CN109913425A - A kind of recombinant influenza rescue method and its application in oncotherapy - Google Patents

Abstract

The invention discloses a kind of recombinant influenza rescue method and its applications in oncotherapy.The present invention provides it is a kind of prepare expression anti-tumor protein antibody or containing its fusion protein encoding gene recombinant virus method, include the following steps: the recombinant virus of the extracellular domain encoding gene of the single-stranded of antibody, heavy chain and/or light chain that the single-stranded encoding gene, heavy chain encoding gene and/or light chain encoding gene of the antibody of anti-tumor protein are obtained to expressing by virus rescue anti-tumor protein and other transforming growth factor receptors or this receptor.The present invention devises the recombinant influenza that expression immunologic test point inhibits antibody.Heavy chain and light chain that encoding immune checkpoint inhibits antibody are constructed on the HA section of PR8 virus and on NA section respectively, so that the recombination oncolytic influenza virus of construction, expresses anti-PD1 antibody, anti-PD-L1 antibody.Oncolytic recombinant influenza provided by the invention can target killing tumor cell, and normal host cell is had no significant effect, can be used for neoplasm targeted therapy.

Description

A kind of recombinant influenza rescue method and its application in oncotherapy

Technical field

The invention belongs to field of biotechnology more particularly to a kind of recombinant influenza rescue method and its in oncotherapy In application.

Background technique

Cancer is one, whole world underlying cause of death, causes within 2015 8800000 death.In terms of global picture, nearly six/ One death is caused by cancer.Most commonly seen cancer types are as follows: lung cancer (1,690,000 death), liver cancer (78.8 ten thousand death), Colorectal cancer (77.4 ten thousand death), gastric cancer (75.4 ten thousand death) and breast cancer (57.1 ten thousand death).Current routine Treatment means mainly include operation excision, transplanting, local ablation therapy, trans-hepatic artery chemoembolization, radiotherapy, molecular targeted therapy Deng, but therapeutic effect is unsatisfactory, and causes serious side reaction to human body and be difficult to eradicate.Therefore a great problem for the treatment of cancer Be, how efficiently, specific killing tumour cell while, reduce damage to normal cell tissue.With molecular biology Development and people in gene level to the completely new understanding of tumour, oncolytic virus targeting therapy for tumor mentions in recent years for treatment of cancer New Research Thinking is supplied.

First generation gene therapy vectors of tumor is imported tumor suppressor gene using viral vectors in the tumour cell of human body, Tumor suppressor gene is set to play the effect of inhibition, killing tumor cell.But tumour mechanism is complicated, gene mutation multiplicity, and one or two The importing of a tumor suppressor gene is not enough to inhibit the growth of tumour cell, and therapeutic effect is not obvious.Therefore a kind of new disease is designed Poison can overcome lacking for first generation gene therapy vectors of tumor by being purposefully transformed and making its selective killing tumour cell It falls into.This viral vectors is referred to as oncolytic virus.

The key of oncolytic virus treatment tumour is the targeting of raising killing tumour, utilizes tumour cell and normal cell Difference (such as the activation of oncogene, the inactivation of tumor suppressor gene, cellular defence mechanisms in terms of hereditary capacity and physiological property Defect, the high expression of specific receptor, abnormality proliferation of tumour cell etc.) target killing tumor cell.The classification of oncolytic virus: Oncolytic virus can be divided into RNA virus and DNA virus from source.RNA virus can be divided into single strand RNA virus and double-stranded RNA again Virus, the RNA virus that common are oncolysis have influenza virus, poliovirus, vesicular stomatitis virus, newcastle disease Virus etc..DNA virus as oncolytic virus mainly has, adenovirus, vaccinia virus, small DNA virus, herpes simplex virus etc..

Tumour immunity targeted therapy receives significant attention in recent years, and especially immune detection point blocking treatment has entered clinic Test and be used for the treatment of kinds of tumors.Immune detection point includes cytotoxic t lymphocyte-associated antigen (CTLA-4), program Property death factors and its ligand (PD-1/PD-L1), they by conjunction with tumor cell surface respective ligand with inducing T cell Immunosupress reaction, promote T cell function failure, promote tumour cell escape immune system monitoring.Immunologic test point Therapy is to trigger a kind of method of anti-tumor immune response, is the immunosupress access activated by antibody blocking by cancer cell Therapy.CTLA-4, a kind of Inhibitory receptor for lowering the T cell activation initial stage, is the initial target of checkpoint antibody.? In oncotherapy using the theoretical basis of anti-CTLA-4 antibody be the release of existing anticancer t cell responses and may triggering it is new Antitumor response.PD1 expression of receptor is another Inhibitory receptor in the T cell surface by antigenic stimulus, and signal presses down downstream T cell processed proliferation, the release of cell factor, lethal effect.In tumor model, PD1 is once tied to the PD- expressed in tumour L1 will inhibit T cell and block anti-tumor immune response.

People H1N1 Influenza virus strain A/PR/8/34 (abbreviation PR8) is that one plant of chicken embryo adapts to Strain, can be had in chicken embryo The duplication of effect ground.PR8 is sub-thread minus strand, segmented RNA, shares 8 independent RNA segment compositions, encodes 10 kinds of protein.Piece The RNA polymerase that section 1-3 coding RNA relies on, 1 encoded polymerase subunit PB2 of segment, 2 encoded polymerase subunit PB1 of segment, piece 3 encoded polymerase subunit PA of section；Segment 4 encodes hemagglutinin HA, is a kind of surface glycoprotein with viral attachment infected with pass；Piece 5 encoding nuclear proteins NP of section, are the dominant structural moieties of viral RNA；6 encoding nerve propylhomoserin enzyme NA of segment, is a kind of coating sugar egg It is white；Segment 7 encodes two kinds of matrix prote m1s and M2, is nonglycosylated structural proteins；Segment 8 encodes two kinds of non-structural proteins NS1 and NS2.

Summary of the invention

It is an object of the present invention to provide a kind of methods of the recombinant virus of antibody for preparing expression anti-tumor protein.

Method provided by the invention includes the following steps: by anti-tumor protein antibody-encoding genes or containing its fusion Protein coding gene obtains expression anti-tumor protein antibody or the recombinant virus of its encoding gene by virus rescue.

In the above method,

The anti-tumor protein antibody fusion protein includes the anti-tumor protein antibody and other protein antibodies；

And/or other described protein antibodies are the extracellular domain of transforming growth factor receptor or this receptor；

And/or the anti-tumor protein antibody is immune globulin antibody or single-chain antibody or chimeric antibody；

And/or the virus is influenza virus or adenovirus or poxvirus or other oncolytic virus；

And/or the influenza virus is human influenza virus；

And/or the human influenza virus is specifically people H1N1 influenza virus.

In the above method,

The virus rescue is to be coupled the anti-tumor protein antibody or the fusion with encoding gene in viral genome The encoding gene of albumen, then remove remaining encoding gene being used for other than the gene of coupling with the viral genome and be transferred to jointly In cell, packaging obtains recombinant virus.

In the above method, the anti-tumor protein antibody is anti-tumor protein single-chain antibody, and the method includes walking as follows It is rapid: by melting for 1 encoding gene in encoding gene and the viral genome containing the anti-tumor protein single-chain antibody Gene third is closed to be transferred in cell jointly with remaining encoding gene other than gene of the removing for merging in the viral genome, Packaging obtains recombinant virus, the expression of recombinant virus anti-tumor protein single-chain antibody；

Or, the anti-tumor protein antibody is immune globulin antibody, described method includes following steps: will be containing immune The fusion first of 1 encoding gene in the heavy chain encoding gene and the viral genome of globulin antibody, containing described The light chain encoding gene of immune globulin antibody and the fusion second of another encoding gene in the viral genome, with It removes remaining encoding gene other than the gene for fusion in the viral genome to be transferred in cell jointly, packaging is weighed Group virus, the expression of recombinant virus immune globulin antibody；

Or, the anti-tumor protein antibody fusion protein includes the immune globulin antibody and transforming growth factor receptor Extracellular domain；Described method includes following steps: by the heavy chain fusion gene containing immune globulin antibody and described The fusion first of 1 encoding gene in viral genome, the light chain encoding gene containing the immune globulin antibody and The fusion second of another encoding gene in the viral genome is used to merge with removing in the viral genome Remaining encoding gene other than gene is transferred in cell jointly, and packaging obtains recombinant virus, the expression of recombinant virus immune globulin The extracellular domain of Bai Kangti and transforming growth factor receptor；The heavy chain fusion gene of the immune globulin antibody includes exempting from The heavy chain encoding gene of epidemic disease globulin antibody and the extracellular structure domain encoding sequence of transforming growth factor receptor.

