that the natural and acquired immunity of these activation can be used to suppress the growth of tumour.We herein propose and utilize plasmodium It is have certain foundation as the immunization therapy carrier of HCC：1, Infected With Plasmodium can swash intravital Th1 reactions, and it is right It is most important for the CTL of tumour antigen in activation；2, although red blood cell lacks major histocompatibility antigen I (major Histocompatibility complex class I, MHC I), this has no effect on it and produces the CTL for tumour antigen anti- Should, research shows that DCs and macrophage are involved in and activate ctl response in vivo for plasmodium antigens；3, plasmodium is in itself In parasitic red blood cell in vivo, what it can be long-term is present in vivo, this long-term expression for being conducive to antigen and release, for a long time Swash intravital ctl response in ground.

CN 101838611A disclose a kind of recombinant plasmodium of expression alien gene, the external source in the recombinant plasmodium Gene includes antigen gene, therapeutic gene, immunomodulator gene or peptide gene, and the plasmid that wherein invention is used is pL0015 Plasmid, pL0015 plasmids mainly use single endonuclease digestion (Bam HI) insert genes of interest, this insert genes of interest when, easily There is the reverse insertion of genes of interest, be unfavorable for that the recombinant plasmodium in the structure of plasmid vector, and the patent can not be while table Gene up to multiple foreign genes, and expression cannot be secreted into outside plasmodium polypide.

The content of the invention

For the problem that presently, there are, the present invention provides a kind of recombinant plasmid, its recombinant plasmodium for building and its application, The pL0017 plasmids for being used use double digestion method (Bam HI/Xba I) to insert genes of interest, in the absence of in plasmid construction Gene reversely insertion and numerous and diverse means such as sequencing identification again, while introducing a new P. berghei elongation factors (EF) -1 α promoters can simultaneously express two even more than two foreign genes.

It is that, up to this purpose, the present invention uses following technical scheme：

On the one hand, the present invention provides a kind of recombinant plasmid, and the recombinant plasmid is to insert liver in pL0017 plasmids to swell Knurl specific antigen gene.

In the present invention, double digestion method (Bam HI/Xba I) is used to insert genes of interest from pL0017 plasmids, this Sample is just reversely inserted and numerous and diverse means such as sequencing identification again in the absence of the gene in plasmid construction.

According to the present invention, the liver neoplasm specific antigen gene is that (glypican-3 expresses base to gpc3 Cause), first embryo protein AFP expressing genes, in MAGE expressing genes or NY-ESO-1 expressing genes any one or at least two Combination, preferably gpc3 antigen genes, the GPC3 albumen of the gpc3 expression is the very strong liver neoplasm of an immunogenicity Antigen, for the antibody of GPC3, polypeptide vaccine and CAR-T treatments have come into in-depth study, wherein GPC3 polypeptides epidemic disease Seedling has been completed the clinical II phases, and its overall survival has with the GPC3 specific immune responses of machine vivo activation and significantly associates.

According to the present invention, transformed on the basis of pL0017 plasmids are original, inserted a new independent gene work( Energy Expression element, it includes new promoter, genes of interest ORFs insertion point and terminator, the restructuring matter Promoter pbeef1a α (the P. berghei elongation factors that new promoter in grain is expressed for the full worm phase of P. berghei (EF) -1 α promoters), new terminator is 3 ' UTR, and the recombinant plasmid genes of interest ORFs insertion point is essence Single restriction enzyme site Not I and Cla I on grain.

In the present invention, it is strong in the whole life cycle of plasmodium that the promoter pbeef1 α a can start downstream gene Expression, is capable of the GPC3 albumen of continuous expression introducing.Simultaneously as improved plasmid includes 2 independent gene expression units Part, this makes the strain of restructuring worm can be while express a foreign gene, and two the even ability of more than two foreign genes are described Foreign gene can be tumour specific antigen or tumor associated antigen.

In the present invention, two new single restriction enzyme site Not I and Cla I are introduced, can thus be formed outside two The ORFs of source gene.

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

Second aspect, the present invention provides a kind of recombinant plasmodium, and the recombinant plasmodium is included as described in relation to the first aspect Recombinant plasmid.

According to the present invention, plasmodium long-term existence in vivo is so conducive to long-term existence and the release of antigen, and this has The Survival of inhibition and tumour model animal beneficial to tumour growth, this is that many expression vectors are exempted from HCC at present Institute is irrealizable in epidemic disease treatment.

The expression that the present invention passes through fluorescin and luciferase, be conducive to positive restructuring plasmodium in vivo with it is external The steps such as differentiability, spike and the further separation of combination flow cytometer, screening, monoclonal in positive restructuring plasmodium In reach the effect of optimization, what the recombinant plasmodium also included green fluorescent protein (GFP) and renilla luciferase merges egg White gRluc and/or secretory protein IBIS1.

In the present invention, the preparation method of the recombinant plasmodium is prepared using this area conventional technique, herein not Special restriction is done, the specific present invention is used liver neoplasm specific antigen gene expression on carrier, obtain recombinating matter Grain, extracts and purifies after being replicated in Escherichia coli, in the Transfected Recombinant Plasmid that extraction is obtained to plasmodium, obtains expressing liver The recombinant plasmodium of dirty tumor associated antigen.

Preferably, the amino acid sequence of the fusion protein gRluc is SEQ ID NO.2, and nucleotides sequence is classified as SEQ ID NO.3。

Amino acid sequence shown in the SEQ ID NO.2 is as follows：

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPD HMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIM ADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT LGMDELYKMTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEP VARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESV VDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREI PLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIK SFVERVLKNEQ.

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

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCA CAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCG GCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGAC CACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGA CGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCA TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATG GCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGC CGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGT CCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACT CTCGGCATGGACGAGCTGTACAAGATGACTTCGAAAGTTTATGATCCAGAACAAAGGAAACGGATGATAACTGGTCC GCAGTGGTGGGCCAGATGTAAACAAATGAATGTTCTTGATTCATTTATTAATTATTATGATTCAGAAAAACATGCAG AAAATGCTGTTATTTTTTTACATGGTAACGCGGCCTCTTCTTATTTATGGCGACATGTTGTGCCACATATTGAGCCA GTAGCGCGGTGTATTATACCAGACCTTATTGGTATGGGCAAATCAGGCAAATCTGGTAATGGTTCTTATAGGTTACT TGATCATTACAAATATCTTACTGCATGGTTTGAACTTCTTAATTTACCAAAGAAGATCATTTTTGTCGGCCATGATT GGGGTGCTTGTTTGGCATTTCATTATAGCTATGAGCATCAAGATAAGATCAAAGCAATAGTTCACGCTGAAAGTGTA GTAGATGTGATTGAATCATGGGATGAATGGCCTGATATTGAAGAAGATATTGCGTTGATCAAATCTGAAGAAGGAGA AAAAATGGTTTTGGAGAATAACTTCTTCGTGGAAACCATGTTGCCATCAAAAATCATGAGAAAGTTAGAACCAGAAG AATTTGCAGCATATCTTGAACCATTCAAAGAGAAAGGTGAAGTTCGTCGTCCAACATTATCATGGCCTCGTGAAATC CCGTTAGTAAAAGGTGGTAAACCTGACGTTGTACAAATTGTTAGGAATTATAATGCTTATCTACGTGCAAGTGATGA TTTACCAAAAATGTTTATTGAATCGGACCCAGGATTCTTTTCCAATGCTATTGTTGAAGGTGCCAAGAAGTTTCCTA ATACTGAATTTGTCAAAGTAAAAGGTCTTCATTTTTCGCAAGAAGATGCACCTGATGAAATGGGAAAATATATCAAA TCGTTCGTTGAGCGAGTTCTCAAAAATGAACAATAA.

Preferably, the secretory protein IBIS1 amino acid sequences are SEQ ID NO.4, and nucleotides sequence is classified as SEQ ID NO.5。

Amino acid sequence shown in the SEQ ID NO.4 is as follows：

MARNFECKKINSDDMTSSKKYSKNVGEKFNLISCTKLFALSMLFLICQNYENSPQSTSSHQEYQYNGLVLGNRILSE LDQAENHTISYKTNNYEDSSVENPNQQTSDSSSLQTDEDKKKDDSDATSIGETSPTTETTSVEETVTIEDTESVEET ESVEETPSTSTEETSSTGKKTYVDRIASILNPLINGEKKSTEKKSSEKKSSEKKSSDEQSSSDEQNSSDDQNSFDDQ KLFEDIDNLINGIKSRYQEFSAKIKSPEFQNKCKSYMNTAKEMIEERRNCAMSFISRNLNALGIDKIFEDEFGGYAL LGKMMLTKVFIDNMFIPDFLRNSSTIILTIVYFLIMMFIVGSYLDINQDTKTERRNTNESKLFNRTQPPM.

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

ATGGCTCGTAATTTTGAATGCAAAAAGATAAATAGTGATGATATGACATCATCCAAGAAATATTCCAAAAATGTTGG CGAGAAATTTAATTTGATTTCTTGTACAAAATTGTTTGCGCTAAGCATGTTATTTTTGATATGCCAAAATTATGAAA ATAGCCCACAAAGCACATCTTCACACCAAGAATACCAATATAATGGTTTGGTTTTAGGAAACAGAATATTATCAGAA TTGGATCAAGCTGAAAATCATACTATAAGTTATAAAACAAACAATTATGAAGACTCTTCGGTTGAAAATCCAAATCA GCAAACCTCCGATAGTTCATCATTACAAACTGATGAAGATAAAAAAAAAGATGACAGTGATGCAACATCCATTGGAG AAACATCACCAACTACAGAAACGACATCAGTTGAAGAAACAGTAACAATTGAAGACACAGAATCAGTTGAAGAAACA GAATCAGTTGAAGAAACACCATCAACATCAACTGAAGAAACATCATCAACTGGAAAAAAAACATATGTTGATAGAAT AGCTTCCATTTTAAATCCATTAATCAATGGCGAAAAAAAATCTACCGAAAAAAAATCTAGTGAAAAAAAATCTAGTG AAAAAAAATCTTCTGATGAACAAAGCTCTTCTGATGAACAAAATTCTTCTGATGACCAAAATTCTTTTGATGACCAA AAACTATTTGAAGATATTGATAATCTAATAAATGGAATCAAATCACGTTATCAAGAGTTCAGCGCTAAAATAAAATC ACCAGAATTCCAAAACAAATGTAAAAGCTATATGAATACTGCAAAAGAAATGATTGAAGAACGTAGAAACTGTGCTA TGAGCTTTATATCTAGAAATTTAAATGCCTTAGGTATTGATAAGATATTCGAAGATGAGTTTGGTGGTTATGCACTC CTTGGAAAAATGATGTTAACAAAAGTCTTTATTGACAATATGTTCATTCCTGACTTCTTAAGAAATAGCTCAACAAT AATTTTAACTATAGTTTATTTCTTAATAATGATGTTCATCGTAGGAAGCTATCTTGATATCAATCAAGATACTAAAA CTGAAAGAAGAAATACAAATGAATCTAAATTGTTCAATAGAACACAACCACCTATGTAA.

