A kind of recombinant plasmid, its recombinant plasmodium for building and its applicationTechnical field
The present invention relates to the cellular immunotherapy field of tumour, more particularly to a kind of recombinant plasmid, its restructuring malaria for buildingProtozoon and its application, the structure of recombinant plasmodium specially based on recombinant plasmid and its in the treatment of anti-liver neoplasmUsing.
Background technology
Hepatocellular carcinoma (Hepatocellular carcinoma, HCC), account for primary carcinoma of liver ratio 85% is arrived90%.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 newIncrease 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 treatmentIncluding surgery excision, orthotopic liver transplantation, TACE and local RF therapy etc., these methods are to early stage HCC for artPatient has certain curative effect.But patient often has found too late, 50% liver cancer patient is not appropriate for using these conventional methods.TogetherWhen, 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, easilyRecurrence etc..In addition, the medicine of targeted therapy liver neoplasm, including Sorafenib and Bevacizumab etc., there is also treatment secondaryThe 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 masterTumour cell is killed by activating in patient's body specific C D8+T lymphocytes.Adoptive treatment includes chimericAntigen receptor T cells (CAR-T) treatments are widely studied and participate in clinical practice, but this for individualTherapeutic modality spend time and money it is all very huge, not all people is receptible.At present, by vector expressionTumour specific antigen, and it is considered as one feasible honest and clean to activate internal specific cellular immunity come the method to neoplasm growthThe 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 rawRate 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 vaccinesMuch progress.However, these vaccines there is also a universal problem, the exactly caused cell for liver tumor antigens is exempted fromEpidemic 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 wellCertainly this problem.
HCC specific antigens are widely found and for the immunization therapy of tumour.Including phosphatidylinositols albumenThe 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 cellReacted to killing.For the antibody of GPC3, polypeptide vaccine and CAR-T treatments have come into in-depth study, wherein GPC3Polypeptide vaccine has been completed the clinical II phases, and its overall survival has significantly pass with the GPC3 specific immune responses of machine vivo activationConnection.
One immunotherapy of tumors based on antigenic activation vivo immuning system, generally requires a good expression and carriesBody.The wherein more carriers of application include DNA, RNA, defective virus and bacterium.They can activate strong in vivo exempting fromEpidemic 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 goodGood Antigen Expression Vectors should include following two abilities：One is to swash the intravital strong specific CTL for antigenReaction；Two is the presence that this specific CTL reaction can be lasting in vivo and performance tumor-killing effect.Numerous studies tableBright, some viruses or bacterium infection can well suppress tumour growth.Meanwhile, it is also wide that protozoan is used for oncotherapyGeneral research, including Infection of Toxoplasma Gondii and taper worm.Meanwhile, plasmodium can activate very strong internal innate immune reaction.PlasmodiumCan be used as a kind of natural adjuvant, some compositions such as GPI (the glycosyl phosphatidylinositol of itselfAnchors), genomic DNA and malarial pigment can be used as pathogen associative mode molecule (pathogen-associatedMolecular 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.AndIt 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 plasmodiumIt is have certain foundation as the immunization therapy carrier of HCC：1, Infected With Plasmodium can swash intravital Th1 reactions, and it is rightIt is most important for the CTL of tumour antigen in activation；2, although red blood cell lacks major histocompatibility antigen I (majorHistocompatibility 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 itselfIn 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 timeSwash intravital ctl response in ground.
CN 101838611A disclose a kind of recombinant plasmodium of expression alien gene, the external source in the recombinant plasmodiumGene includes antigen gene, therapeutic gene, immunomodulator gene or peptide gene, and the plasmid that wherein invention is used is pL0015Plasmid, pL0015 plasmids mainly use single endonuclease digestion (Bam HI) insert genes of interest, this insert genes of interest when, easilyThere 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 tableGene 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 constructionGene 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 swellKnurl specific antigen gene.