The heavy chain fusion gene of the immune globulin antibody is by the heavy chain encoding gene of immune globulin antibody, link peptide The extracellular structure domain encoding sequence of encoding gene (amino acid sequence of link peptide is sequence 10) and transforming growth factor receptor Composition.

In the above method, the virus is influenza virus,

Encoding gene is PB2 protein coding gene, PB1 protein coding gene, PA albumen in the influenza virus gene group Encoding gene, HA protein coding gene, NP protein coding gene, NA protein coding gene, M protein coding gene and NS albumen are compiled Code gene；

Or, the fusion or remaining described encoding gene are transferred to cell by recombinant vector importing.

Or,

The fusion first is from successively including PB1 protein coding gene and described in the viral genome the end 5' The heavy chain encoding gene of the immune globulin antibody of anti-tumor protein or the heavy chain fusion gene of the immune globulin antibody；

And the fusion second from the end 5' successively include the viral genome in PA protein coding gene and institute State the light chain encoding gene of the immune globulin antibody of anti-tumor protein；

And remaining described encoding gene is the PB2 protein coding gene, the HA protein coding gene, the NP egg White encoding gene, the NA protein coding gene, the M protein coding gene and the NS protein coding gene.

Above-mentioned introduction method specifically comprises the following steps: the expression vector containing fusion first, contains fusion The expression vector of second, expression vector, the HA containing the influenza virus of the PB2 protein coding gene containing the influenza virus The expression vector of protein coding gene, NP protein coding gene containing the influenza virus expression vector, contain the stream The expression of the expression vector of the NA protein coding gene of Influenza Virus, M protein coding gene containing the influenza virus carries The expression vector of body and the NS protein coding gene containing the influenza virus is transferred in cell jointly, and packaging obtains recombinant influenza Virus；

The fusion first passes through recombinant vector pHW-PB1-HUPD1 or pHW-PB1-HUPD1-linker-TGF β RII ECD imports aim cell；

The fusion second imports aim cell by recombinant vector pHW-PA-HUPD1；

The HA protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-HA；

The NA protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-NA；

The NP protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-NP；

The PB2 protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-PB2；

The M protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-M；

The NS protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-NS.

Or,

The fusion first is from successively including HA protein coding gene and described in the viral genome the end 5' The heavy chain encoding gene of the immune globulin antibody of anti-tumor protein or the heavy chain fusion gene of the immune globulin antibody；

And the fusion second from the end 5' successively include the viral genome in NA protein coding gene and institute State the light chain encoding gene of the immune globulin antibody of anti-tumor protein；

And remaining described encoding gene is the PB2 protein coding gene, the PB1 protein coding gene, the PA egg White encoding gene, the NP protein coding gene, the M protein coding gene and the NS protein coding gene；

The introduction method specifically comprises the following steps: the expression vector containing fusion first, contains fusion The expression vector of second, the expression vector of the PB2 protein coding gene containing the influenza virus contain the influenza virus The expression vector of PB1 protein coding gene, the expression vector of PA protein coding gene containing the influenza virus, containing described The expression vector of the expression vector of the NP protein coding gene of influenza virus, M protein coding gene containing the influenza virus It is transferred in cell jointly with the expression vector of the NS protein coding gene containing the influenza virus, it is susceptible that packaging obtains recombined streams Poison；

In an embodiment of the present invention,

The fusion first passes through recombinant vector pHW-HA-HUPD1 or pHW-HA-HUPD1-linker-TGF β RII ECD imports aim cell；

The fusion second imports aim cell by recombinant vector pHW-NA-HUPD1；

The PB2 protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-PB2；

The PB1 protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-PB1；

The PA protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-PA；

The NP protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-NP；

The M protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-M；

The NS protein coding gene of the influenza virus imports aim cell by recombinant vector pHW-NS.

In the above method, the immune globulin antibody is immune detection point antibody；

And/or the immune detection point antibody is PD1 antibody or PDL1 antibody or CCR4 antibody or CTLA4；

And/or the immune detection point antibody is people's source protein antibody or source of mouse protein antibodies；

And/or the extracellular domain of the transforming growth factor receptor is the cell of transform growth factor-beta receptor II Extracellular portion, specially transforming growth factor-β (transforming growth factor- β, TGF-β) receptor II (TGF β RIIECD the ligand binding sequence (sequence 11) of extracellular domain).

It is also the scope of protection of the invention by recombinant virus prepared by the above method.

Or, the antibody of the anti-tumor protein prepared by the recombinant virus is also the scope of protection of the invention；

Or, a kind of method for the antibody for preparing anti-tumor protein, for animal is immunized in the recombinant virus, what is obtained is anti-swollen The antibody of tumor albumen.

The animal is humanization immune system mouse, is specifically people CD34⁺Cellular reconstitution humanization immune system mouse.

The antibody of above-mentioned recombinant virus or anti-tumor protein is also this hair resisting or treating or inhibiting the application in tumour The range of bright protection；

Or the antibody of above-mentioned recombinant virus or anti-tumor protein is also the present invention inhibiting the application in tumor cell proliferation The range of protection；

Or the antibody of above-mentioned recombinant virus or anti-tumor protein resists or treats or inhibits answering in tumour product in preparation With being also the scope of protection of the invention；

Or the antibody of above-mentioned recombinant virus or anti-tumor protein inhibits the application in tumor cell proliferation product in preparation；

Or, a kind of antitumor product, active constituent is the antibody of above-mentioned recombinant virus or anti-tumor protein.

Among the above, the tumour is other tumours such as melanoma, liver cancer, lung cancer.

The carrier of gene or expressing said gene in viral genome is preparing answering in antineoplastic genetic engineering drug With being also the scope of protection of the invention.

In above-mentioned influenza virus preparation,

The people H1N1 influenza virus is A/PR/8/34.

If the antibody is source of people PD1, the nucleotides sequence of the fusion first is classified as shown in 2 887-3689 of sequence DNA fragmentation shown in 6 887-3176 of DNA fragmentation or sequence, and the nucleotides sequence of the fusion second is classified as sequence 3 DNA fragmentation shown in 7 887-2880 of DNA fragmentation shown in 887-3699 or sequence；

If the antibody is source of people PDL1, the nucleotides sequence of the fusion first is classified as shown in 4 887-3720 of sequence 8 887-3196 of DNA fragmentation or sequence shown in DNA fragmentation, and the nucleotides sequence of the fusion second is classified as sequence 5 DNA fragmentation shown in 9 887-2877 of DNA fragmentation shown in 887-3696 or sequence.

The cell is the nephrocyte (COS-1) and dog kidney cells (MDCK) of cercopithecus aethiops SV40 conversion.

The expression vector is pHW2000.

With constantly improve for Reverse Genetics, the transformation and utilization of influenza virus are possibly realized.Influenza virus is changed The influenza vectors of foreign gene-carrying are caused, the advantage with carrier abundance, while being conducive to be transfected into purpose thin Born of the same parents, especially tumour cell provide new means for therapy of tumor.Simultaneously as influenza virus has tumour cell Apoptotic effect is expected to be developed into tumor vaccine and the viral vectors with gene delivery effect is used for oncogene and controls It treats.

Immune " brake of the immunologic test point inhibitor for treating by inhibiting immunologic test point activity, in release tumor microenvironment Vehicle " reactivates T cell to the immune response effect of tumour, to reach antineoplastic action.

The present invention exactly uses influenza vectors expression anti-PD1 antibody or anti-PDL1 antibody, to reach anti- The effect of tumour.The present invention is carrier by influenza virus, can produce a large amount of antibody by the duplication of recombinant influenza, It can solve existing many drawbacks that monoclonal antibody is produced by hybridoma technology.

Detailed description of the invention

The dynamics that Fig. 1 is FLU-HUPD1 (A), antibody generates during FLU-HUPDL1 (B) infection.