In the present invention, tumour specific antigen can be divided into two kinds of situations, i.e. secreting, expressing and overstepping one's bounds in the expression of plasmodium Secrete expression.Research shows that the GPC3 albumen of nonsecreting type can significantly activate CTLs in vivo for GPC3 albumen and significantly press down The growth of tumour processed, and the GPC3 albumen of secreting type can not significantly activate CTLs in vivo for GPC3 albumen and suppress swollen The growth of knurl.Illustrate that the expression of tumour specific antigen should be preferential when being treated for the biological immune of carrier with plasmodium Nonsecreting type expression is considered, rather than secreting, expressing.But if the gene therapy with plasmodium as carrier, such as expression P53 or During toxic protein, it is contemplated that with the protein expression form of secreting type.

The third aspect, the present invention provides a kind of vaccine, the vaccine include recombinant plasmid as described in relation to the first aspect and/or Recombinant plasmodium described in second aspect.

In the present invention, there is the specific for tumour antigen cytotoxic T of activation in the biological immune treatment of liver neoplasm always Lymphocyte (cytotoxic T-lymphocyte, CTL) strong reaction but not lasting problem, this causes the effect of immunization therapy Fruit is had a greatly reduced quality, therefore therapeutic effect is undesirable.First, it is that the premise environment for producing ctl response raising good in vivo is assessment One of expression vector pays the utmost attention to condition.One important aspect of immunization therapy of tumour is exactly activation special for tumour in vivo The CD8+T cell effects of the opposite sex.With plasmodium as carrier, only expression tumour antigen is inadequate, in addition it is also necessary to detect that malaria is former Can worm carrier provide good precondition to swash intravital CD8+T cell effects.Here, the present invention is main by detection The tendency differentiation of Th1 reactions (reaction of the type of t helper cell 1) and DCs of Vaccins of plasmodium infection early stage mid-term, they are special tumour The CTLs and its suppression tumour growth of Specific Antigen activation provide good condition.

Fourth aspect, the invention provides a kind of recombinant plasmid as described in relation to the first aspect, the weight as described in second aspect Group plasmodium or the vaccine as described in the third aspect are used for the treatment of liver neoplasm.

According to the present invention, the treatment of the liver neoplasm is specifically included：By the recombinant plasmodium knot as described in second aspect Close the erythrocytic stage treatment liver neoplasm of plasmodium；Or by the vaccine organism as described in the third aspect.

5th aspect, the invention provides a kind of medicine for liver neoplasm treatment, the medicine includes such as first party Recombinant plasmid described in face and/or the recombinant plasmodium as described in second aspect.

Compared with prior art, the present invention has the advantages that：

(1) pL0017 plasmids of the present invention use double digestion method (Bam HI/Xba I) to insert purpose base Cause, in the absence of the reverse insertion of gene in plasmid construction and numerous and diverse means such as sequencing identification again, while introducing one new primary Family name plasmodium elongation factors (EF) -1 α promoters can simultaneously express two even more than two foreign genes；

(2) present invention is provided in plasmodium secreting, expressing and the method for non-secreting expression alien gene, is related to IBIS1 albumen Application.Function and immunization therapy purpose according to foreign protein flexibly select different protein expression modes；

(3) a kind of recombinant plasmodium that the present invention is provided, compares with DNA and RNA carriers, and it can realize internal A large amount of amplifications, are conducive to antigen increasing in vivo, compare with defective virus and bacteria carrier, and it survives in body red blood cell Time it is more long, will not be removed by body immune system in a short time, can the exogenous tumour antigen of permanently effective expression, be conducive to The long-term existence and immunostimulation of antigen；

(4) a kind of recombinant plasmodium that the present invention is provided, is not only a kind of expression vector, and itself can be adjusted and activated The innate immunity of body and acquired immunity, the Th1 reactions raised after it is immune and the increasing proportion of CD8 α+DC, these have Beneficial to the generation for tumour antigen CTL；

(5) a kind of recombinant plasmodium that the present invention is provided, can activate the product in vivo for tumour antigen (GPC3) CTL Raw, this is conducive to being lifted the antitumous effect of vaccine, and the Vaccins of plasmodium for expressing tumour antigen suppresses tumour growth and improves lotus The effect of knurl mouse survival rate is fairly obvious.

Brief description of the drawings

Fig. 1 is the improvement and the functional verification with fluorescin as representative of pL0017 plasmids.Wherein, Fig. 1 (A) is double external sources The schematic diagram of gene expression element insertion method；Fig. 1 (B) is the correct structure that pL0017-gRluc-X plasmids are identified in digestion, its In, passage 1 is Bam HI/Xba I double digestion results, and passage 2 is Bam HI/Cla I double digestion results, and passage 3 is Cla I/ Not I double digestion results；Fig. 1 (C) be by gRluc (gfp-Rluc) and mCherry/gpc3 be inserted into ORFs ORF1 with The electrophoresis the result of ORF2, illustrates that mCherry or gpc3 can be successively inserted into ORF2 frames.Wherein, plasmid pL0017- GRluc-mCherry identifies middle passage 1 for Bam HI/Xba I double digestion results, and passage 2 is Bam HI/Cla I double digestion knots Really, passage 3 is Cla I/Not I double digestion results, and passage 4 is Cla I/Xba I double digestion results.Plasmid pL0017- GRluc-gpc3 identifies middle passage 1 for Cla I/Xba I double digestion results, and passage 2 is Not I/Cla I double digestion results, is led to Road 3 is Bam HI/Not I double digestion results, and passage 4 is Bam HI/Xba I double digestion results；Fig. 1 (D) is small animal living body Detect recombinant plasmodium P.y-gRluc-mCherry, P.y-gRluc-ex_mCherry in C57BL/6 mouse under imager Distribution situation；Fig. 1 (E) is the confocal laser microscopy images figure of the recombinant plasmodium for building；Fig. 1 (F) is the restructuring for building The Western blot result figure of the mCherry of expression is inserted in plasmodium in ORF2；

Fig. 2 is that mouse GPC3 albumen stabilization can be expressed in P. yeolii, including secretion and non-secreting expression.Its In, Fig. 2 (A) is that different xgpc3 genes insert pL0017 plasmids site and wild type plasmodium chromosome is inserted into c-rrna The schematic diagram in region；Fig. 2 (B) is that the gpc3 genes after genetic recombination is integrated are arranged and be correctly inserted into plasmodium gene group The design diagram of primer during identification；Fig. 2 (C) for PCR cloned from recombinant plasmodium genome 5 ' int, 3 ' int, gRluc and Gpc3 genetic fragments；Fig. 2 (D) is Western blot result figure of the GPC3 albumen in the internal expression of recombinant plasmodium strain；Fig. 2 (E) It is confocal laser scanning microscope secreting type (P.y-eGPC3:2F) with nonsecreting type (P.y-GPC3:2F) GPC3 albumen is two Distribution situation in individual recombinant plasmodium and red blood cell；Fig. 2 (F) is the laser co-focusing microexamination under GPC3 antibody labelings GPC3 is in P.y-GPC3:(ring bodies of plasmodium, trophozoite, schizont and gamete in the 2F worms strain erythrocytic stage difference polypide stage The body phase) expression and distribution situation；

Fig. 3 is that Infected With Plasmodium influences on mouse spleen DC cell differentiations, including CD11c+CD8 α+DC and CD11c+CD8 The cell proportion of α-DC and its expression of CD80, CD86 protein molecular.Wherein, Fig. 3 (A) is P.y-WT and P.y-GPC3: 2F is immune and the ratio and significant difference of CD8 α+DC in mouse spleen are not immunized；Fig. 3 (B) is in CD8 α+DC and CD8 α-DC The expression ratio of middle CD80 and CD86, significant difference is * P≤0.05, * * P≤0.01, * * * P≤0.001 in figure；

Fig. 4 is for after Infected With Plasmodium C57BL/6 mouse, Th1 relevant cell factors are in different time points in internal serum Concentration Testing, including IL-2, TNF-α, IFN-γ, wherein significant difference be * P≤0.05, * * P≤0.01, * * * P≤ 0.001；

Fig. 5 IFN-γs-ELISAPOT detects the cell toxicant for GPC3 albumen in the immune Mice Body of each recombinant plasmodium Property T lymphocytes (CTL) reaction and significant difference be * P≤0.05, * * P≤0.01, * * * P≤0.001；

The inhibition of Fig. 6 difference plasmodium immune mouse liver neoplasms after 17 days, wherein, Fig. 6 (A) be P.y-WT, P.y-eGPC3:2F、P.y-GPC3:2F immunoinfectives tumor-bearing mice is after 17 days, the growing state of mouse hypodermic tumour, and normal Erythrocyte immune tumor-bearing mice be experimental comparison group, left side be tumor size in fact shine, right side for tumor size volume and system Meter learns diversity ratio pair, and all statistical methods are T inspections (unpaired two-tailed Student ' s t-tests), wherein It is expressed as * P≤0.05, * * P≤0.01, * * * P≤0.001；Fig. 6 (B) is that the SABC detection subcutaneous tumor-bearing mice of each group is subcutaneous Ki67 expressions in tumour；

Fig. 7 is P.y-GPC3:2F and the immune influence to mice tumors grew and Survival of P.y-WT plasmodiums, control Group normocytic mouse for injection has, Fig. 7 is left for significant difference is marked；P.y-GPC3:2F can significantly improve mouse Life span and survival rate, significant difference in Fig. 7；It is infection rate curve, statistical discrepancy that Fig. 7 is right：*P≤0.05,**P≤ 0.01,***P≤0.001。

Specific embodiment

Further to illustrate technological means and its effect that the present invention is taken, below in conjunction with accompanying drawing and by specific real Mode is applied to further illustrate technical scheme, but the present invention is not limited in scope of embodiments.