In the present invention, double digestion method (Bam HI/Xba I) is used to insert genes of interest from pL0017 plasmids, thisSample 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 gpc3Cause), first embryo protein AFP expressing genes, in MAGE expressing genes or NY-ESO-1 expressing genes any one or at least twoCombination, preferably gpc3 antigen genes, the GPC3 albumen of the gpc3 expression is the very strong liver neoplasm of an immunogenicityAntigen, for the antibody of GPC3, polypeptide vaccine and CAR-T treatments have come into in-depth study, wherein GPC3 polypeptides epidemic diseaseSeedling 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 matterPromoter 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 essenceSingle 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 geneExpression, is capable of the GPC3 albumen of continuous expression introducing.Simultaneously as improved plasmid includes 2 independent gene expression unitsPart, 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 describedForeign 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 twoThe 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 aspectRecombinant 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 hasThe Survival of inhibition and tumour model animal beneficial to tumour growth, this is that many expression vectors are exempted from HCC at presentInstitute 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 externalThe steps such as differentiability, spike and the further separation of combination flow cytometer, screening, monoclonal in positive restructuring plasmodiumIn reach the effect of optimization, what the recombinant plasmodium also included green fluorescent protein (GFP) and renilla luciferase merges eggWhite 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 notSpecial restriction is done, the specific present invention is used liver neoplasm specific antigen gene expression on carrier, obtain recombinating matterGrain, extracts and purifies after being replicated in Escherichia coli, in the Transfected Recombinant Plasmid that extraction is obtained to plasmodium, obtains expressing liverThe 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 IDNO.3。
Amino acid sequence shown in the SEQ ID NO.2 is as follows：
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKMTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQ.
Nucleotide sequence shown in the SEQ ID NO.3 is as follows：
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGATGACTTCGAAAGTTTATGATCCAGAACAAAGGAAACGGATGATAACTGGTCCGCAGTGGTGGGCCAGATGTAAACAAATGAATGTTCTTGATTCATTTATTAATTATTATGATTCAGAAAAACATGCAGAAAATGCTGTTATTTTTTTACATGGTAACGCGGCCTCTTCTTATTTATGGCGACATGTTGTGCCACATATTGAGCCAGTAGCGCGGTGTATTATACCAGACCTTATTGGTATGGGCAAATCAGGCAAATCTGGTAATGGTTCTTATAGGTTACTTGATCATTACAAATATCTTACTGCATGGTTTGAACTTCTTAATTTACCAAAGAAGATCATTTTTGTCGGCCATGATTGGGGTGCTTGTTTGGCATTTCATTATAGCTATGAGCATCAAGATAAGATCAAAGCAATAGTTCACGCTGAAAGTGTAGTAGATGTGATTGAATCATGGGATGAATGGCCTGATATTGAAGAAGATATTGCGTTGATCAAATCTGAAGAAGGAGAAAAAATGGTTTTGGAGAATAACTTCTTCGTGGAAACCATGTTGCCATCAAAAATCATGAGAAAGTTAGAACCAGAAGAATTTGCAGCATATCTTGAACCATTCAAAGAGAAAGGTGAAGTTCGTCGTCCAACATTATCATGGCCTCGTGAAATCCCGTTAGTAAAAGGTGGTAAACCTGACGTTGTACAAATTGTTAGGAATTATAATGCTTATCTACGTGCAAGTGATGATTTACCAAAAATGTTTATTGAATCGGACCCAGGATTCTTTTCCAATGCTATTGTTGAAGGTGCCAAGAAGTTTCCTAATACTGAATTTGTCAAAGTAAAAGGTCTTCATTTTTCGCAAGAAGATGCACCTGATGAAATGGGAAAATATATCAAATCGTTCGTTGAGCGAGTTCTCAAAAATGAACAATAA.
Preferably, the secretory protein IBIS1 amino acid sequences are SEQ ID NO.4, and nucleotides sequence is classified as SEQ IDNO.5。
Amino acid sequence shown in the SEQ ID NO.4 is as follows：
MARNFECKKINSDDMTSSKKYSKNVGEKFNLISCTKLFALSMLFLICQNYENSPQSTSSHQEYQYNGLVLGNRILSELDQAENHTISYKTNNYEDSSVENPNQQTSDSSSLQTDEDKKKDDSDATSIGETSPTTETTSVEETVTIEDTESVEETESVEETPSTSTEETSSTGKKTYVDRIASILNPLINGEKKSTEKKSSEKKSSEKKSSDEQSSSDEQNSSDDQNSFDDQKLFEDIDNLINGIKSRYQEFSAKIKSPEFQNKCKSYMNTAKEMIEERRNCAMSFISRNLNALGIDKIFEDEFGGYALLGKMMLTKVFIDNMFIPDFLRNSSTIILTIVYFLIMMFIVGSYLDINQDTKTERRNTNESKLFNRTQPPM.