The stability that Fig. 2 is FLU-HUPD1 (A), antibody is expressed at any time during FLU-HUPDL1 (B) infection.

Fig. 3 is the work for being used to assess FLU-HUPD1 (A), FLU-HUPDL1 (B) with bilateral melanocyte tumor model Property, wherein figure A/B: the mouse for injecting FLU-HUPD1/FLU-HUPDL1 is compared in PR8 group with PBS group, right side of mice tumour body The variation of product increment.Scheme C/D: the mouse for injecting FLU-HUPD1/FLU*-HUPDL1 is compared in PR8 group with PBS group, and mouse is left The variation of side gross tumor volume increment.

Fig. 4 is the melanocyte tumor model for injecting FLU-HUPD1 (A), FLU-HUPDL1 (B), in PR8 group and PBS Group is compared, the variation of the survival rate of mouse.

Fig. 5 is HepG2 rat liver cancer tumor model for assessing FLU-HUPD1/FLU-HUPDL1/FLU*-HUPD1/ Internal inhibiting effect of the FLU*-HUPDL1 to rat liver cancer；Figure A: the mouse of FLU-HUPD1/FLU*-HUPD1 is injected in PR8 group It is compared with PBS group, the variation of right side of mice gross tumor volume increment.Figure B: the mouse of FLU-HUPDL1/FLU*-HUPDL1 is injected It is compared in PR8 group with PBS group, the variation of right side of mice gross tumor volume increment.

The dynamics that Fig. 6 is FLU*-HUPD1 (A), antibody generates during FLU*-HUPDL1 (B) infection.

The stability that Fig. 7 is FLU*-HUPD1 (A), antibody is expressed at any time during FLU*-HUPDL1 (B) infection.

Fig. 8 is that FLU-HUPD1-TGF β RII ECD (A), FLU-HUPDL1-TGF β RII ECD (B) treat mouse tumor mould Type tumor volume change.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

PHW-PB2, pHW-PB1, pHW-PA, pHW-NP, pHW-M, pHW-NS, pHW-HA and pHW-NA are documented in as follows In offering: Erich Hoffmann, A DNA transfection system for generation of influenza A virus from eight plasmids.(2000)J.PNAS,vol.97:6108–6113。

ELISA detects anti-PD1/anti-PDL1 antibody in following embodiments and its method of content is specific as follows:

1, Protein G purified antibody

Anti-PD1/anti-PDL1 antibody is isolated from the chicken embryo of recombinant virus infection first, specific method: recombination Virus carries out 10 in PBS^-4Dilution.10 age in days SPF chicken embryos are inoculated in through allantoic cavity, each dilution is inoculated with 10 pieces of chicken embryos, often Piece chicken embryo 0.2mL.Chicken embryo is incubated for 72h at 37 DEG C, then separates allantoic fluid, passes through vacuum 0.22um filter (Falcon) Filtering, filtrate is added in 1.5 × 30cm chromatographic column (Bio-Rad) equipped with Protein G.G-protein/agarose is filled in chromatographic column (Invivogen).It collects effluent and is added in chromatographic column again.Permeate is discarded, is washed chromatographic column 2 times with PBS, uses 45ml 0.1M glycine (pH 2.7) is by antibody elution in 5ml Tris-HCl (pH 10).It is filtered using Amicon Ultra-15 single Buffer is simultaneously changed to PBS by first (EMD Millipore) concentrated antibody.Finally by NanoDrop (Thermo Fisher) Measure antibody concentration.

2, ELISA detects anti-PD1/anti-PDL1 antibody content

Elisa plate is coated with PD1/PDL1 albumen (Abcam), 4 DEG C overnight, are then washed 3 times with PBS at room temperature, 1%BSA closes 2h.Measuring samples are added, are incubated overnight or are incubated at room temperature 1h for 4 DEG C, PBS is washed 3 times, and anti-mouse is added The secondary antibody of HRP conjugation incubates 30 minutes together, and substrate colour developing, 450nm reading is added.Use the anti-PD1/ of known concentration Anti-PDL1 (Abcam) draws standard curve.

The rescue of embodiment 1, recombinant influenza

1, the building of recombinant vector

Recombinant vector shown in sequence 2 is named as pHW-PB1-HUPD1, DNA shown in 887-3187 of sequence 2 Molecule is PB1 gene；Sequence shown in 3188-3253 of sequence 2 is PTV-1 2A sequence；3254-3313 of sequence 2 Shown sequence is signal peptide sequence；Sequence shown in 3314-3525 of sequence 2 is HUPD1 (HEAVY CHAIN) sequence；Sequence Sequence shown in 3526-3689 of column 2 is PB1-PS packaging sequence；Keep the other sequences of carrier pHW2000 (sequence 1) not Become, obtains recombinant vector, which is named as pHW-PB1-HUPD1.The recombinant vector is by 2 887-3689 of sequence Shown in DNA fragmentation (fusion first) insertion pHW2000 carrier BsmbI restriction enzyme site between obtained carrier, expression PB1 and Source of people PD1 heavy chain.

Recombinant vector shown in sequence 3 is named as pHW-PA-HUPD1, DNA shown in 887-3064 of sequence 3 Molecule is PA gene；Sequence shown in 3065-3130 of sequence 3 is PTV-1 2A sequence；3131-3190 of sequence 3 Shown sequence is signal peptide sequence；Sequence shown in 3191-3511 of sequence 3 is HUPD1 (LIGHT CHAIN) sequence；Sequence Sequence shown in 3512-3699 of column 3 is PA-PS packaging sequence；It keeps the other sequences of carrier pHW2000 constant, obtains The recombinant vector is named as pHW-PA-HUPD1 by recombinant vector.The recombinant vector is will be shown in 3 887-3699 of sequence The carrier obtained between the BsmbI restriction enzyme site of DNA fragmentation (fusion second) replacement pHW2000 carrier, expresses PA and source of people PD1 Light chain.

Recombinant vector shown in sequence 4 is named as pHW-PB1-HUPDL1, shown in 887-3187 of sequence 4 DNA molecular is PB1 gene；Sequence shown in 3188-3253 of sequence 4 is PTV-1 2A sequence；The 3254- of sequence 4 3313 shown sequences are signal peptide sequence；Sequence shown in 3314-3556 of sequence 4 is HUPDL1 (HEAVY CHAIN) Sequence；Sequence shown in 3557-3720 of sequence 4 is PB1-PS packaging sequence；Keep the other sequences of carrier pHW2000 not Become, obtains recombinant vector, which is named as pHW-PB1-HUPDL1.The recombinant vector is by 4 887- of sequence The carrier obtained between the BsmbI restriction enzyme site of DNA fragmentation shown in 3720 (fusion first) replacement pHW2000 carrier, expression PB1 and source of people PDL1 heavy chain.

Recombinant vector shown in sequence 5 is named as pHW-PA-HUPDL1, DNA shown in 887-3064 of sequence 5 Molecule is PA gene；Sequence shown in 3065-3130 of sequence 5 is PTV-1 2A sequence；3131-3190 of sequence 5 Shown sequence is signal peptide sequence；Sequence shown in 3191-3508 of sequence 5 is HUPDL1 (LIGHT CHAIN) sequence；Sequence Sequence shown in 3509-3696 of column 5 is PA-PS packaging sequence；It keeps the other sequences of carrier pHW2000 constant, obtains The recombinant vector is named as pHW-PA-HUPDL1 by recombinant vector.The recombinant vector is will be shown in 5 887-3696 of sequence The carrier obtained between the BsmbI restriction enzyme site of DNA fragmentation (fusion second) replacement pHW2000 carrier, expresses PA and source of people PDL1 light chain.