1) experiment material

1.1 plasmid.PL0017 plasmids used by the experiment are by MR4 (Malaria Research and Reference Reagent Resource Center) give.Toxoplasm dihyrofolate reductase (tg containing mouse on pL0017 plasmids Dhfr/ts) gene, the gene has pyrimethamine resistance.We are by exogenous gene cloning and insert Bam H I with Xba I Point, replaces the gfp genes on original plasmid, and the ORFs ORF for inserting gene is opened by P. berghei transcriptional elongation factor Mover (pbeef1 α a) starts transcription；PEGFP-N1 (Clontech, #6085-1) plasmid is used to clone egfp genes；pRL- SV40vector (Promega, #E2231) is used to clone rRluc genes；PmCherry-C1 plasmids (Clontech, # 632524), for mCherry gene clonings.

1.2 primer.Gfp (green fluorescence protein gene), rRluc are separately designed with the softwares of Primer premier 5.0 (renilla luciferase gene), mCherry (red fluorescent protein gene), ibis1, gpc3 (glypican-3) Primer, synthesized by Huada gene company.Plasmid with Laboratories Accession amplifies required gene ORF, needed for experiment as template Primer can be searched in embodiment one.

1.3 clones main enzyme and related reagent used.Bam H I(Thermo,FD0054)、Not I(Thermo, FD0594), Cla I (Thermo, FD0144), Xba I (Thermo, FD0684), Eco31I (Thermo, ER0291).

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

1.5 mouse, plasmodium and Hepa1-6 cells.No-special pathogen (Specific Pathogen Free SPF) Level female C57BL/6 mouse, 5 week old or so.Purchased from Shanghai Rider your experimental animal Co., Ltd (license:SCXK (HU)2007-0003,China).Mouse is raised by Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health Experimental Animal Center Support, rule illumination in 12 hours, 12 hours dark.The operation of zoopery meets the relevant regulations that experimental animal uses, Suo Youshi Test and approved using administration committee through Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health experimental animal.The non-lethal type of mouse is about Family name plasmodium Plasmodium yoelii 17XNL (MRA-593) is by MR4 (the Malaria Research and Reference Reagent Resource Center) give.Hepa1-6 is from Shanghai Chinese Academy of Sciences cell bank (the Chinese Academy of Sciences Cell Bank, Shanghai, China).

1.6 other main agents and experiment equipment.AxyPrep DNA small volume of reagent boxes (Axygen, #AP-MN-P- 50), AxyPrep glue reclaims kit (Axygen, AP-GX-50), peptone, dusty yeast, NaCl, agarose, pyrimethamine； PCR instrument (AM General biotech firm), agarose gel electrophoresis instrument (company of Beijing 6 1), UV detector (Beckman Company), light microscope (Lycra company), cryogenic freezing centrifuge (German Eppendorf companies), biochemical cultivation case (Shanghai One permanent instrument company), half dry type transferring film instrument (Bio-Red companies), electrotransfection instrument (German Amaxa companies), C6, Arial, Fortessa flow cytometers (BD), laser confocal microscope (Lycra company).

The Prepare restructuring plasmid pL0017-gRluc-X of embodiment 1

First, the basic molecule clone technology that this experiment is used：

1) extraction of plasmid.

Enter with reference to the specification (Axygen, #AP-MN-P-50) of Axygen company AxyPrep DNA small volume of reagent boxes OK.

2) gel DNA fragment is extracted.

Carried out with reference to Axygen companies AxyPrep DNA gel QIAquick Gel Extraction Kit specifications.

3) digestion of DNA and carrier are identified.

DNA is configured to suitable reaction system with corresponding restriction enzyme and its supporting buffer solution, 37 DEG C of 3~4h of water-bath, identify digestion result.

The system that DNA reactions are reclaimed in digestion is as follows：10 × the buffer of 3 μ l, the digestion carrier DNA of 20 μ l, enzyme E1/E2 is each 0.5 μ l (according to experiment purpose selectional restriction restriction endonuclease), 6 μ l ddH₂O, the μ l of cumulative volume 30.

4) purpose fragment and the connection of carrier, convert.

With reference to TaKaRa T Vector specifications, by the amount of carrier and target DNA in proportion about 1：4 mode is mixed, plus Enter the Solution I (2 ×) containing ligase or quick T4 ligases and buffer solution, 37 DEG C of connections 15-30min, Ran Houjin Row is heat-shock transformed, and wherein E. coli competent is bought in Transgen companies.

Specific step of converting：(1) take the competent cell that two pipes freeze, a pipe add 1 μ l DNAs to be transformed or 10 μ l DNA connection products, mix, and another pipe makees negative control, is not added with DNA, and other treatment are identical；

(2) ice bath 30min, 42 DEG C of heat shock 90sec, immediately ice bath 5min；

(3) add the new LB37 DEG C of shaking table culture of non-resistant of 500 μ l 1 hour, LB is coated into the LB containing correspondence antibiotic and is consolidated On body culture medium flat plate；

(4) plate is inverted, 37 DEG C of constant incubator 10~16h of culture are placed in, the growing state of bacterium colony is observed；

(5) picking individual colonies number as needed, and expands in the LB of correspondence antibiotic and accompany, part is taken in 15% glycerine, guarantor Bacterium deposits -80 DEG C of refrigerators, meanwhile, plasmid is extracted as needed.

5) measure of DNA sequence dna.

The Huada gene company is sent to carry out determined dna sequence DNA fragmentation, carrier or bacterium solution, software detection sequence, clone Correctly.

Primer needed for clone：

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’- GGTCTAGAGAGACCTTACTTATCGTCGTCATCCTTGTAATCCTTATCGTCGTCATCCTTGTAATCGTGCACCAGGAA AAAAAAGCACGCC-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- GGTCTAGAGTTACTTATCGTCGTCATCCTTGTAATCCTTATCGTCGTCATCCTTGTAATCGGTGATCTCGTTGTCCT TCTGATTT-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’。

2nd, Trizol extracts total mRNA of Hepa1-6 cells and P.y17XNL-WT plasmodiums, clone gpc3 and Ibis1 genes.

(1) reagent is prepared：Chloroform, isopropanol, 75% ethanol (DEPC water is matched somebody with somebody), the water or 0.5%SDS of RNAase-free Solution (adds water to the vial of RNase-free, plus DEPC is to final concentration 0.01% (V/V), overnight and high pressure, SDS is also used Treated DEPC water configuration)；

(2) the cell Hepa1-6 of adherent growth：3.5cm diameter dishes add 1ml Trizol, blow and beat repeatedly, (1ml Trizol is used for 10cm²Area, Trizol not enough amount may cause DNA pollution), room temperature place 5min, to ensure nucleoprotein Complex will be completely dissociated；P.y17XNL-WT plasmodium worm blood of the infection rate more than 30%, the exposed malaria that erythrocyte cracked liquid is obtained Protozoon polypide, Trizol dissolvings；

(3) in ready Hepa1-6 samples and exposed plasmodium polypide, 0.2ml chloroforms are added per 1mL Trizol, Lid is covered, mixing 15s is overturned, room temperature places 2-3min, 4 DEG C of centrifugation 15min, no more than 12000g；

(4) taking upper strata aqueous phase can retain organic phase in new pipe, such as separating DNA or protein, add per 1ml Trizol 0.5ml isopropanols, are incubated at room temperature 10min；

(5) 12000g × 15min, 4 DEG C.Supernatant is removed, at least adds the ethanol of 1ml 75%, spiral to mix per 1ml Trizol, 7500g × 5min, removes supernatant by 4 DEG C, and RNA precipitate is air-dried in short-term, is dissolved with the water or 0.5%SDS of RNase-free, obtains Obtain Hepa1-6 cells and the total mRNA of P.y17XNL-WT plasmodiums；

(6) for several times, 55-60 DEG C is incubated 10min, -80 DEG C of preservations for piping and druming；

(7) RT-PCR obtains total cDNA of Hepa1-6 cells and plasmodium；

A) to the total serum IgE for adding 2 μ l to extract in a PCR pipe without RNase, 2 μ l M-MLV Reverse The Transcriptase μ l RNasin of buffer, the dNTP of 2 μ L, 1, it is 16 μ to add 9 ultra-pure waters of the μ l without RNase to cumulative volume L, fully mixes；

B) the two new PCR pipes without RNase are taken, upper RT+, RT- is marked respectively, divide above-mentioned 16 μ l mixed liquors equally so far two Pipe；

C) often pipe plus 1 μ l gpc3 or ibis1 reverse primer, plus 1 μ l M-MLV Reverse Transcriptase Into RT+ pipes, plus in the ultra-pure water without RNase to the RT- pipes of 1 μ l, mix.

D) reverse transcription 1 hour in 42 DEG C of waters；

E) 3min inactivations reverse transcriptase at 93 DEG C；

F) cDNA with reverse transcription enters performing PCR as template, and electrophoresis detection clones gpc3 and ibis1 genes；

3rd, mCherry (Cla I/Xba I), ibis1-mCherry (Cla I/Xba I), gpc3 (Cla I/Xba I), 3’UTR-pbeef1aa(Not I/Cla I)、gRluc(BamH I/Not I)、gpc3:2F (BamH I/Xba I) and ibis1- gpc3:The gene cloning of 2F (Bam H I/Xba I).

Gene cloning primer according to above-mentioned described molecule clone technology and synthesis (be shown in experiment material by primer sequence Primer portion), by Reverse Transcriptase kit (II 1st strand cDNA Synthesis kit, Takara after) obtaining total cDNA of Hepa1-6 cells and P.y17XNL-WT plasmodiums, gpc3 (Cla are cloned as template I/Xba I) gene, the gene is connected on pMD18T carriers (TAKARA), heat shock is transferred in Escherichia coli, obtains monoclonal Bacterial strain and sequencing identification.Same method, we with pL0017, pEGFP-N1, pRL-TK, pmCherry-C1 as template, we (gfp's and rLuc melts to have cloned mCherry, ibis1-mCherry, gRluc by regular-PCR or overlapping PCR method achievement Close gene), gpc3:2F、ibis1-gpc3:2F、3’UTR-pbeef1αa.