Nucleotide sequence shown in the SEQ ID NO.5 is as follows：
ATGGCTCGTAATTTTGAATGCAAAAAGATAAATAGTGATGATATGACATCATCCAAGAAATATTCCAAAAATGTTGGCGAGAAATTTAATTTGATTTCTTGTACAAAATTGTTTGCGCTAAGCATGTTATTTTTGATATGCCAAAATTATGAAAATAGCCCACAAAGCACATCTTCACACCAAGAATACCAATATAATGGTTTGGTTTTAGGAAACAGAATATTATCAGAATTGGATCAAGCTGAAAATCATACTATAAGTTATAAAACAAACAATTATGAAGACTCTTCGGTTGAAAATCCAAATCAGCAAACCTCCGATAGTTCATCATTACAAACTGATGAAGATAAAAAAAAAGATGACAGTGATGCAACATCCATTGGAGAAACATCACCAACTACAGAAACGACATCAGTTGAAGAAACAGTAACAATTGAAGACACAGAATCAGTTGAAGAAACAGAATCAGTTGAAGAAACACCATCAACATCAACTGAAGAAACATCATCAACTGGAAAAAAAACATATGTTGATAGAATAGCTTCCATTTTAAATCCATTAATCAATGGCGAAAAAAAATCTACCGAAAAAAAATCTAGTGAAAAAAAATCTAGTGAAAAAAAATCTTCTGATGAACAAAGCTCTTCTGATGAACAAAATTCTTCTGATGACCAAAATTCTTTTGATGACCAAAAACTATTTGAAGATATTGATAATCTAATAAATGGAATCAAATCACGTTATCAAGAGTTCAGCGCTAAAATAAAATCACCAGAATTCCAAAACAAATGTAAAAGCTATATGAATACTGCAAAAGAAATGATTGAAGAACGTAGAAACTGTGCTATGAGCTTTATATCTAGAAATTTAAATGCCTTAGGTATTGATAAGATATTCGAAGATGAGTTTGGTGGTTATGCACTCCTTGGAAAAATGATGTTAACAAAAGTCTTTATTGACAATATGTTCATTCCTGACTTCTTAAGAAATAGCTCAACAATAATTTTAACTATAGTTTATTTCTTAATAATGATGTTCATCGTAGGAAGCTATCTTGATATCAATCAAGATACTAAAACTGAAAGAAGAAATACAAATGAATCTAAATTGTTCAATAGAACACAACCACCTATGTAA.
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 plasmodiumSecrete expression.Research shows that the GPC3 albumen of nonsecreting type can significantly activate CTLs in vivo for GPC3 albumen and significantly press downThe growth of tumour processed, and the GPC3 albumen of secreting type can not significantly activate CTLs in vivo for GPC3 albumen and suppress swollenThe growth of knurl.Illustrate that the expression of tumour specific antigen should be preferential when being treated for the biological immune of carrier with plasmodiumNonsecreting type expression is considered, rather than secreting, expressing.But if the gene therapy with plasmodium as carrier, such as expression P53 orDuring 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/orRecombinant 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 alwaysLymphocyte (cytotoxic T-lymphocyte, CTL) strong reaction but not lasting problem, this causes the effect of immunization therapyFruit 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 assessmentOne of expression vector pays the utmost attention to condition.One important aspect of immunization therapy of tumour is exactly activation special for tumour in vivoThe 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 formerCan worm carrier provide good precondition to swash intravital CD8+T cell effects.Here, the present invention is main by detectionThe 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 tumourThe 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 aspectGroup 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 aspectClose 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 partyRecombinant 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 baseCause, 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 primaryFamily 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 albumenApplication.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 internalA 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 cellTime 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 toThe 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 activatedThe innate immunity of body and acquired immunity, the Th1 reactions raised after it is immune and the increasing proportion of CD8 α+DC, these haveBeneficial 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) CTLRaw, 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 lotusThe 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 sourcesThe schematic diagram of gene expression element insertion method；Fig. 1 (B) is the correct structure that pL0017-gRluc-X plasmids are identified in digestion, itsIn, 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 withThe 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 knotsReally, 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 toRoad 