2, the rescue of recombinant influenza

By pHW-PB1-HUPD1, pHW-PA-HUPD1 of step 1 and recombinant vector pHW-PB2, pHW-NP, pHW-M, 10 μ L transfection reagents (Effectene, Qiagen Products) are added after each 0.2 μ g mixing of pHW-NS, pHW-HA and pHW-NA Middle room temperature acts on 10min, and cotransfection is into the nephrocyte (COS-1) and dog kidney cells (MDCK) converted containing cercopithecus aethiops SV40 In six orifice plates of (COS-1 cell is purchased from Beijing consonance cell bank, and mdck cell is purchased from ATTC, CCL-34), wherein COS-1 cell 2×10⁵A/hole, mdck cell 1 × 10⁵A/hole places it in 37 DEG C, cultivates 60-72h under 5%CO2, it is outstanding to obtain cell after transfection Liquid, by cell suspension inoculation 9-11 age in days SPF chicken embryo, 37 DEG C of culture 72h harvest chick embryo allantoic liquid and according to OIE standard to it It carries out blood clotting (HA) test and blood clotting inhibits (HI) test.HA and HI test result is that positive sample contains rescue successfully Recombinant influenza (is named as FLU-HUPD1), obtains FLU-HUPD1 by amplification, concentration, purifying, -70 DEG C freeze.

Also according to the above method, by pHW-PB1-HUPD1, pHW-PA-HUPD1 replace with pHW-PB1-HUPDL1, PHW-PA-HUPDL1, other steps are constant, carry out the rescue of recombinant influenza, obtain influenza virus FLU- after purification HUPDL1, -70 DEG C freeze.

3, the identification of recombinant influenza

The recombinant influenza FLU-HUPD1 and FLU-HUPDL1 that step 2 obtains transmission electron microscope observing (reference after negative staining Document: example of transmission electron microscope technology of preparing (one) film sample technology of preparing, J, physical testing, 1995:42-44) disease Malicious form, the results showed that FLU-HUPD1 and FLU-HUPDL1 meets influenza virus representative configuration feature, there is coating, surface spinosity Lug structure, virion size is between 80-120nm.

By recombinant influenza FLU-HUPD1 and FLU-HUPDL1 the inoculation 9-11 age in days SPF chicken embryo passage of step 2, take Second generation chick embryo allantoic liquid extracts viral RNA, by RT-PCR (PCR method and amplimer reference papers: Yang Penghui, reconfiguration The research .2006 of H1N1 subtype influenza virus attenuated vaccine strain), amplify correct PB2, NP, HA, NA and M gene of sequence Segment and improved PB1-PD1, PA-PD1, PB1-PDL1, PA-PDL1 gene.

By FLU-HUPD1, FLU-HUPDL1 of step 2 through chicken embryo mass propgation, it is concentrated by ultrafiltration, sucrose gradient centrifugation purifying After (specific steps reference papers: the research .2006 of H1N1 subtype influenza virus attenuated vaccine strain is reconfigured in Yang Penghui), carry out NP, HA1, HA2, NS1 albumen (NP egg of corresponding size can be detected after gel-colored, decoloration in polyacrylamide gel electrophoresis White about 60KD, HA1, HA2 albumen are about 55KD, and NS1 albumen is about 26KD), show that the main component of antigen is not lost.

Blood clotting identification: by the 6 orifice plates multigelation of transfection, 10 age in days SPF chick embryo allantoic cavities, each egg inoculation are inoculated with 0.2ml sets 37 DEG C, 72~96h of 5%CO2 incubator culture, and 4 DEG C of chicken embryos of freezing to death collect allantoic fluid and measure hemagglutinative titer, positive Strain expanded for 3 generations in chicken embryo, collected chick embryo allantoic liquid, surveyed hemagglutinative titer.In 96 hole micro-reaction plates, 50 μ l are added in first hole Viral allantoic fluid, successively doubling dilution, last 1 hole discard 50 μ l after mixing, and add 1% chicken red blood cell of 50ul, after 15min Observe result.Criterion: it is the positive that agglutination phenomenon, which occurs, for red blood cell, and red blood cell forms 1 small ring, and surrounding has blood clot, and person is Terminal.The hemagglutinative titer of FLU-HUPD1 and FLU-HUPDL1 can reach 2 as the result is shown⁸。

Chicken embryo 50 3nfective dose (EID₅₀) measurement:

10 times of the 1st generation allantoic fluid work of FLU-HUPD1, FLU-HUPDL1 and PR8 virus inoculation chicken embryo of step 2 is progressive Dilution is inoculated in 10 age in days SPF chicken embryos through allantoic cavity, and each dilution is inoculated with 4 embryos, and every embryo 0.2mL, 37 DEG C of cultures take out For dead germ and culture after for 24 hours to the embryo of 72h, hemagglutinative titer is that egg infectious is positive in the judgement of 1:16 or more, uses Reed- Muench method calculates EID₅₀.As a result the EID of FLU-HUPD1, FLU-HUPDL1₅₀It is 10^-6/0.2mL。

Embodiment 2 has evaluated the dynamics that antibody generates during FLU-HUPD1, FLU-HUPDL1 infect

The recombinant influenza FLU-HUPD1, FLU-HUPDL1 (- 10PFUs) inoculated into chick embryo prepared respectively with embodiment 1, Each time point chooses 3 piece of 9 age in days SPF chicken embryo (being purchased from: Beijing Bo Linge Yin Ge Hanweitong Bioisystech Co., Ltd), simultaneously PR8 (purchased from Chinese Center for Disease Control and Prevention) control is set up, with PR8 (- 10PFUs) inoculated into chick embryo, each time point chooses 3 Piece chicken embryo, while PBS control is set up, and with there are the amounts of anti-CTLA4 antibody in ELISA method measurement allantois.

As a result as shown in Figure 1, it can be seen that 2 days detectable antibody after inoculation FLU-HUPD1, FLU-HUPDL1, and Reach within 4-5 days peak value, after inoculation antibody is all not detected in third day in PR8 group and PBS group.

The stability that assessment antibody is expressed at any time, specific as follows: dilution FLU-HUPD1, FLU-HUPDL1 and PR8 are extremely 10^-6, be inoculated with 3 pieces of chicken embryos, every piece of egg inoculation 100ul, 33 DEG C incubator culture 3 days.After collecting allantoic fluid, it is diluted to 10^-6, connect Kind of 3 pieces of chicken embryos, every piece of egg inoculation 100ul, 33 DEG C incubator culture 3 days.Repetition is passaged to forth generation.

Measure in different passage allantois that there are the amounts of antibody with ELISA method, it is as a result as shown in fig. 2, it can be seen that different The loss for not observing the amount of antibody of generation in passage allantois shows that antibody gene is stablized in influenza gene group and maintains.

Embodiment 3, recombinant influenza FLU-HUPD1 and FLU-HUPDL1 are to the internal inhibiting effect of melanoma

FLU-HUPD1 and FLU-HUPDL1 PBS prepared by embodiment 1 (are weighed into NaCl8g, KCl0.2g, Na₂HPO₄· 12H₂O 3.63g,KH₂PO₄0.24g is dissolved in 900ml distilled water, with hydrochloric acid tune pH value to 7.4, water is added to be settled to 1L, room temperature Save backup) it is resuspended, obtaining titre is 5.5 × 10⁵The FLU-HUPD1 suspension and titre of pfu/100 μ l is 5.5 × 10⁵pfu/ The FLU-HUPDL1 suspension of 100 μ l；

Strains of influenza viruses A/PR/8/34 (being purchased from Chinese Center for Disease Control and Prevention) is resuspended with PBS, obtaining titre is 5.5×10⁵The strains of influenza viruses A/PR/8/34 suspension of pfu/100 μ l.

1, mouse subcutaneous tumor model is prepared

The employment CD34 on NSG mouse⁺Cellular reconstitution humanization immune system mouse is (by tieing up the sensible limited public affairs of biotechnology Department provides), (note: following experiment mouse used is humanization immune system mouse) every inoculates in right side back part 2×10⁵A B16-F10 cell (being purchased from ATCC, catalog number (Cat.No.): 30-2002), injects 4 times respectively at 5/7/9/11d.5th day every Mouse inoculates 1 × 10 in left side back part⁵A B16-F10 cell, respectively at the 5/7/9/11/13/15/17/19/21st Tumour major diameter and minor axis, gross tumor volume=(longest diameter × most short diameter are measured every three days²)/2。

2, FLU-HUPD1 is immune

When above-mentioned 1 right side of mice gross tumor volume is 40mm³When, mouse is grouped at random, respectively control group, A/ PR/8/34 group, FLU-HUPD1 treatment group, specific as follows:

FLU-HUPD1 treatment group (9 mouse): being 5.5 × 10 by titre⁵The FLU-HUPD1 suspension of pfu/100 μ l is right Side tumour intratumor injection C57BL/6 mouse；

A/PR/8/34 group (8 mouse): being 5.5 × 10 by titre⁵The strains of influenza viruses of pfu/100 μ l

A/PR/8/34 suspension right side tumor intratumor injection C57BL/6 mouse；

Control group (PBS；13 mouse): by 100ulPBS right side tumor intratumor injection C57BL/6 mouse；

The/7/9/11/13/15/17/19/21 measures tumour major diameter and minor axis every three days after immune, and calculates Gross tumor volume.