4th, build containing two novel plasmid pL0017-gRluc-X plasmids of independent expression cassette element, and on this basis PL0017-gRluc-ibisi-mCherry plasmids, pL0017-gRluc-gpc3 plasmids, pL0017-gpc3 plasmids are constructed And pL0017-ibis1-gpc3 plasmids.

By genetic fragment mCherry, ibis1-mCherry of above-mentioned successful clone, gRluc, (gfp merges base with Rluc's Cause), gpc3:2F、ibis1-gpc3:2F, 3 ' UTR-pbeef1 α a are according to the Bam H that pL0017 plasmids are inserted Fig. 1 (A) Suo Shi Between I/XbaI restriction enzyme sites, wherein, first ORFs ORF1 can be inserted into gRluc genes, in order that second opening is read Frame ORF2 successful expression genes of interest, we add a new terminator (3 ' UTR) before ORF2, and it is used to terminate The normal transcription of ORF1 genes, we are inserted into a new promoter pbeef1 α a, the base for starting ORF2 after 3 ' UTR Because of expression, new plasmid pL0017-gRluc-X (Fig. 1 (B)) is built into, in the new plasmids of Fig. 1 (B), is not inserted in ORF2 Enter any foreign gene, the fragment cut out from passage 1Bam HI/Xba I double digestion results includes gRluc-3 ' UTR-pbeef1 α A, ORF2 are not inserted into any gene；Passage 2Bam HI/Cla I double digestion results, the fragment for cutting out includes gRluc-3 ' UTR- pbeef1αa；Passage 3Cla I/Not I double digestion results, the fragment for cutting out includes 3 ' UTR-pbeef1 α a.It is connected to pL0017 On carrier between Bam H I/Xba I sites, and successfully construct pL0017-gRluc-mCherry plasmids (Fig. 1 (C)), PL0017-gRluc-ibisi-mCherry plasmids, pL0017-gRluc-gpc3 plasmids (Fig. 1 (C)), pL0017-gpc3:2F matter Grain (Fig. 2 (C)) and pL0017-ibis1-gpc3:2F plasmids, illustrate that mCherry or gpc3 can be successively inserted into ORF2 frames.

The structure of the recombinant plasmodium of embodiment 2

First, pL0017-gRluc-mCherry plasmids, pL0017-gRluc-ibisi-mCherry plasmids, pL0017- GRluc-gpc3 plasmids, pL0017-gpc3:2F plasmids and pL0017-ibis1-gpc3:Heavy dose of extracting of 2F plasmids.

1) plasmid is carried greatly

(1) line activation is containing correct restructuring pL0017 plasmids are built, have pL0017-gRluc-mCherry plasmids, PL0017-gRluc-ibisi-mCherry plasmids, pL0017-gRluc-gpc3 plasmids, pL0017-gpc3:2F plasmids and pL0017-ibis1-gpc3:The bacterial strain of 2F plasmids, cultivates 16h in incubator；

(2) random 2 monoclonals of picking, transfer in cultivating 8-12h in 15ml centrifuge tubes；

(3) according to 1:The amplification of above-mentioned nutrient solution is seeded to equipped with 200ml ammonia benzyl resistance LB culture mediums 1 by 500 inoculum concentrations Rise in triangular flask, 37 DEG C of shaking culture 12h collect bacterium solution and carry plasmid kit (QIfilter greatly with QIAGEN companies Plasmid Maxi kit) plasmid is extracted, by specification operation is carried out, and the UV detector that will be extracted determines the dense of plasmid Degree and purity, be stored in -80 DEG C it is standby.

2nd, the activation of plasmodium P.yoelii 17XNL, electricity turn plasmodium preparation and the acquisition of recombinant plasmodium, including P.y-gRluc-mCherry、P.y-gRluc-ex_mCherry、P.y-GPC3、P.y-GPC3:2F and P.y-eGPC3:2F.

(1) the non-lethal type plasmodium strain P.yoelii 17XNL of Yue Shi will be frozen to be taken out from liquid nitrogen container, 37 DEG C of water-baths are melted Change；

(2) in 1ml syringes Intraperitoneal injection C57BL/6 Mice Bodies, two mouse are injected, every worm blood is 0.5ml, dilution It is 1% infection rate；

(3) inoculation starts on the 3rd day, and daily tail point part takes blood film, observes infection rate, and methyl alcohol is fixed after air-drying, with dilute The Giemsa stain dyeing 30min for releasing, clear water slowly to be washed away and count infection rate, every slice, thin piece meter under microscope oil mirror after dye liquor Average rate under 10 visuals field of number is the protozoan infection rate of the mouse.When infection rate reaches more than 10%, eyeball is plucked Take blood, conservation, 3.8% trisodium citrate liquid or liquaemin liquid anti-freezing；

(4) blood sampling is completed, and is mixed with the PBS solution of isometric plasmodium frozen stock solution or 10% glycerine, overturns mixed repeatedly It is even, it is sub-packed in cell cryopreservation tube, it is put into liquid nitrogen container and preserves；

(5) blood of 20-50 μ L is taken from the Mouse Tail-tip that infection rate is 10% or so, 0.6ml PBS, intraperitoneal injection is dissolved in To 3 C57BL/6 mouse, 0.2ml every is designated as Day0；

(6) the 4th days, start painting blood and observe every mouse infection rate, typically in Day 7-Day 10 or so, in 10%- 20% then then walks down, if less than this infection rate, operated again to this infection rate after waiting；

(7) it is general to operate this step at night, it is equipped with the complete medium 200ml of RPMI 1640 of 10%FBS.It is wherein every 25mM HEPES (5.96g), 0.85g NaHCO3,50mg neomycinsulphates are added in 100ml culture mediums, 0.22 μM of mistake is filtered Bacterium；

(8) 5ml complete mediums are added in 50ml centrifuge tubes, liquaemin anti-freezing is contained within.Above-mentioned 3 C57BL/6 is small Mouse carries out heart sterile blood sampling, is added in above-mentioned 50ml centrifuge tubes, centrifugation 450g/min 8min；

(9) erythrocyte sedimentation rate forms sediment resuspended with 50ml complete mediums, and this 50ml is divided equally to two 250ml Tissue Culture Flasks, every bottle The complete medium of 25ml is added again, is filled with containing 5%CO through 0.22 μm of filter in blake bottle₂, 5%O₂, 90%N₂Gaseous mixture Body, culture is kept flat be placed in 37 DEG C of incubator after covering tightly, and slowly shakes overnight incubation；

(10) blake bottle is opened, is taken in 0.5ml nutrient solutions to 1.5ml EP pipes, maximum velocity centrifugation 5s takes after abandoning supernatant Precipitation smear staining, it is then lower to walk when major part is such as shown under field of microscope for mature schizont, if being also not reaching to Smear observation again after 1-2 hours can be waited, typically completes in vitro culture in 12-16 hours after incubation；

(11) the 100ml plasmodium cultures that will be cultivated, and divide equally to 3 50ml centrifuge tubes, will with aseptic Bath pipe The PBS of 30ml Nycodenz and 20ml is configured to 60% careful the addition by bottom of Nycodenz liquid after mixing is equipped with plasmodium The centrifuge tube of culture, 450g/min 20min are centrifuged after trim with swing bucket rotor room temperature, and centrifugation starting loop is 3, centrifugation Brake acceleration is 0.

(12) one layer of centre after being centrifuged with Pasteur's pipe careful collection, 3 pipes can about collect 20-25ml, add fresh Insect complete medium to cumulative volume is 40ml, 450g centrifugation 8min, abandons supernatant, and the electricity that insect is resuspended in 1ml carefully is turned into liquid In；

(13) in packing to 10 aseptic 1.5mL EP pipes, the μ g-30 μ g linearization plasmids of 10 μ L 10, they include PL0017-gRluc-mCherry plasmids, pL0017-gRluc-ibisi-mCherry plasmids, pL0017-gRluc-gpc3 matter Grain, pL0017-gpc3:2F plasmids and pL0017-ibis1-sgpc3:2F, Sac II or the Apa I of the plasmid linearization Enzyme；

(14) it is transferred in electric shock cup after mixing, has been careful not to bubble, plasmid is incubated 5min, uses Amaxa with plasmodium Company's nucleotides electroporation electricity turns, and electric carryover sequence is T-16；

(15) after electricity turns, carefully suctioned out in the protozoon after electricity turns to 1.5ml EP pipes with miniature Bath pipe, add 50 μ l complete Full culture medium, 150 μ l altogether, with insulin syringe by this 150 μ l mixed liquor through tail vein injection to C57BL/6 Mice Bodies It is interior；

(16) to the μ l of mouse peritoneal injection pyrimethamine 200, (PH=4.0, DMSO dissolve and are prepared with PBS, agent daily after 24h Amount is less than or equal to 1mg/kg), for screening restructuring worm strain.Cutting tail point blood film has seen whether plasmodium daily after four days Occur；

(17) when infection rate reaches 10%, new mouse of transferring, and this mouse blood eyeball is taken out, use anticoagulant heparin Mix with isometric plasmodium frozen stock solution after blood sampling, be sub-packed in cell cryopreservation tube after mixing, be stored in liquid nitrogen container and preserve.

3rd, the extraction and identification of Plasmodium yoelii genomic DNA are recombinated

(1) we are negative control with the genomic DNA of wild type P.y 17XNL-WT in this experiment.Work as P.y- GPC3、P.y-GPC3:2F and P.y-eGPC3:When infection rate of the 2F worms strain in mouse reaches 10%, pluck eyeball and take blood, 3.8% trisodium citrate or liquaemin anti-freezing, are collected in 1.5ml EP pipes, 450g/min centrifugation 8min, abandon supernatant blood plasma, use Once, 450g/min is centrifuged 8min to the resuspended cleanings of PBS；

(2) cracked with erythrocyte cracked liquid, put 5min on ice, overturned mix 2-3 times therebetween, 600g/min centrifugation 8min, Supernatant is abandoned, with the resuspended cleanings of PBS once, 450g/min centrifugation 8min abandon supernatant, and precipitation had both been the exposed malaria from red blood cell release Protozoon, it is resuspended with 200 μ l PBS, extract plasmodium gene group DNA with the QIAamp DNA Blood Kits of QIAGEN companies；

(3) after quantifying and determine concentration through UV detector, -20 DEG C are stored in.