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 bodyDetect recombinant plasmodium P.y-gRluc-mCherry, P.y-gRluc-ex_mCherry in C57BL/6 mouse under imagerDistribution situation；Fig. 1 (E) is the confocal laser microscopy images figure of the recombinant plasmodium for building；Fig. 1 (F) is the restructuring for buildingThe 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.ItsIn, Fig. 2 (A) is that different xgpc3 genes insert pL0017 plasmids site and wild type plasmodium chromosome is inserted into c-rrnaThe 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 groupThe design diagram of primer during identification；Fig. 2 (C) for PCR cloned from recombinant plasmodium genome 5 ' int, 3 ' int, gRluc andGpc3 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 twoDistribution situation in individual recombinant plasmodium and red blood cell；Fig. 2 (F) is the laser co-focusing microexamination under GPC3 antibody labelingsGPC3 is in P.y-GPC3:(ring bodies of plasmodium, trophozoite, schizont and gamete in the 2F worms strain erythrocytic stage difference polypide stageThe 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+CD8The 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-DCThe 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 serumConcentration 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 plasmodiumProperty 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 normalErythrocyte immune tumor-bearing mice be experimental comparison group, left side be tumor size in fact shine, right side for tumor size volume and systemMeter learns diversity ratio pair, and all statistical methods are T inspections (unpaired two-tailed Student ' s t-tests), whereinIt 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 subcutaneousKi67 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, controlGroup normocytic mouse for injection has, Fig. 7 is left for significant difference is marked；P.y-GPC3:2F can significantly improve mouseLife 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 realMode 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 ReferenceReagent Resource Center) give.Toxoplasm dihyrofolate reductase (tg containing mouse on pL0017 plasmidsDhfr/ts) gene, the gene has pyrimethamine resistance.We are by exogenous gene cloning and insert Bam H I with Xba IPoint, replaces the gfp genes on original plasmid, and the ORFs ORF for inserting gene is opened by P. berghei transcriptional elongation factorMover (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 templatePrimer 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 lexaFluor@488donkey anti-mouse IgG (H+L) (Life technologies, #1423052), Anti-mouseIgG-HRP antibody (Cell Signaling Technology, #7076), Anti-Rabbit IgG-HRP antibody (CellSignaling 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 CenterSupport, rule illumination in 12 hours, 12 hours dark.The operation of zoopery meets the relevant regulations that experimental animal uses, Suo YoushiTest 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 aboutFamily name plasmodium Plasmodium yoelii 17XNL (MRA-593) is by MR4 (the Malaria Research andReference Reagent Resource Center) give.Hepa1-6 is from Shanghai Chinese Academy of Sciences cell bank (theChinese 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 (BeckmanCompany), light microscope (Lycra company), cryogenic freezing centrifuge (German Eppendorf companies), biochemical cultivation case (ShanghaiOne 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 boxesOK.
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 each0.5 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, plusEnter the Solution I (2 ×) containing ligase or quick T4 ligases and buffer solution, 37 DEG C of connections 15-30min, Ran HoujinRow 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 or10 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 consolidatedOn 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, guarantorBacterium 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, cloneCorrectly.