3, FLU-HUPDL1 is immune

When above-mentioned 1 right side of mice gross tumor volume is 40mm³When, mouse is grouped at random, respectively control group, A/ PR/8/34 group, FLU-HUPD1 treatment group, specific as follows:

FLU-HUPD1 treatment group (9 mouse): being 5.5 × 10 by titre⁵The FLU-HUPDL1 suspension of pfu/100 μ l Right side tumor intratumor injection C57BL/6 mouse；

A/PR/8/34 group (8 mouse): being 5.5 × 10 by titre⁵The strains of influenza viruses A/PR/8/34 of pfu/100 μ l is outstanding Supernatant liquid right side tumor intratumor injection C57BL/6 mouse；

Control group (PBS；13 mouse): by 100ulPBS right side tumor intratumor injection C57BL/6 mouse；

5/7/9/11/13/15/17/19/21st measures tumour major diameter and minor axis every three days after immune, and counts Calculate gross tumor volume.

As a result as shown in Figure 3, wherein Fig. 3 A can be seen that the mouse of injection FLU-HUPD1 in PRB group and PBS group phase Than right side of mice gross tumor volume increases most slow variation；Fig. 3 B can be seen that injection FLU-HUPD1 mouse and PRB group and PBS group is compared, and right side of mice gross tumor volume increases most slow variation.The mouse also table of FLU-HUPD1 and FLU-HUPDL1 treatment The left side gross tumor volume growth for revealing untreated slows down that (it is complete to show that FLU-HUPD1 and FLU-HUPDL1 treatment is supported by Fig. 3 C, D Body anti-tumor immune response.

The death condition of observation mouse in the 5-31 days, counts each group mouse and treats in different survival rates away from first time Time.

As a result as shown in figure 4, compared with the individually mouse of injection PR8 or PBS, FLU-HUPD1 and FLU-HUPDL1 treatment The survival of mouse can be extended.

The above results show that recombinant influenza FLU-HUPD1 and FLU-HUPDL1 can inhibit melanoma.

Embodiment 4, recombinant influenza FLU-HUPD1, FLU-HUPDL1 are to the internal inhibiting effect of liver cancer

FLU-HUPD1 and FLU-HUPDL1 prepared by embodiment 1 are resuspended with PBS, obtaining titre is 5.5 × 10⁵pfu/ The FLU-HUPD1 suspension of 100 μ l；

Strains of influenza viruses A/PR/8/34 (being purchased from Chinese Center for Disease Control and Prevention) is resuspended with PBS, obtaining titre is 5.5×10⁵The strains of influenza viruses A/PR/8/34 suspension of pfu/100 μ l.

1, mouse tumor model is prepared

PBS adjust HepG2 cell (by the 5th medical center of Chinese People's Liberation Army General Hospital provide), trypan blue count and Cell viability is measured, HepG2 cell concentration 1 × 10 is adjusted⁶A/ml works as body by 200 μ l cell infusions in the right back part of mouse Product grows to (cubature formula V=4/3 × π × S when diameter is 4~6mm²/ 2 × L/2L is longest diameter, and S is shortest straight Diameter), it is grouped, every group 12, is administered once every two days at random according to gross tumor volume and mouse weight, totally 4 times.

2, FLU-HUPD1 is immune

When above-mentioned 1 right side of mice gross tumor volume is 40mm³When, mouse is grouped at random, respectively control group, A/ PR/8/34 group, FLU-HUPD1 treatment group, specific as follows:

FLU-HUPD1 treatment group (12 mouse): being 7 × 10 by titre⁵The FLU-HUPD1 suspension of pfu/100 μ l is right Side tumour intratumor injection C57BL/6 mouse；

A/PR/8/34 group (12 mouse): being 7 × 10 by titre⁵The strains of influenza viruses A/PR/8/34 of pfu/100 μ l is outstanding Supernatant liquid right side tumor intratumor injection C57BL/6 mouse；

Control group (PBS；12 mouse): by 100ulPBS right side tumor intratumor injection C57BL/6 mouse；

The/7/9/11/13/15/17/19/21 measures tumour major diameter and minor axis every three days after immune, and calculates Gross tumor volume.

3, FLU-HUPDL1 is immune

When above-mentioned 1 right side of mice gross tumor volume is 40mm³When, mouse is grouped at random, respectively control group, A/ PR/8/34 group, FLU-HUPDL1 treatment group, specific as follows:

FLU-HUPDL1 treatment group (12 mouse): being 7 × 10 by titre⁵The FLU-HUPDL1 suspension of pfu/100 μ l Right side tumor intratumor injection C57BL/6 mouse；

A/PR/8/34 group (12 mouse): being 7 × 10 by titre⁵The strains of influenza viruses A/PR/8/34 of pfu/100 μ l is outstanding Supernatant liquid right side tumor intratumor injection C57BL/6 mouse；

Control group (PBS；12 mouse): by 100ulPBS right side tumor intratumor injection C57BL/6 mouse；

The/7/9/11/13/15/17/19/21 measures tumour major diameter and minor axis every three days after immune, and calculates Gross tumor volume.

Mouse state, every two days record mouse states, gross tumor volume and changes of weight are observed after treatment.

As a result as shown in Figure 5, it can be seen that compared with the mouse for receiving PBS injection, after slowing down right side with PR8 treatment The growth of dorsal tumors；However, the mouse of injection FLU-HUPD1, FLU-HUPDL1 show that gross tumor volume growth is most slow.

Show that recombinant influenza FLU-HUPD1 and FLU-HUPDL1 can inhibit liver cancer.

The rescue of embodiment 5, recombinant influenza

1, the building of recombinant vector

Recombinant vector shown in sequence 6 is named as pHW-HA-HUPD1, DNA shown in 887-2617 of sequence 6 Molecule is HA gene；Sequence shown in 2618-2684 of sequence 6 is PTV-1 2A sequence；2685-2745 of sequence 6 Shown sequence is signal peptide sequence；Sequence shown in 2746-3095 of sequence 6 is HUPD1 (HEAVY CHAIN) sequence；Sequence Sequence shown in 3096-3176 of column 6 is HA-PS packaging sequence；Keep the other sequences of carrier pHW2000 (sequence 1) not Become, obtains recombinant vector, which is named as pHW-HA-HUPD1.The recombinant vector is by 6 887-3176 of sequence Shown in DNA fragmentation insertion pHW2000 carrier BsmbI restriction enzyme site between obtained carrier, express HA and source of people PD1 heavy chain.

Recombinant vector shown in sequence 7 is named as pHW-NA-HUPD1, DNA shown in 887-2272 of sequence 7 Molecule is NA gene；Sequence shown in 2273-2339 of sequence 7 is PTV-1 2A sequence；2340-2400 of sequence 7 Shown sequence is signal peptide sequence；Sequence shown in 2401-2722 of sequence 7 is HUPD1 (LIGHT CHAIN) sequence；Sequence Sequence shown in 2723-2880 of column 7 is NA-PS packaging sequence；It keeps the other sequences of carrier pHW2000 constant, obtains The recombinant vector is named as pHW-NA-HUPD1 by recombinant vector.The recombinant vector is will be shown in 7 887-2880 of sequence The carrier obtained between the BsmbI restriction enzyme site of DNA fragmentation replacement pHW2000 carrier, expresses NA and source of people PD1 light chain.