Restructuring Plasmodium yoelii P.y-GPC3, P.y-GPC3:2F and P.y-eGPC3:The PCR identifications of 2F.

(1) with wild type plasmodium and restructuring P.y-GPC3, P.y-GPC3:2F and P.y-eGPC3:2F genomes are mould Plate, takes the template that 500ng reacts as PCR, prepares PCR reaction systems, right as feminine gender with wild type Plasmodium yoelii genome According to；

(2) including checking gpc3 (contain ibis1-gpc3), gRluc, 5 ' int and 3 ' int clone, wherein, 5 ' int and 3 ' Clone's success of int, illustrates that pL0017 sequence successful stabilizations are inserted into (Fig. 2 (A) and figure in plasmodium gene group c-rrna elements 2(B))。

The primer used in the identification is as shown in embodiment one, wherein identifying that gpc3 the primers are gpc3-Cla I-F (SEQ ID NO.10) and gpc3-Xba I-R (SEQ ID NO.11)；Identify that ibis1-gpc3 the primers are ibis1-Bam H I-F (SEQ ID NO.16) and gpc3-Xba I-R (SEQ ID NO.11)；Identify that gRluc the primers are egfp-Bam H I-F (SEQ ID NO.6) and Rluc-Not I-R (SEQ ID NO.9)；5 ' int the primers of identification are L635 (SEQ ID ) and PyL739 (SEQ ID NO.22) NO.25；Identify that 3 ' int the primers are:PyL740 (SEQ ID NO.23) and L665 (SEQ ID NO.24)。

Pcr amplification reaction system is as follows：

Wherein, one group of negative control with water as sample.

Reaction condition is as follows：

(3) after the completion of PCR, 1% Ago-Gel is prepared, loading electrophoresis, in being imaged in gel imaging instrument, identifies that electricity turns Whether there are gpc3 genes in plasmodium gene group afterwards.

Shown in result such as Fig. 2 (C), it is seen then that compare with negative control P.y-WT, 5 ' int and 3 ' int are in P.y-GPC3:2F、 P.y-GPC3 and P.y-eGPC3:Be successfully be detected in 2F, the pL0017 plasmids of this various transformation of explanation are successfully embedded into malaria original In the genome of worm.

5) extraction of recombinant plasmodium albumen

(1) when the mouse infection rate for passing worm again reaches more than 10%, pluck eyeball and take blood, anticoagulant heparin is collected in In 1.5mlEP pipes, 450g/min centrifugation 8min abandon supernatant blood plasma, with the resuspended cleanings of PBS once, 450g/min centrifugations 8min；

(2) cracked with erythrocyte cracked liquid, put 5min on ice, overturned mix 2-3 times therebetween, 600g/min centrifugation 8min, Supernatant is abandoned, with the resuspended cleanings of PBS once, 450g/min centrifugation 8min abandon supernatant；

(3) precipitation had both been the exposed plasmodium from red blood cell release, added the RIPA of 300 μ l in plasmodium, rapid to use Rifle piping and druming of exerting oneself is mixed, and is positioned over and is cracked 20min on ice, and period beats tube wall every 2-3min bullets of exerting oneself, 4 DEG C of 12000g/min from Heart 20min；

(4) supernatant is transferred to new pipe, with the concentration of protein quantification kit measurement albumen, and is recorded.To in each sample 5 × loading buffer of 1/4 volume are added, is mixed with rifle and is boiled 5min, 4 DEG C of maximum velocity centrifugations after boiling water bath 10min。

(5) supernatant is transferred to and newly manages and dispense, and is stored in -80 DEG C of refrigerators.

6) Western blot identifications GPC3 expresses the plasmodium of GPC3 albumen and expression mCherry in recombinant plasmodium The expression of middle mCherry

(1) glass plate is cleaned：One hand buckles glass plate, and another hand dips in a washing powder and gently cleans.All clean on two sides Later rushed with running water, then with distilled water flushing it is clean after stand on and dried in basket；

(2) encapsulating and loading：Clamping in folder is put into after glass plate alignment, then vertical card prepares encapsulating on the top of the shelf：Press Formula prepares 10% separation gel, and encapsulating by being shaken up immediately after addition TEMED during encapsulating, can draw 5ml glue along glass with 10ml rifles Glass is released, when glue surface is raised to greenbelt medium line height.Accelerated to be gelled with isopropanol fluid-tight.Room temperature places 30min, works as sight After observing gelling, isopropanol is removed, rinsed with clear water, and blotted only with blotting paper.With 4% concentration glue, stood after adding TEMED Encapsulating by shaking up.Remaining space is filled into concentration glue and then by comb insertion concentration glue.Due to volume meeting when gelling is solid Shrink and reduce, so that the loading volume of well reduces, so will be often in both sides glue during concentration gelling is solid. Until after concentration gelling is solid, the both sides that two hands pinch comb respectively are gently pulled out straight up.Rinsed with water and concentrated Glue, in putting it into electrophoresis tank；

(3) the protein sample loading of extraction is taken, per the μ g of electrophoresis hole loading 30 (including P.y-GPC3, P.y-GPC3:2F、 P.y-gRluc-mCherry, P.y-gRluc-ex_mCherry), loading is begun preparing for after filling up enough electrophoresis liquids, (electrophoresis liquid At least to cover the small glass plate of interior survey) use the adherent pipette samples of microsyringe, by sample suction out should not inspiration bubble, will plus Sample device syringe needle is slowly added to sample in being inserted to well, (sample-adding sample can be made very much to go out well soon, if having bubble it is also possible that Sample is overflowed, and when adding next sample, injector need to be washed for several times in water jacket electrophoretic buffer, in order to avoid cross pollution)；

(4) electrophoresis：General 4~the 5h of electrophoresis time, voltage is that 40V is preferable, it is also possible to which 60V (in order to accelerate speed, concentrates glue When run with 80V, run with 120-140V to after separation gel, as a result also good, or so total time 2h) electrophoresis to bromjophenol blue just runs out of Electrophoresis can be terminated, transferring film is carried out；

(5) transferring film：Pvdf membrane is moistened in methyl alcohol before transferring film is then dipped in transferring film buffer solution, cut the concentration of gel Glue, gel is also steeped in transferring film buffer solution, and two panels thickness filter paper is moistened with transferring film buffer solution, transferring film sandwich of layers is assembled, under To filter paper is above followed successively by, pvdf membrane, gel, filter paper is connected with the mains, constant current 60mA transferring films 1 hour；

(6) close：After film is washed with PBST, film is soaked in the confining liquid with the 5% of PBST preparations skimmed milk power Interior, 4 DEG C of closings are overnight；

(7) primary antibody hybridization：After the film that will have been closed is with PBST washes cleans, film is soaked in the rabbit GPC3 prepared with PBST Antibody (1：2000) or in mouse Flag antibody, the primary antibody hybridization solution of mCherry room temperature jog hybridizes 2 hours；

(8) film is washed：The film hybridized by primary antibody is washed 6 times with PBST, each 4min, shaking washing at a high speed on shaking table；

(9) secondary antibody hybridization：The film that will have been washed is soaked in the anti-rabbit of the horseradish peroxidase-labeled prepared with confining liquid IgG(1：Or anti-mouse IgG (1 2000)：2000) room temperature jog hybridizes 1 hour in secondary antibody hybridization solution；

(10) film is washed：The film hybridized by secondary antibody is washed 6 times with PBST, each 4min, shaking washing at a high speed on shaking table；

(11) chemiluminescence, development is fixed：By two kinds of reagents of A and B in the first-class volume mixture of preservative film；After 1min, by film Albumen faces down and this mixed liquor is fully contacted；After 1min, film is moved on another preservative film, remove most raffinate, wrapped, be put into X- In mating plate folder.In darkroom, 1 × developer solution and fixing solution are poured into vinyl disc respectively；X- mating plates are taken out under red light, is used Hand papercutter is cut out appropriately sized (long and wide than film is both needed to big 1cm)；X- mating plates folder is opened, X- mating plates are placed on film, once Put, it is just immovable, X- mating plates folder is shut, start timing；The power appropriate adjustment time for exposure according to signal, generally 10min, also may be selected different time multiple compressing tablet, with up to optimum efficiency；After the completion of exposure, X- mating plates folder is opened, take out X- light Piece, develops in rapid immersion developer solution, after obvious band to appear, development is terminated at once.Developing time is generally 1~2min (20~25 DEG C), (being less than 16 DEG C) when temperature is too low needs proper extension developing time；After development terminates, X- mating plates are immersed at once In fixing solution, fixing time is generally 5~10min, with film it is transparent；After the fixing solution of residual is washed away with running water, room Dried under temperature.

Shown in result figure 1 (F), it is seen then that mCherry albumen is in P.y-gRluc-mCherry, P.y-gRluc-ex_ Successful expression in the strain of mCherry worms, shows that the mCherry albumen of IBIS1 albumen connection has Partial Shear, part mCherry Albumen can exist in independent protein form, and IBIS1 albumen mainly exercises the function toward the outer transport protein of polypide.Meanwhile, such as Fig. 2 (D) shown in, it is seen then that the P.y-GPC3 (worm strain expresses gRluc) of either double ORF expression GPC3 or list ORF expression The P.y-GPC3 of GPC3:2F (worm strain do not express gRluc) can successful expression GPC3, GPC3 albumen in P.y-GPC3:2F And successful expression in the strain of P.y-GPC3 worms, control group is protein sample prepared by the cracking of P.y-WT plasmodiums.

7) distribution situation of confocal laser scanning microscope mCherry and GPC3 albumen in plasmodium

(1) in C57BL/6 mouse activate P.y-gRluc-mCherry, P.y-gRluc-ex_mCherry, P.y-WT, P.y-GPC3:2F and P.y-eGPC3:2F；

(2) the different time take blood in mouse tail and be coated on the slide of Concanavalin A (Sigma)-treatment. PBS containing 4%PFA and 0.0075% glutaraldehyde is fixed or normal temperature 4%PFA fixes 20min, then fixes 10s with ice methyl alcohol；

(3) 5% hyclones close sample, 1%Triton X-100 permeabilized cells samples 15min；

(4) PBS containing 2%FBS is washed 3 times, each 5min.Resisting GPC 3 antibody or anti-Flag antibody incubations sample, 4 DEG C of mistakes Night；

(5) PBS containing 2%FBS is washed 3 times, each 5min, and the secondary antibody of fluorescence labeling is incubated 1 hour under room temperature condition, notes Lucifuge；

(6) PBS containing 2%FBS is washed 3 times, each 5min, DAPI working solutions (the green skies) samples of incubation 15min；

(7) PBS containing 2%FBS is washed 3 times, each 5min, anti-fluorescent quenching mounting liquid (the green skies) mounting；

(8) expression and distribution situation of laser confocal fluorescence microscope (Lycra) the observation GPC3 albumen in plasmodium.