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’-GGTCTAGAGAGACCTTACTTATCGTCGTCATCCTTGTAATCCTTATCGTCGTCATCCTTGTAATCGTGCACCAGGAAAAAAAAGCACGCC-3’
ibis1-Bam H I–F(SEQ ID NO.16):5’-AGGATCCATGGCTCGTAATTTTGAATGCAAAA-3’
ibis1-linker-R(SEQ ID NO.17):5’-CCGCCAGATCCACCTCCACCACTTCCGCCACCTCCCATAGGTGGTTGTGTTCTATTGAA-3’
gpc3-F-linker(SEQ ID NO.18):5'-GGAGGTGGCGGAAGTGGTGGAGGTGGATCTGGCGGTGGAGGAAGCATGGCCGGGACCGTGCGCACCGCGT-3'
gpc3-Xba I-2Flag-R(SEQ ID NO.19):5-GGTCTAGAGTTACTTATCGTCGTCATCCTTGTAATCCTTATCGTCGTCATCCTTGTAATCGGTGATCTCGTTGTCCTTCTGATTT-3’
mCherry-Cla I-F(SEQ ID NO.20):5'-AATCGATATGGTGAGCAAGGGCGAGGAGGATA-3'
mCherry-XbaI-R(SEQ ID NO.21):5'-AGTCTAGATTACTTGTACAGCTCGTCCATGCCGCCG-3'
PyL739(SEQ ID NO.22):5'-ATGTAATATTTGGATATTTC-3’
PyL740(SEQ ID NO.23):5'-TCACCTACGGAAACCTTGTTAC-3’
L665(SEQ ID NO.24):5'-GTTGAAAAATTAAAAAAAAAC-3’
L635(SEQ ID NO.25):5'-TTTCCCAGTCAGTCACGACGTTG-3’。
2nd, Trizol extracts total mRNA of Hepa1-6 cells and P.y17XNL-WT plasmodiums, clone gpc3 andIbis1 genes.
(1) reagent is prepared：Chloroform, isopropanol, 75% ethanol (DEPC water is matched somebody with somebody), the water or 0.5%SDS of RNAase-freeSolution (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 usedTreated DEPC water configuration)；
(2) the cell Hepa1-6 of adherent growth：3.5cm diameter dishes add 1ml Trizol, blow and beat repeatedly, (1mlTrizol is used for 10cm2Area, Trizol not enough amount may cause DNA pollution), room temperature place 5min, to ensure nucleoproteinComplex 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 obtainedProtozoon 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 Trizol0.5ml 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, obtainsObtain 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 ReverseThe 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 volumeL, 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 twoPipe；
C) often pipe plus 1 μ l gpc3 or ibis1 reverse primer, plus 1 μ l M-MLV Reverse TranscriptaseInto 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 sequencePrimer 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 templateI/Xba I) gene, the gene is connected on pMD18T carriers (TAKARA), heat shock is transferred in Escherichia coli, obtains monoclonalBacterial 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 achievementClose 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 basisPL0017-gRluc-ibisi-mCherry plasmids, pL0017-gRluc-gpc3 plasmids, pL0017-gpc3 plasmids are constructedAnd pL0017-ibis1-gpc3 plasmids.
By genetic fragment mCherry, ibis1-mCherry of above-mentioned successful clone, gRluc, (gfp merges base with Rluc'sCause), 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 ShiBetween I/XbaI restriction enzyme sites, wherein, first ORFs ORF1 can be inserted into gRluc genes, in order that second opening is readFrame ORF2 successful expression genes of interest, we add a new terminator (3 ' UTR) before ORF2, and it is used to terminateThe normal transcription of ORF1 genes, we are inserted into a new promoter pbeef1 α a, the base for starting ORF2 after 3 ' UTRBecause 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 ORF2Enter any foreign gene, the fragment cut out from passage 1Bam HI/Xba I double digestion results includes gRluc-3 ' UTR-pbeef1 αA, 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 pL0017On 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 matterGrain (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 andpL0017-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 concentrationsRise in triangular flask, 37 DEG C of shaking culture 12h collect bacterium solution and carry plasmid kit (QIfilter greatly with QIAGEN companiesPlasmid Maxi kit) plasmid is extracted, by specification operation is carried out, and the UV detector that will be extracted determines the dense of plasmidDegree 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, includingP.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 meltedChange；
(2) in 1ml syringes Intraperitoneal injection C57BL/6 Mice Bodies, two mouse are injected, every worm blood is 0.5ml, dilutionIt 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 diluteThe 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 liquorAverage rate under 10 visuals field of number is the protozoan infection rate of the mouse.When infection rate reaches more than 10%, eyeball is pluckedTake 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 repeatedlyIt 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 inTo 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 every25mM HEPES (5.96g), 0.85g NaHCO3,50mg neomycinsulphates are added in 100ml culture mediums, 0.22 μM of mistake is filteredBacterium；
(8) 5ml complete mediums are added in 50ml centrifuge tubes, liquaemin anti-freezing is contained within.Above-mentioned 3 C57BL/6 is smallMouse 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 bottleThe 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 mixtureBody, 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 supernatantPrecipitation 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 toSmear 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 pipeThe PBS of 30ml Nycodenz and 20ml is configured to 60% careful the addition by bottom of Nycodenz liquid after mixing is equipped with plasmodiumThe centrifuge tube of culture, 450g/min 20min are centrifuged after trim with swing bucket rotor room temperature, and centrifugation starting loop is 3, centrifugationBrake 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 freshInsect 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 liquidIn；
(13) in packing to 10 aseptic 1.5mL EP pipes, the μ g-30 μ g linearization plasmids of 10 μ L 10, they includePL0017-gRluc-mCherry plasmids, pL0017-gRluc-ibisi-mCherry plasmids, pL0017-gRluc-gpc3 matterGrain, pL0017-gpc3:2F plasmids and pL0017-ibis1-sgpc3:2F, Sac II or the Apa I of the plasmid linearizationEnzyme；