Recombinant vector shown in sequence 8 is named as pHW-HA-PDL1, DNA shown in 887-2617 of sequence 8 divides Son is HA gene；Sequence shown in 2618-2684 of sequence 8 is PTV-1 2A sequence；2685-2745 institutes of sequence 8 Show that sequence is signal peptide sequence；Sequence shown in 2746-3115 of sequence 8 is HUPDL1 (HEAVY CHAIN) sequence；Sequence Sequence shown in 3116-3196 of 8 is HA-PS packaging sequence；It keeps the other sequences of carrier pHW2000 constant, obtains weight Group carrier, is named as pHW-HA-HUPDL1 for the recombinant vector.The recombinant vector is by DNA shown in 8 887-3196 of sequence The carrier obtained between the BsmbI restriction enzyme site of segment replacement pHW2000 carrier, expresses HA and source of people PDL1 heavy chain.

Recombinant vector shown in sequence 9 is named as pHW-NA-HUPDL1, DNA shown in 887-2272 of sequence 9 Molecule is NA gene；Sequence shown in 2273-2339 of sequence 9 is PTV-1 2A sequence；2340-2400 of sequence 9 Shown sequence is signal peptide sequence；Sequence shown in 2401-2719 of sequence 9 is HUPDL1 (LIGHT CHAIN) sequence；Sequence Sequence shown in 2720-2877 of column 9 is NA-PS packaging sequence；It keeps the other sequences of carrier pHW2000 constant, obtains The recombinant vector is named as pHW-NA-HUPDL1 by recombinant vector.The recombinant vector is will be shown in 9 887-2877 of sequence The carrier obtained between the BsmbI restriction enzyme site of DNA fragmentation replacement pHW2000 carrier, expresses NA and source of people PDL1 light chain.

2, the rescue of recombinant influenza

By pHW-HA-HUPD1, pHW-NA-HUPD1 of step 1 and recombinant vector pHW-PB2, pHW-PB1, pHW-PA, It is added in 10 μ L transfection reagents (Effectene, Qiagen Products) after each 0.2 μ g mixing of pHW-NP, pHW-M, pHW-NS Room temperature acts on 10min, and cotransfection is into the nephrocyte (COS-1) and dog kidney cells (MDCK) converted containing cercopithecus aethiops SV40 In six orifice plates of (COS-1 cell is purchased from Beijing consonance cell bank, and mdck cell is purchased from ATTC, CCL-34), wherein COS-1 cell 2×10⁵A/hole, mdck cell 1 × 10⁵A/hole places it in 37 DEG C, cultivates 60-72h under 5%CO2, it is outstanding to obtain cell after transfection Liquid, by cell suspension inoculation 9-11 age in days SPF chicken embryo, 37 DEG C of culture 72h harvest chick embryo allantoic liquid and according to OIE standard to it It carries out blood clotting (HA) test and blood clotting inhibits (HI) test.HA and HI test result is that positive sample contains rescue successfully Recombinant influenza (is named as FLU*-HUPD1), obtains FLU*-HUPD1, -70 DEG C of jellies by amplification, concentration, purifying It deposits.

Also according to the above method, pHW-HA-HUPD1, pHW-NA-HUPD1 are replaced with into pHW-HA-HUPDL1, pHW- NA-HUPDL1, other steps are constant, carry out the rescue of recombinant influenza, obtain influenza virus FLU*- after purification HUPDL1, -70 DEG C freeze.

3, the identification of recombinant influenza

The recombinant influenza FLU*-HUPD1 and FLU*-HUPDL1 that step 2 obtains transmission electron microscope observing (ginseng after negative staining Examine document: example of transmission electron microscope technology of preparing (one) film sample technology of preparing, J, physical testing, 1995:42-44) Morphology of virus, the results showed that FLU*-HUPD1 and FLU*-HUPDL1 meets influenza virus representative configuration feature, there is coating, surface Spinosity lug structure, virion size is between 80-120nm.

Recombinant influenza FLU*-HUPD1 and FLU*-HUPDL1 the inoculation 9-11 age in days SPF chicken embryo of step 2 are passed on, Second generation chick embryo allantoic liquid is taken to extract viral RNA, by RT-PCR (PCR method and amplimer reference papers: Yang Penghui, weight Research .2006 with H1N1 subtype influenza virus attenuated vaccine strain), amplify sequence correct PB2, PB1, PA, NP, M, NS Genetic fragment and improved HA-PD1, NA-PD1, HA-PDL1, NA-PDL1 gene.

By FLU*-HUPD1, FLU*-HUPDL1 of step 2 through chicken embryo mass propgation, ultrafiltration concentration, sucrose gradient centrifugation are pure After changing (specific steps reference papers: the research .2006 of H1N1 subtype influenza virus attenuated vaccine strain is reconfigured in Yang Penghui), into NP, HA1, HA2, NS1 albumen (NP of corresponding size can be detected after gel-colored, decoloration in row polyacrylamide gel electrophoresis Albumen is about 60KD, and HA1, HA2 albumen are about 55KD, and NS1 albumen is about 26KD), show that the main component of antigen is not lost.

Blood clotting identification: by the 6 orifice plates multigelation of transfection, 10 age in days SPF chick embryo allantoic cavities, each egg inoculation are inoculated with 0.2ml sets 37 DEG C, 72~96h of 5%CO2 incubator culture, and 4 DEG C of chicken embryos of freezing to death collect allantoic fluid and measure hemagglutinative titer, positive Strain expanded for 3 generations in chicken embryo, collected chick embryo allantoic liquid, surveyed hemagglutinative titer.In 96 hole micro-reaction plates, 50 μ l are added in first hole Viral allantoic fluid, successively doubling dilution, last 1 hole discard 50 μ l after mixing, and add 1% chicken red blood cell of 50ul, after 15min Observe result.Criterion: it is the positive that agglutination phenomenon, which occurs, for red blood cell, and red blood cell forms 1 small ring, and surrounding has blood clot, and person is Terminal.The hemagglutinative titer of FLU*-HUPD1 and FLU*-HUPDL1 can reach 2 as the result is shown⁸。

Chicken embryo 50 3nfective dose (EID₅₀) measurement:

The 1st generation allantoic fluid of FLU*-HUPD1, FLU*-HUPDL1 and PR8 virus inoculation chicken embryo of step 2 is made 10 times to pass Into dilution, 10 age in days SPF chicken embryos are inoculated in through allantoic cavity, each dilution is inoculated with 4 embryos, and every embryo 0.2mL, 37 DEG C of cultures take Out for 24 hours after dead germ and culture to the embryo of 72h, hemagglutinative titer is that egg infectious is positive in the judgement of 1:16 or more, uses Reed- Muench method calculates EID₅₀.As a result the EID of FLU*-HUPD1, FLU*-HUPDL1₅₀It is 10^-7/0.2mL。

Embodiment 6 has evaluated the dynamics that antibody generates during FLU*-HUPD1, FLU*-HUPDL1 infect

Chicken is inoculated with recombinant influenza FLU*-HUPD1, FLU*-HUPDL1 (- 10PFUs) prepared by embodiment 1 respectively Embryo, each time point choose 3 piece of 9 age in days SPF chicken embryo (being purchased from: Beijing Bo Linge Yin Ge Hanweitong Bioisystech Co., Ltd), PR8 (purchased from Chinese Center for Disease Control and Prevention) control is set up simultaneously, with PR8 (- 10PFUs) inoculated into chick embryo, each time point choosing 3 pieces of chicken embryos are taken, while setting up PBS control, and with there are anti-HUPD1 and anti-in ELISA method measurement allantois The amount of HUPDL1 antibody.

As a result as shown in Figure 6, it can be seen that 2 days detectable antibody after inoculation FLU*-HUPD1, FLU*-HUPDL1, and Reached peak value at 4-5 days, after inoculation antibody is all not detected in third day in PR8 group and PBS group.

The stability that assessment antibody is expressed at any time, specific as follows: dilution FLU*-HUPD1, FLU*-HUPDL1 and PR8 are extremely 10^-6, be inoculated with 3 pieces of chicken embryos, every piece of egg inoculation 100ul, 37 DEG C incubator culture 3 days.It receives after allantoic fluid, is diluted to 10^-6, connect Kind of 3 pieces of chicken embryos, every piece of egg inoculation 100ul, 37 DEG C incubator culture 3 days.Repetition is passaged to forth generation.It is measured with ELISA method There are the amounts of antibody in difference passage allantois, as a result as shown in Figure 7, it can be seen that do not observe in difference passage allantois The loss of the amount of antibody of generation shows that antibody gene is stablized in influenza gene group and maintains.