Result such as Fig. 1 (E), mCherry and GFP is in P.y-gRluc-mCherry, P.y-gRluc-ex_mCherry Successful expression, and positioning is it can be seen that IBIS1 albumen successfully draws mCherry albumen in the strain of P.y-gRluc-ex_mCherry worms Derive plasmodium polypide and enter in red blood cell.Shown in 2 (E) and Fig. 2 (F), it is seen then that Fig. 2 (E) is illustrated in P.y-eGPC3:2F worms GPC3 Protein transports are successfully gone out plasmodium and are secreted into red blood cell by IBIS1 albumen in strain, the result and Fig. 1 (E) result Unanimously；Fig. 2 (F) then indicates GPC3 albumen in P.y-GPC3:Stabilization under 2F recombinant plasmodiums erythrocytic stage difference polypide state Expression.

8) distribution situation of the small animal living body imaging system observation plasmodium in Mice Body

(1) plasmodium P.y-WT, P.y-gRluc-mCherry, P.y-gRluc-ex_mCherry injection that will be activated are small Mouse is intraperitoneal, and every mouse injects the red blood cell 5*10 of Infected With Plasmodium⁵Individual/only.

(2) mouse infection plasmodium is after 7 days, intraperitoneal injection D- fluorescein sylvite, dosage 15mg/ml, every μ of mouse 200 L, carries out living imaging observation after 10 minutes.

Shown in result such as Fig. 1 (D), the sea pansy in the strain of P.y-gRluc-mCherry, P.y-gRluc-ex_mCherry worm is glimmering Light element zymoprotein successful expression, and can show that these worms of erythrocytic stage strain distribution situation in vivo.

The immune detection of the P. yeolii immune mouse of embodiment 3

Including CD8 α+DC, Th1 relevant cell factors and the ctl response detection for GPC3 albumen.

1st, experiment material：C57BL/6 mouse, Hepa1-6 cells；Penicillin and streptomysin are Bio Basic Inc companies Product；Pancreatin is Amresco Products.Anti- CD11c-FITC, anti-CD8 α-PE, anti-CD86-APC, anti-CD80-PerCP- Cy5.5 antibody (is purchased from eBioscience, San Diego, CA, USA), BD FACSAria flow cytometers, FlowJo analyses Software (Tree Star, Inc.), mouse Th correlations multiple-factor detection kit (BioLegend), BDTM ELISPOT Mouse IFN-γ-Kit(BD Biosciences,#552569).Fortessa flow type analyzers (BD), Legendplex analysis softwares (BioLegend)；1 × erythrocyte cracked liquid (BD), 200 mesh cell filtering nets, 1mL syringes, cryogenic freezing centrifuge (Germany Eppendorf companies), biochemical cultivation case (the permanent instrument company in Shanghai one), tweezers, scissors, alcohol, glass capillary；DMEM and The culture mediums of RPMI 1640 (GIBCO), calf serum FBS (GIBCO), GM-CSF (PeproTech), 2-mercaptoethanol (2-ME, Sigma-Aldrich), Tissue Culture Dish；Mouse CD8 α+magnetic bead (U.S. day Ni).10 × Giemsa dye liquors (5g/L Giemsa stain powder, 25% glycerine, 25% methyl alcohol)

2nd, the immune tumor-bearing mice of restructuring P. yeolii, detects CD8 α+DC cellular changes situations and CD80 in its spleen With CD86 expressions.

1) the Hepa1-6 cells for freezing are taken out in liquid nitrogen, 37 DEG C of defrostings, iuntercellular activate and Amplification Culture；

2) P.y-WT, the P.y-GPC3 for freezing are taken out in liquid nitrogen:The strain of 2F plasmodiums worm, dissolves and two mouse of Intraperitoneal injection In vivo；

3) after 3 days, mouse Infected With Plasmodium rate is observed, when infection rate is in 3%-5%, counts protozoan infection rate and red thin Born of the same parents' concentration (individual/ml)；

4) immune mouse is divided into three groups, every group 4, first group of inoculation P. yeolii P.y-WT, inoculum concentration is 5*10⁵Individual/only；Second group of inoculation transfects the mouse P. berghei P.y-GPC3 of empty carrier:2F, inoculum concentration is 5*10⁵Individual/only；The Three groups of red blood cells of the normal mouse of inoculation equal number, as blank control group.The day is set to D0；

5) while, give three groups of experiment mice dorsal scs inoculation Hepa1-6 cells 5*10⁵Individual/only；

6) fortnight after mouse inoculation plasmodium and tumour cell, takes out spleen, separates and obtains spleen cell. 200 mesh filter screens filter spleen cell；

7) erythrocyte cracked liquid (BD) is added, 20min, 300g centrifugations 5min is cracked on ice；

8) twice of PBS of the use containing 2%FBS, 300g centrifugations 5min.If erythrocyte splitting is insufficient, then red blood cell Lysate is cracked once on ice；

9) 200 mesh filter screens refilter spleen cell once；

10) dye, 106 spleen cells are taken out, with streaming antibody CD11c-FITC, CD8 α-PE, CD86-APC, CD80- PerCP-Cy5.5 (being purchased from eBioscience, San Diego, CA, USA) stained specimens, wherein the sample that is unstained, CD11c- The mono- dyes of FITC, the double dyes of CD11c-FITC and CD8 α-PE are used as negative control group, each sample lucifuge dyeing 10min；

11) twice of PBS of the use containing 2%FBS, 300g centrifugations 5min；

12) machine testing cell sample in streaming.Streaming instrument is BD FACSAria, and flow cytometer showed software is FlowJo (Tree Star,Inc.)；

Shown in result such as Fig. 3 (A)-Fig. 3 (B), Fig. 3 (A)-Fig. 3 (B) causes the ratio of CD8 α+DC to increase for Infected With Plasmodium Plus, the CD80 and CD86 of CD8 α+DC expression are higher than expression on the CD8 α-DC in Mice Body, the immune mice spleen of plasmodium The ratio of dirty interior CD8 α+DC will be significantly improved than do not have plasmodium immune, while in each group mouse, P.y-GPC3:2F exempts from CD80 and CD86 has the raising of conspicuousness compared with other two groups in CD8 α+DC in epidemic disease group, and CD80 and CD86 are in CD8 α+DC Expression is high, and the expression ratio of CD80 and CD86 is reduced in CD8 α-DC, particularly the expression of CD80.

3rd, the immune tumor-bearing mice of restructuring P. yeolii, the table of Th1 relevant cell factors in detection immune serum Up to situation.

5) while, give three groups of experiment mice dorsal scs inoculation Hepa1-6 cells 5*10⁵Individual/only.

First day, three days, the 7th day and fortnight, i.e. D1, D3, D7, D14, blood 150 is taken with glass capillary eye μ l, the physiological saline of intraperitoneal injection same volume.Sample blood normal temperature is stood into 1h, after after blood coagulation, 1000rpm/min is centrifuged 10min, takes out supernatant serum, and packing 2 is managed, and -80 DEG C of refrigerators freeze.

The concentration of Cytokine of Serum is detected using BioLegend multiple-factors detection kit, the kit can be examined Survey the concentration of Th1 relevant cell factors.Specific kit detection operation refer to product description and technical support.

Result as shown in figure 4, plasmodium can effectively activate Th1 relevant cell factors for the worm strain vaccine of carrier, including IL-2, IFN-γ, and TNF-α, testing result show that their concentration reaches peak on 7th day immune, and are deposited with control group In significant difference, statistical difference specific analysis P≤0.05, *, P≤0.01, * *；P≤0.001, * * *.

4th, after ELISAPOT detections restructuring Plasmodium yoelii immune mouse, the CTL for GPC3 albumen is detected in vivo

1) in vitro culture of mouse BM-DC cells

(1) takeC57BL/6 mouse 4, cervical dislocation puts to death mouse, mouse is put into 70% ethanol and is soaked 10min, for sterilizing；

(2) in aseptic operating platform, two back legs of mouse are removed the peel with sterilizing tweezers and scissors is cut from getting off, be put into diameter In 10cm culture dishes, cell culture medium RPMI 1640 is soaked, and the muscle on thigh bone is peeled off with scissors；

(3) back leg femur is obtained, femur two ends joint is cut off with scissors, syringe notes the complete mediums of RPMI 1640 Enter in thigh bone cavity, repeatedly rinse and obtain bone cavity inner cell；

The cell liquid that the aseptic strainer filtering of (4) 200 mesh is obtained, 300g centrifugations 8min；

(5) 15mL centrifuge tubes are taken, adds 2ml erythrocyte cracked liquids resuspended and cracking centrifugation bottom of the tube cell, cracked on ice 8min, adds 5ml PBS, and 300g centrifugations 8min, is washed one time again with PBS again；

(6) in cell being spread into 10cm culture dishes, RPMI RPMI-1640s are added, containing 10%FBS, GM-CSF (50g/l) And 2-Me (50 μM) carries out Fiber differentiation；

(7) after culture 24h-48h, adherent growth is removed partial suspended cell by BM-DC cells.Culture 7 days.

2) acquisition of plasmodium immune mouse spleen cell and CD8 α+T cell.