(14) it is transferred in electric shock cup after mixing, has been careful not to bubble, plasmid is incubated 5min, uses Amaxa with plasmodiumCompany'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 completeFull culture medium, 150 μ l altogether, with insulin syringe by this 150 μ l mixed liquor through tail vein injection to C57BL/6 Mice BodiesIt 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 24hAmount 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 daysOccur；
(17) when infection rate reaches 10%, new mouse of transferring, and this mouse blood eyeball is taken out, use anticoagulant heparinMix 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, useOnce, 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 releaseProtozoon, 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 mouldPlate, takes the template that 500ng reacts as PCR, prepares PCR reaction systems, right as feminine gender with wild type Plasmodium yoelii genomeAccording 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 elements2(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-BamH I-F (SEQ ID NO.16) and gpc3-Xba I-R (SEQ ID NO.11)；Identify that gRluc the primers are egfp-Bam HI-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 turnsWhether 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 originalIn 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 inIn 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 useRifle 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 fromHeart 20min；
(4) supernatant is transferred to new pipe, with the concentration of protein quantification kit measurement albumen, and is recorded.To in each sample5 × 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 bath10min。
(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 plasmodiumThe 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 sidesLater 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：PressFormula prepares 10% separation gel, and encapsulating by being shaken up immediately after addition TEMED during encapsulating, can draw 5ml glue along glass with 10ml riflesGlass 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 sightAfter observing gelling, isopropanol is removed, rinsed with clear water, and blotted only with blotting paper.With 4% concentration glue, stood after adding TEMEDEncapsulating by shaking up.Remaining space is filled into concentration glue and then by comb insertion concentration glue.Due to volume meeting when gelling is solidShrink 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 concentratedGlue, 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 liquidAt 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 plusSample 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 thatSample 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 glueWhen 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 ofElectrophoresis 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 gelGlue, 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, underTo 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 powerInterior, 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 PBSTAntibody (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 liquidIgG(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 filmAlbumen 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 usedHand 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, oncePut, it is just immovable, X- mating plates folder is shut, start timing；The power appropriate adjustment time for exposure according to signal, generally10min, 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- lightPiece, 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 onceIn 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, roomDried 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 mCherryAlbumen 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 expressionThe P.y-GPC3 of GPC3:2F (worm strain do not express gRluc) can successful expression GPC3, GPC3 albumen in P.y-GPC3:2FAnd 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 mistakesNight；
(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, notesLucifuge；
(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_mCherrySuccessful expression, and positioning is it can be seen that IBIS1 albumen successfully draws mCherry albumen in the strain of P.y-gRluc-ex_mCherry wormsDerive 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 wormsGPC3 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) resultUnanimously；Fig. 2 (F) then indicates GPC3 albumen in P.y-GPC3:Stabilization under 2F recombinant plasmodiums erythrocytic stage difference polypide stateExpression.
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 smallMouse 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 200L, 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 glimmeringLight 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 companiesProduct；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 analysesSoftware (Tree Star, Inc.), mouse Th correlations multiple-factor detection kit (BioLegend), BDTM ELISPOT MouseIFN-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 (GermanyEppendorf companies), biochemical cultivation case (the permanent instrument company in Shanghai one), tweezers, scissors, alcohol, glass capillary；DMEM andThe 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/LGiemsa 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 spleenWith 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 injectionIn 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 thinBorn 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 is5*105Individual/only；Second group of inoculation transfects the mouse P. berghei P.y-GPC3 of empty carrier:2F, inoculum concentration is 5*105Individual/only；TheThree 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 cellLysate 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 (TreeStar,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 PlasmodiumPlus, 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 plasmodiumThe 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 fromCD80 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+DCExpression 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 serumUp to situation.