Embodiment 7, recombinant influenza FLU*-HUPD1, FLU*-HUPDL1 are to the internal inhibiting effect of liver cancer

FLU*-HUPD1 and FLU*-HUPDL1 prepared by embodiment 5 are resuspended with PBS, obtain titre be 5.5 × 10⁵FLU*-HUPD1 the and FLU*-HUPDL1 suspension of pfu/100 μ l；

Strains of influenza viruses A/PR/8/34 (being purchased from Chinese Center for Disease Control and Prevention) is resuspended with PBS, obtaining titre is 5.5×10⁵The strains of influenza viruses A/PR/8/34 suspension of pfu/100 μ l.

1, mouse tumor model is prepared

PBS adjusts HepG2 cell concentration 5*10⁶/ ml, trypan blue counts and measurement cell viability.It is dense to adjust HepG2 cell Degree 1 × 10⁶A/ml, by 200 μ l cell infusions in right side of mice back part, when volume is grown to when diameter is 4~6mm, (volume is public Formula V=4/3 × π × S²/ 2 × L/2 L is longest diameter, and S is shortest diameter), according to gross tumor volume and mouse weight with Machine grouping, is administered once every two days by every group 12, and totally 4 times.

2, FLU*-HUPD1 is immune

When above-mentioned 1 right side of mice gross tumor volume is 40mm³When, mouse is grouped at random, respectively control group, A/ PR/8/34 group, FLU-HUPD1 treatment group, specific as follows:

FLU*-HUPD1 treatment group (12 mouse): being 7 × 10 by titre⁵The FLU-HUPD1 suspension of pfu/100 μ l is right Side tumour intratumor injection C57BL/6 mouse；

A/PR/8/34 group (12 mouse): being 7 × 10 by titre⁵The strains of influenza viruses A/PR/8/34 of pfu/100 μ l is outstanding Supernatant liquid right side tumor intratumor injection C57BL/6 mouse；

Control group (PBS；12 mouse): by 100ulPBS right side tumor intratumor injection C57BL/6 mouse；

The/7/9/11/13/15/17/19/21 measures tumour major diameter and minor axis every three days after immune, and calculates Gross tumor volume.

3, FLU*-HUPDL1 is immune

When above-mentioned 1 right side of mice gross tumor volume is 40mm³When, mouse is grouped at random, respectively control group, A/ PR/8/34 group, FLU-HUPDL1 treatment group, specific as follows:

FLU*-HUPDL1 treatment group (12 mouse): being 7 × 10 by titre⁵The FLU-HUPDL1 suspension of pfu/100 μ l Right side tumor intratumor injection C57BL/6 mouse；

A/PR/8/34 group (12 mouse): being 7 × 10 by titre⁵The strains of influenza viruses A/PR/8/34 of pfu/100 μ l is outstanding Supernatant liquid right side tumor intratumor injection C57BL/6 mouse；

Control group (PBS；12 mouse): by 100ulPBS right side tumor intratumor injection C57BL/6 mouse；

5/7/9/11/13/15/17/19/21st measures tumour major diameter and minor axis every three days after immune, and counts Calculate gross tumor volume.

Mouse state, every two days record mouse states, gross tumor volume and changes of weight are observed after treatment.

As a result as shown in Figure 5, it can be seen that compared with the mouse for receiving PBS injection, after slowing down right side with PR8 treatment The growth of dorsal tumors；However, the mouse of injection FLU*-HUPD1, FLU*-HUPDL1 show that gross tumor volume growth is most slow.Table Bright recombinant influenza FLU*-HUPD1 and FLU*-HUPDL1 can inhibit liver cancer.

From in Fig. 5 it can also be seen that in rat liver cancer model, in the weight of the HA/NA building PD1/PDL1 of PR8 Strain Recombinant influenza FLU*-HUPD1 and FLU*-HUPDL1 is to the inhibiting effect of tumour than in PR8 obtained from chain and light chain Recombinant influenza FLU-HUPD1 and FLU- obtained from the heavy chain and light chain of the PB1/PA building PD1/PDL1 of Strain HUPDL1 is obvious to hepatic carcinoma inhibiting effect.

Comparative example:

The heavy chain of source of people PD1/PDL1 and light chain are connected to PR8 strains of influenza viruses not with same method by the present invention With on albumen, attempt following combination altogether: the heavy chain and light chain of 1. source of people PD1/PDL1 is connected to PR8 strains of influenza viruses On PB2 and PB1；2. the heavy chain and light chain of source of people PD1/PDL1 are connected on the PB2 and PA of PR8 strains of influenza viruses；3. people The heavy chain and light chain of source PD1/PDL1 are connected to the PA/PB1 of PR8 strains of influenza viruses；4. the heavy chain of source of people PD1/PDL1 and Light chain is connected to the NA/HA of PR8 strains of influenza viruses；As a result: above-mentioned 1,2 combinations do not obtain recombination oncolytic virus.On It is lower to state 3,4 combination recombination oncolytic virus efficacies, lower than the embodiment of front.

The heavy chain of source of people PD1 (PDL1) and light chain are connected to PR8 strains of influenza viruses PB1/PA (HA/NA) by the present invention Different location, that is, be connected to PB1/PA (HA/NA) sequence 3 ' end packaging sequence (PS) after, specific construction method: PS Sequence+heavy chain (light chain) sequence+signal peptide sequence+PTV-1 2A sequence；

Wherein PB1/PA/HA/NA segment GenBanK is numbered: EF467819.1/EF467820.1/EF467821.1/ AF389120.1；

The packaging sequence that wherein PB1/PA/HA/NA segment 3 ' is held is respectively 60bp/12bp/45bp/187bp；

As a result: the heavy chain of source of people PD1 (PDL1) and light chain are connected to PR8 strains of influenza viruses PB1/PA (HA/NA) 3 ' end packaging sequences (PS) after, do not obtain recombinant influenza according to above-mentioned construction method.

Therefore, the present invention successfully obtains recombinant influenza in the heavy chain and light chain of the HA/NA building PD1/PDL1 of PR8 Strain Viral FLU*-HUPD1 and FLU*-HUPDL1 is successfully obtained in the heavy chain and light chain of the PB1/PA building PD1/PDL1 of PR8 Strain To recombinant influenza FLU-HUPD1 and FLU-HUPDL1, FLU*-HUPD1, FLU*-HUPDL1, FLU-HUPD1 and FLU- HUPDL1 has apparent inhibiting effect to tumour especially liver cancer, and in rat liver cancer model, in the HA/ of PR8 Strain NA constructs recombinant influenza FLU*-HUPD1 and FLU*-HUPDL1 obtained from the heavy chain and light chain of PD1/PDL1 to tumour Inhibiting effect is than the recombinant influenza obtained from the heavy chain and light chain of the PB1/PA of PR8 Strain building PD1/PDL1 FLU-HUPD1 and FLU-HUPDL1 is obvious to hepatic carcinoma inhibiting effect.

The rescue of embodiment 8, recombinant influenza

By merging the C-terminal of the heavy chain of people anti-PD1/anti-PDL1 antibody, pass through flexible joint peptide Linker (GGGGSGGGGSGGGGS) with transforming growth factor-β (transforming growth factor- β, TGF-β) receptor II The ligand binding sequence of the extracellular domain of (TGF β RIIECD) connects.Using PR8 influenza virus as carrier, TGF β RII ECD It is co-expressed in conjunction with anti-PD1 antibody.

1, the building of recombinant vector

Recombinant vector pHW-PB1-HUPD1-linker-TGF β RII ECD is by the coding DNA of bifunctional fusion proteins A The carrier obtained between the BsmbI restriction enzyme site of molecule insertion pHW2000 carrier, expresses bifunctional fusion proteins.

Bifunctional fusion proteins A is from N-terminal by PB1, source of people PD1 heavy chain, flexible joint peptide GGGGSGGGGSGGGGS (sequence Column 10) and TGF β RIIECD (sequence 11) composition.

The non-coding DNA molecules of bifunctional fusion proteins A fusion as shown in 2 887-3689 of sequence from 5 ' ends First, flexible joint peptide GGGGSGGGGSGGGGS coded sequence and TGF β RIIECD coded sequence composition.Recombinant vector pHW-PB1- HUPDL1-linker-TGF β RII ECD is that the non-coding DNA molecules of bifunctional fusion proteins B are inserted into pHW2000 carrier The carrier obtained between BsmbI restriction enzyme site expresses bifunctional fusion proteins.