(1) the Hepa1-6 cells for freezing are taken out in liquid nitrogen, 37 DEG C of defrostings, iuntercellular activate and Amplification Culture；

(2) P.y-WT, the P.y-GPC3 for freezing are taken out in liquid nitrogen:2F and P.y-eGPC3:The strain of 2F plasmodiums worm, dissolving is simultaneously In two Mice Bodies of Intraperitoneal injection；

After (3) 3 days, mouse Infected With Plasmodium rate is observed, when infection rate is in 3%-5%, count protozoan infection rate and red Cell concentration (individual/ml)；

(4) immune mouse is divided into four groups, every group 5, first group of inoculation P. yeolii P.y-WT, inoculum concentration is 5*105/only；Second group of inoculation plasmodium P.y-GPC3:2F, inoculum concentration is 5*10⁵Individual/only；3rd group of inoculation transfection is unloaded The mouse P. berghei P.y-eGPC3 of body:2F, inoculum concentration is 5*10⁵Individual/only；The 4th group of normal mouse of inoculation equal number Red blood cell, as blank control group.The day is set to D0；

(5) while, give three groups of experiment mice dorsal scs inoculation Hepa1-6 cells 5*10⁵Individual/only；

(6) the 17th day of mouse Infected With Plasmodium, puts to death mouse, and peels off spleen, separates and obtains spleen cell；

(7) the aseptic spleen that takes is put into the sterilized petri dishes containing the complete mediums of RPMI 1640, is rinsed and is carefully removed muscle Film and adipose tissue；

(8) 200 mesh steel meshes of a sterilizing are placed on small sterile plate, it is spleen is placed on it and drip 1ml containing 2% 1640 culture mediums of FBS；

(9) RPMI 1640 culture mediums of the 1ml containing 2%FBS is extracted with 2ml syringes, from the injection of spleen side, makes spleen Swelling；

(10) spleen is cut into the fritter of 1-2mm with curved scissors, then with the plunger gentle abrasion of 2ml, 5-6ml is used The culture mediums of RPMI 1640 containing 2%FBS rinse grinding spleen cell, and cell is collected into a centrifuge tube of 50ml, will manage It is put into ice；

(11) after the spleen of required treatment has been operated successively as stated above, 4 DEG C, 300g × 10min centrifugations are abandoned Clearly, scattered cell precipitation is gently shaken；

(12) add in 10ml erythrocyte cracked liquids, slow shake makes cell resuspended, and action is gentle, in order to avoid form cell Aggegation block, is stored at room temperature 4-5min；

(13) RPMI 1640 culture mediums of the 25mL containing 2%FBS is added to terminate cracking, 4 DEG C, 200g × 10min centrifugations are abandoned Supernatant, adds 15ml nutrient solutions, slowly shakes re-suspended cell；

(14) pipe is stood into 2-3min, the cell for taking top 12ml is resuspended, in moving into another new pipe；

(15) 4 DEG C, supernatant is abandoned in 200g × 10min centrifugations, adds the complete mediums of 6ml RPMI 1640, re-suspended cell；

(16) the CD8 α+T cell in U.S. day Ni mouse CD8 α+magnetic bead sorting splenocyte.Concrete operations are with reference to U.S.'s day Ni magnetic beads Sorting kit specification.

3) CD8 α+T cell is co-cultured with BM-DC, the IFN-γ feelings of IFN-γ ELISPOT detection CD8 α+T cell secretion Condition.

(1) the CD8 α+T lymphocytes for obtaining every mouse are calculated, and go 2 × 10⁶Individual CD8 α+T lymphocytes are put into 24 In orifice plate.5 × 10 are added per hole⁵The BM-DC of individual advance culture, adds the complete medium 400 of the μ g/ml of the albumen of GPC3 containing mouse 30 μ l, are incubated culture 7 days in cell culture incubator；

(2) prepare the orifice plates of ELISPOT 96 of pre-coated IFN-γ antibody, 2 × 10 are added per hole⁵Individual advance incubation The BM-DC and 5 × 10 of mouse GPC3 albumen cultures⁵Individual CD8 α+T cell, adds the μ l of complete medium 100, in cell culture incubator It is incubated culture 2 days；

(3) culture medium is removed, deionized water is cleaned 2 times, each 5min (film should not be encountered during washing)；

(4) 200 μ l Wash Buffer are added dropwise per hole, clean 3 times, each 5min；

(5) 100 μ l antibody tests liquid (Dectection Antibody Solution) are added dropwise per hole, close the lid room temperature It is incubated 2 hours；

(6) antibody test liquid is removed, 200 μ l Wash Buffer is added dropwise per hole, cleaned 3 times, each 2min；

(7) 100 μ l HRP are added to mark streptomysin Avidin (streptavidin-HRP) per hole.Cover lid, room temperature It is incubated 1 hour；

(8) removal HRP marks streptomysin Avidin, and 200 μ l Wash Buffer are added dropwise per hole, cleaning 4 times, every time 2min, PBS 2 times, per the μ l of hole 200；

(9) AEC solution (AEC substrate solution) dyeing is added dropwise per hole, when determining colour developing according to colour developing situation Between, generally 5-60min；

(10) it is washed with deionized water, terminating reaction.After last time is washed, by plate back-off on clean blotting paper；

(11) lucifuge air dried overnight, removes outer layer plastic sheet frame, keeps in dark place, and uses plate reader read plate.

Result is as shown in Figure 5, it is seen that the recombinant plasmodium P.y-eGPC3 of GPC3 secreting types:2F and wild type plasmodium After P.y-WT immune mouses, mouse body can not be all set to produce the specific CTL for GPC3 to react.And P.y-GPC3:2F malarias Protozoon possesses the ability reacted for the specific CTL of GPC3 in activation mouse body, and possess significant difference (P≤ 0.001, * * *), it is seen then that P.y-GPC3:2F immune mouse is able to detect that the strong CTL for GPC3, and secreting type GPC3 Expression worm strain P.y-eGPC3:The CTL for GPC3 is not all detected in 2F and control group.

Embodiment 4 recombinates inhibition of the Plasmodium yoelii to the subcutaneous liver neoplasm of tumor-bearing mice

1st, experiment material.Plasmodium yoelii, Hepa1-6 cells, slide measure, insulating box, 4% paraformaldehyde, dimethylbenzene, Ethanol, arrests rafter acid sodium, PBS, 3%H₂O₂Deionized water, haematine, immunologic combined detection reagent kit (BD).Ki67 antibody (abcam, #ab16667).

2nd, the 14th day restructuring immune inhibition to tumor-bearing mice hypodermic tumour of P. yeolii

2) P.y-WT, the P.y-GPC3 for freezing are taken out in liquid nitrogen:2F、P.y-eGPC3:The strain of 2F plasmodiums worm, dissolves and abdomen Chamber is injected in two Mice Bodies；

4) immune mouse is divided into four groups, every group 5, first group of inoculation P. yeolii P.y-WT, inoculum concentration is 5 ×10⁵Individual/only；Second group of inoculation plasmodium P.y-GPC3:2F, inoculum concentration is 5 × 10⁵Individual/only；3rd group of inoculation transfection is unloaded The mouse P. berghei P.y-eGPC3 of body:2F, inoculum concentration is 5 × 10⁵Individual/only；The 4th group of normal mouse of inoculation equal number Red blood cell, as blank control group.The day is set to D0；

5) while, every group of every mouse hypodermic inoculation Hepa1-6 cell 5 × 10⁵Individual/only；

6) the 7th day starts, and measures the size of mouse hypodermic tumour, worm blood infection rate and Survival；

7) the 14th day, mouse tumor is taken out, and taken pictures together with ruler.

Result is as shown in fig. 6, gross tumor volume computing formula is：V=(ab²)/2, wherein a are the length of tumour, and b is tumour It is wide.The expression of Ki67 in SABC detection each group tumour.Result shows in the immune mouse of plasmodium, P.y-eGPC3: The immune control groups immune with plasmodium is not carried out of 2F or P.y-WT compares, and tumour has certain inhibition (P≤0.05, *), And Hepa1-6-P.y-GPC3:2F compares with control group and suppresses the effect of tumour most substantially (P≤0.001, * * *), it and P.y- eGPC3:2F or P.y-WT treatment group tumor sizes compare, and its inhibition is also fairly obvious (P≤0.01, * *).Immune group Change result to also indicate that, Ki67 receives effective suppression in the expression of plasmodium treatment group intra-tumor.

3rd, the immune tumor-bearing mice hypodermic tumour of plasmodium, distributions of the detection Ki67 in tumour

1) the mouse hypodermic tumour peeled off, 4% paraformaldehyde fixes tumor tissues, and SABC detects Ki67 in tumour Interior distribution situation；

2) wash.After fixation, the fixer in tissue must be rinsed well material, because residual fixation in the tissue Liquid, what is had is unfavorable for dyeing, some generation precipitations or crystallization influence observation；

4) it is dehydrated.30%th, 50%, 70%, 80%, 90% ethanol solutions at different levels are dehydrated each 40min, are put into 95%, 100% Respectively twice, each 20min (after various materials are through fixed and washing, contains large quantity of moisture, because water can not be mutual with paraffin in tissue It is molten, so the moisture in tissue must be removed)；

5) it is transparent.100% alcohol, dimethylbenzene equivalent mixed liquor 15min, dimethylbenzene 0.5h (or to transparent), must change Dimethylbenzene.(because ethanol is immiscible with paraffin, and dimethylbenzene can be dissolved in ethanol and can be dissolved in paraffin, so after dehydration Will also be by dimethylbenzene with transition, when all being occupied by dimethylbenzene in tissue, light can be passed through, and tissue shows different journeys The pellucidity of degree)；

6) saturating wax.Be put into the mixed liquor 15min of dimethylbenzene and paraffin half and half, place into paraffin I, the saturating waxes of paraffin II each 20~ 30min.(purpose of saturating wax is clarifier such as dimethylbenzene in removing tissue etc., paraffin infiltration is reached saturation to organization internal Degree is to embed.The saturating wax time is more long according to the saturating wax time of tissue, about needs 1-2day.Saturating wax should be carried out in insulating box, And the temperature inside the box is kept at 55-60 DEG C or so, notice that temperature should not be too high, in order to avoid tissue embrittlement.It is placed in insulating box 0.5h)；

7) embed., together with the paraffin of fusing, poured into by the tissue of saturating wax in container together, cold water is put into immediately after In, it is frozen into wax stone at once.For embed melting point of paraffin wax between 50-60 DEG C, during embedding should according to organization material, The factors such as slice thickness, weather conditions, select the paraffin of different melting points.The conventional paraffin melting point of general animal material is 52-56 ℃；

8) cut into slices；

9) paster.