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 injectionIn 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 thinBorn 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 is5*105Individual/only；Second group of inoculation transfects the mouse P. berghei P.y-GPC3 of empty carrier:2F, inoculum concentration is 5*105Individual/only；TheThree 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.
First day, three days, the 7th day and fortnight, i.e. D1, D3, D7, D14, blood 150 is taken with glass capillary eyeμ l, the physiological saline of intraperitoneal injection same volume.Sample blood normal temperature is stood into 1h, after after blood coagulation, 1000rpm/min is centrifuged10min, 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 examinedSurvey 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, includingIL-2, IFN-γ, and TNF-α, testing result show that their concentration reaches peak on 7th day immune, and are deposited with control groupIn 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 soaked10min, 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 diameterIn 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 1640Enter 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 ice8min, 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 simultaneouslyIn 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 redCell 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 is5*105/only；Second group of inoculation plasmodium P.y-GPC3:2F, inoculum concentration is 5*105Individual/only；3rd group of inoculation transfection is unloadedThe mouse P. berghei P.y-eGPC3 of body:2F, inoculum concentration is 5*105Individual/only；The 4th group of normal mouse of inoculation equal numberRed 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 muscleFilm 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 spleenSwelling；
(10) spleen is cut into the fritter of 1-2mm with curved scissors, then with the plunger gentle abrasion of 2ml, 5-6ml is usedThe culture mediums of RPMI 1640 containing 2%FBS rinse grinding spleen cell, and cell is collected into a centrifuge tube of 50ml, will manageIt 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 abandonedClearly, 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 cellAggegation 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 abandonedSupernatant, 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 beadsSorting 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 secretionCondition.
(1) the CD8 α+T lymphocytes for obtaining every mouse are calculated, and go 2 × 106Individual CD8 α+T lymphocytes are put into 24In 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 30μ 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 incubationThe 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 incubatorIt 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 temperatureIt 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 temperatureIt 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 time2min, 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 situationBetween, 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 plasmodiumAfter 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 malariasProtozoon 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 GPC3Expression 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
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、P.y-eGPC3:The strain of 2F plasmodiums worm, dissolves and abdomenChamber is injected in two Mice Bodies；
3) after 3 days, mouse Infected With Plasmodium rate is observed, when infection rate is in 3%-5%, counts protozoan infection rate and red thinBorn of the same parents' 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 5×105Individual/only；Second group of inoculation plasmodium P.y-GPC3:2F, inoculum concentration is 5 × 105Individual/only；3rd group of inoculation transfection is unloadedThe mouse P. berghei P.y-eGPC3 of body:2F, inoculum concentration is 5 × 105Individual/only；The 4th group of normal mouse of inoculation equal numberRed 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 tumourIt 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 groupChange 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 tumourInterior distribution situation；
2) wash.After fixation, the fixer in tissue must be rinsed well material, because residual fixation in the tissueLiquid, 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 tissueIt 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 changeDimethylbenzene.(because ethanol is immiscible with paraffin, and dimethylbenzene can be dissolved in ethanol and can be dissolved in paraffin, so after dehydrationWill also be by dimethylbenzene with transition, when all being occupied by dimethylbenzene in tissue, light can be passed through, and tissue shows different journeysThe 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 internalDegree 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 afterIn, 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 respectively3min；
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
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 injectionIn 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 thinBorn of the same parents' concentration (individual/ml)；
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 identicalThe red blood cell of the normal mouse of quantity, as blank control group (n=9)；
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) according to animal welfare and ethics, any diameter of mouse tumor is no more than 20mm, or has two tumours to give birth toWhen 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, andMouse 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 mousesBeam；
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 3When, 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 20mmDead 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 malaria25 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-WTThe 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 PlasmodiumNormal growth.Most importantly show in Fig. 7, plasmodium P.y-GPC3:2F immune mouse can not only effectively suppressTumour 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 officeIt 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.ArtTechnical 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 inventionAddition, 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