Using SP (streptavidin-perosidase) method, mainly comprise the following steps：

1) histotomy toasts 20min in 60 DEG C of insulating boxs；

2) histotomy is placed in immersion 10min in dimethylbenzene (I), changes and soaks 10min again to after dimethylbenzene (II)；

3) immersion 5min in absolute ethyl alcohol (I)；

4) immersion 2min in absolute ethyl alcohol (II)；

5) 2min is soaked in 95% ethanol；

6) 2min is soaked in 70% ethanol；

7) 2min is soaked in single steaming water；

8) antigen retrieval：Rafter acid sodium microwave method is arrested with 0.01M to repair；

9) standing 20min makes section recover to room temperature, soaking flushing in tri-distilled water；

10) PBS washes immersion 5min；

11) 3%H₂O₂Deionized water, is stored at room temperature 10min, and TBST washes 2-3 each 5min；

12) Normal Goat Serum confining liquid, room temperature 15min is added dropwise；

13) surplus liquid is got rid of, rabbit-anti CD8a antibody (1 is added respectively：200) 4 night is spent；4 spend night；TBST washes 3 times respectively 3min；

14) biotinylated secondary antibody 40-50 μ l are added dropwise, 30min is stored at room temperature；TBST washes 3 each 3min；

15) three anti-40-50 μ l that are antibiotin being added dropwise and marking horseradish peroxidating compound enzyme, are stored at room temperature 30min, TBST washes 3 each 3min；

16) DAB colour developings, grasp dye levels under the microscope；

17) PBS or running water rinse 10min；

18) haematoxylin redyeing 1min or so, the differentiation of 1% hydrochloride alcohol；

19) running water rinses 10-15min；

20) it is dehydrated：Twice, twice, dimethylbenzene 10 seconds is twice for 100% alcohol 10 seconds for 95% alcohol 10 seconds；

21) piece mounting is dried.

4th, the immune long-term inhibition and survival effect to tumor-bearing mice hypodermic tumour of restructuring P. yeolii

4) immune mouse is divided into three groups, first group of inoculation P. yeolii P.y-WT, inoculum concentration is 5 × 10⁵Individual/ Only (n=8)；Second group of inoculation plasmodium P.y-GPC3:2F, inoculum concentration is 5 × 10⁵Individual/only (n=8)；3rd group of inoculation is identical The red blood cell of the normal mouse of quantity, as blank control group (n=9)；

7) according to animal welfare and ethics, any diameter of mouse tumor is no more than 20mm, or has two tumours to give birth to When long, the maximum gauge of two tumours is added up no more than 20mm.If it exceeds the regulation of the above, is considered as dead mouse, and Mouse is carried out into euthanasia operation；

8) measurement of tumor size terminates for 32 days or so, and Survival record is then tied untill the death of all experimental mouses Beam；

9) when the tumor size otherness of each group mouse is counted, it is ensured that every group of mouse quantity is more than or equal to 3.Less than 3 When, the tumour growth difference analysis between each group are not compared；

Result according to animal welfare and ethics as shown in fig. 7, specify that length of tumor will be put to death and be considered as more than 20mm Dead mouse, tumor size stops measurement when every group of mouse survival is less than 3.Shown according to the left statisticses of Fig. 7, it is former in malaria 25 days after worm is immune, tumour is all subject to significant suppression (P≤0.001, * * *), while P.y-GPC3:2F can more have compared with P.y-WT The growth (P≤0.05, *) of the suppression tumour of effect；Fig. 7 right sides show that GPC3 is expressed in plasmodium and have no effect on Blood-stage Plasmodium Normal growth.Most importantly show in Fig. 7, plasmodium P.y-GPC3:2F immune mouse can not only effectively suppress Tumour growth can also significantly extend the life cycle of mouse.

Applicant states that the present invention illustrates method detailed of the invention by above-described embodiment, but the present invention not office It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Art Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosing.

SEQUENCE LISTING

<110>Guangzhou Zhong Kelanhua bio tech ltd

<120>A kind of recombinant plasmid, its recombinant plasmodium for building and its application

<130> 2016

<160> 25

<170> PatentIn version 3.3

<210> 1

<211> 14873

<212> DNA

<213>Artificial 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 ataaccatga 3000

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 tctacgtgaa cgaccagaag 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 tctttcttcc agagactgca 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>Artificial 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>Artificial 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 tggtaaacct gacgttgtac 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>Artificial 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>Artificial 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>Artificial synthesized sequence

<400> 6

ggatccatgg tgagcaaggg cgaggagctg t 31

<210> 7

<211> 50

<212> DNA

<213>Artificial synthesized sequence

<400> 7

ctggatcata aactttcgaa gtcatcttgt acagctcgtc catgccgaga 50

<210> 8

<211> 50

<212> DNA

<213>Artificial synthesized sequence

<400> 8

tctcggcatg gacgagctgt acaagatgac ttcgaaagtt tatgatccag 50

<210> 9

<211> 35

<212> DNA

<213>Artificial synthesized sequence

<400> 9

gcggccgctt attgttcatt tttgagagaa ctcgc 35

<210> 10

<211> 33

<212> DNA

<213>Artificial synthesized sequence

<400> 10

atatcgatat ggccgggacc gtgcgcaccg cgt 33

<210> 11

<211> 34

<212> DNA

<213>Artificial synthesized sequence

<400> 11

gtctagaggt cagtgcacca ggaaaaaaaa gcac 34

<210> 12

<211> 33

<212> DNA

<213>Artificial synthesized sequence

<400> 12

gcggccgcga tcccgttttt cttacttata tat 33

<210> 13

<211> 33

<212> DNA

<213>Artificial synthesized sequence

<400> 13

atatcgatcc ctatgtttta taaaattttt tat 33

<210> 14

<211> 32

<212> DNA

<213>Artificial synthesized sequence

<400> 14

aggatccatg gccgggaccg tgcgcaccgc gt 32

<210> 15

<211> 90

<212> DNA

<213>Artificial synthesized sequence

<400> 15

ggtctagaga gaccttactt atcgtcgtca tccttgtaat ccttatcgtc gtcatccttg 60

taatcgtgca ccaggaaaaa aaagcacgcc 90

<210> 16

<211> 32

<212> DNA

<213>Artificial synthesized sequence

<400> 16

aggatccatg gctcgtaatt ttgaatgcaa aa 32

<210> 17

<211> 59

<212> DNA

<213>Artificial synthesized sequence

<400> 17

ccgccagatc cacctccacc acttccgcca cctcccatag gtggttgtgt tctattgaa 59

<210> 18

<211> 70

<212> DNA

<213>Artificial synthesized sequence

<400> 18

ggaggtggcg gaagtggtgg aggtggatct ggcggtggag gaagcatggc cgggaccgtg 60

cgcaccgcgt 70

<210> 19

<211> 85

<212> DNA

<213>Artificial synthesized sequence

<400> 19

ggtctagagt tacttatcgt cgtcatcctt gtaatcctta tcgtcgtcat ccttgtaatc 60

ggtgatctcg ttgtccttct gattt 85

<210> 20

<211> 32

<212> DNA

<213>Artificial synthesized sequence

<400> 20

aatcgatatg gtgagcaagg gcgaggagga ta 32

<210> 21

<211> 36

<212> DNA

<213>Artificial synthesized sequence

<400> 21

agtctagatt acttgtacag ctcgtccatg ccgccg 36

<210> 22

<211> 20

<212> DNA

<213>Artificial synthesized sequence

<400> 22

atgtaatatt tggatatttc 20

<210> 23

<211> 22

<212> DNA

<213>Artificial synthesized sequence

<400> 23

tcacctacgg aaaccttgtt ac 22

<210> 24

<211> 21

<212> DNA

<213>Artificial synthesized sequence

<400> 24

gttgaaaaat taaaaaaaaa c 21

<210> 25

<211> 23

<212> DNA

<213>Artificial synthesized sequence

<400> 25

tttcccagtc agtcacgacg ttg 23

Claims

1. a kind of recombinant plasmid, it is characterised in that the recombinant plasmid is to insert liver neoplasm in pL0017 plasmids specifically to resist Protogene.

2. recombinant plasmid according to claim 1, it is characterised in that the liver neoplasm specific antigen gene is phosphatidyl The expressing gene gpc3 of inositol proteoglycans 3, first embryo protein AFP expressing genes, MAGE expressing genes or NY-ESO-1 expressing genes In any one or at least two combination, preferably glypican-3 expressing gene gpc3；

Preferably, recombinant plasmid promoter pbeef1a α and terminator 3 ' that also the full worm phase including P. berghei expresses UTR；

Preferably, the recombinant plasmid also includes restriction enzyme site Not I and Cla I.

3. recombinant plasmid according to claim 1 and 2, it is characterised in that the nucleotide sequence of the recombinant plasmid such as SEQ Shown in ID NO.1.

4. the recombinant plasmid according to any one of claim 1-3, it is characterised in that the recombinant plasmid expresses 2 simultaneously Individual or more than 2 foreign genes.

5. a kind of recombinant plasmodium, it is characterised in that the recombinant plasmodium is included as any one of claim 1-4 Recombinant plasmid.

6. recombinant plasmodium according to claim 5, it is characterised in that the recombinant plasmodium also includes green fluorescence egg The fusion protein gRluc and/or secretory protein IBIS1 of white GFP and renilla luciferase；

Preferably, the amino acid sequence of the fusion protein gRluc is SEQ ID NO.2, and nucleotides sequence is classified as SEQ ID NO.3；

Preferably, the secretory protein IBIS1 amino acid sequences are SEQ ID NO.4, and nucleotides sequence is classified as SEQ ID NO.5.

7. a kind of vaccine, it is characterised in that the vaccine include recombinant plasmid as any one of claim 1-4 with/ Or the recombinant plasmodium described in claim 5.

8. a kind of recombinant plasmid as any one of claim 1-4, recombinant plasmodium as claimed in claim 5 or such as Vaccine described in claim 7 is used for the treatment of liver neoplasm.

9. application according to claim 8, it is characterised in that the treatment of the liver neoplasm includes：Will be such as claim 5 Described recombinant plasmodium and/or vaccine organism as claimed in claim 7；

Preferably, the treatment of the liver neoplasm is specifically included：By recombinant plasmodium combination plasmodium as claimed in claim 5 Erythrocytic stage treatment liver neoplasm；Or by vaccine organism as claimed in claim 7.

10. a kind of medicine for liver neoplasm treatment, it is characterised in that the medicine is included as any in claim 1-4 Recombinant plasmid described in and/or the recombinant plasmodium described in claim 5.