Bifunctional fusion proteins B from N-terminal by PB1, source of people PDL1 heavy chain, flexible joint peptide GGGGSGGGGSGGGGS and TGF β RIIECD composition.

The non-coding DNA molecules of bifunctional fusion proteins B fusion as shown in 4 887-3720 of sequence from 5 ' ends First, flexible joint peptide GGGGSGGGGSGGGGS coded sequence and TGF β RIIECD coded sequence composition.2, recombinant influenza Rescue

By pHW-PB1-HUPD1-linker-TGF β RII ECD, pHW-PA-HUPD1 and the recombinant vector pHW- of step 1 PB2, pHW-NP, pHW-M, pHW-NS, pHW-HA and pHW-NA prepare stream after purification according to 2 method of embodiment 1 Influenza Virus FLU-HUPD1-TGF β RII ECD.

By pHW-PB1-HUPDL1-linker-TGF β RII ECD, pHW-PA-HUPDL1 and the recombinant vector of step 1 PHW-PB2, pHW-NP, pHW-M, pHW-NS, pHW-HA and pHW-NA are prepared after purification according to 2 method of embodiment 1 Influenza virus FLU-HUPDL1-TGF β RII ECD.

3, the identification of recombinant influenza

Detection method is identical as the 3 of embodiment 1, influenza virus FLU-HUPD1-TGF β RII ECD and purifying after purification The EID of influenza virus FLU-HUPDL1-TGF β RII ECD afterwards₅₀It is 10^-6/ 0.2mL and 10^-6.5/0.2mL。

Embodiment 9, recombinant influenza FLU-HUPD1-TGF β RII ECD and FLU-HUPDL1-TGF β RII ECD are to liver The internal inhibiting effect of cancer

1, mouse subcutaneous tumor model is prepared

The employment CD34 on NSG mouse⁺Cellular reconstitution humanization immune system mouse is (by tieing up the sensible limited public affairs of biotechnology Department provides), (note: following experiment mouse used is humanization immune system mouse) every inoculates in right side back part 2×10⁵A HepG2 cell (being provided by the 5th medical center of Chinese People's Liberation Army General Hospital), infuses respectively at 5/7/9/11d It penetrates 4 times.5th day every mouse inoculates 1 × 10 in left side back part⁵A HepG2 cell, respectively at the 5/7/9/11/13/th 15/17/19/21 measures tumour major diameter and minor axis, gross tumor volume=(longest diameter × most short diameter every three days²)/2。

2, it is immunized

When above-mentioned 1 right side of mice gross tumor volume is 40mm³When, mouse is grouped at random, respectively control group, A/ PR/8/34 group, FLU-HUPD1-TGF β RII ECD treatment group, specific as follows:

FLU-HUPD1-TGF β RII ECD treatment group (9 mouse): being 5.5 × 10 by titre⁵The FLU- of pfu/100 μ l HUPD1-TGF β RII ECD suspension right side tumor intratumor injection C57BL/6 mouse；

FLU-HUPDL1-TGF β RII ECD treatment group (9 mouse): being 5.5 × 10 by titre⁵The FLU- of pfu/100 μ l HUPDL1-TGF β RII ECD suspension right side tumor intratumor injection C57BL/6 mouse；

A/PR/8/34 group (8 mouse): being 5.5 × 10 by titre⁵The strains of influenza viruses A/PR/8/34 of pfu/100 μ l is outstanding Supernatant liquid right side tumor intratumor injection C57BL/6 mouse；

Control group (PBS；13 mouse): by 100ulPBS right side tumor intratumor injection C57BL/6 mouse；

The/7/9/11/13/15/17/19/21 measures tumour major diameter and minor axis every three days after immune, and calculates Gross tumor volume.

As a result see Fig. 8,

Show that FLU-HUPD1-TGF β RII ECD and FLU-HUPDL1-TGF β RII ECD can inhibit tumour.

SEQUENCE LISTING

<110>the 5th medical center of Chinese People's Liberation Army General Hospital

<120>a kind of recombinant influenza rescue method and its application in oncotherapy

<160>11

<170> PatentIn version 3.5

<210> 1

<211> 3063

<212> DNA

<213>Artificial sequence

<400> 1

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 60

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 120

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 180

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 240

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 300

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 360

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 420

gacgtcgata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 480

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 540

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 600

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 660

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 720

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa 780

cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt 840

aacgctagca gttaaccgga gtactggtcg acctccgaag ttggggggga ggagacggta 900

ccgtctccaa taacccggcg gcccaaaatg ccgactcgga gcgaaagata tacctccccc 960

ggggccggga ggtcgcgtca ccgaccacgc cgccggccca ggcgacgcgc gacacggaca 1020

cctgtcccca aaaacgccac catcgcagcc acacacggag cgcccggggc cctctggtca 1080

accccaggac acacgcggga gcagcgccgg gccggggacg ccctcccggc ggtcacctaa 1140

atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 1200

gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 1260

aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 1320

tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg 1380

tgggctctat ggcttctgag gcggaaagaa ccagctgcat taatgaatcg gccaacgcgc 1440

ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 1500

ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 1560

cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 1620

gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 1680

tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 1740

ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 1800

atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 1860

gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 1920

tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 1980

cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 2040

cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 2100

tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 2160

cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 2220

cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 2280

gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 2340

gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 2400

gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 2460

ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 2520

atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 2580

agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 2640

ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 2700

tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 2760

ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 2820

caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 2880

gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 2940

atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 3000

accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 3060

aaa 3063

<210> 2

<211> 5840

<212> DNA

<213>Artificial sequence

<400> 2

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 60

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 120

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 180

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 240

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 300

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 360

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 420

gacgtcgata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 480

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 540

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 600

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 660

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 720

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa 780

cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt 840

aacgctagca gttaaccgga gtactggtcg acctccgaag ttggggggga gcgaaagcag 900

gcaaaccatt tgaatggatg tcaatccgac cttacttttc ttaaaagtgc cagcacaaaa 960

tgctataagc acaactttcc cttatactgg agaccctcct tacagccatg ggacaggaac 1020

aggatacacc atggatactg tcaacaggac acatcagtac tcagaaaagg gaagatggac 1080

aacaaacacc gaaactggag caccgcaact caacccgatt gatgggccac tgccagaaga 1140

caatgaacca agtggttatg cccaaacaga ttgtgtattg gaggcgatgg ctttccttga 1200

ggaatcccat cctggtattt ttgaaaactc gtgtattgaa acgatggagg ttgttcagca 1260

aacacgagta gacaagctga cacaaggccg acagacctat gactggactc taaatagaaa 1320

ccaacctgct gcaacagcat tggccaacac aatagaagtg ttcagatcaa atggcctcac 1380

ggccaatgag tctggaaggc tcatagactt ccttaaggat gtaatggagt caatgaacaa 1440

agaagaaatg gggatcacaa ctcattttca gagaaagagg cgggtgagag acaatatgac 1500

taagaaaatg ataacacaga gaacaatggg taaaaagaag cagagattga acaaaaggag 1560

ttatctaatt agagcattga ccctgaacac aatgaccaaa gatgctgaga gagggaagct 1620

aaaacggaga gcaattgcaa ccccagggat gcaaataagg gggtttgtat actttgttga 1680

gacactggca aggagtatat gtgagaaact tgaacaatca gggttgccag ttggaggcaa 1740

tgagaagaaa gcaaagttgg caaatgttgt aaggaagatg atgaccaatt ctcaggacac 1800

cgaactttct ttcaccatca ctggagataa caccaaatgg aacgaaaatc agaatcctcg 1860

gatgtttttg gccatgatca catatatgac cagaaatcag cccgaatggt tcagaaatgt 1920

tctaagtatt gctccaataa tgttctcaaa caaaatggcg agactgggaa aagggtatat 1980

gtttgagagc aagagtatga aacttagaac tcaaatacct gcagaaatgc tagcaagcat 2040

cgatttgaaa tatttcaatg attcaacaag aaagaagatt gaaaaaatcc gatcgctctt 2100

aatagagggg actgcatcat tgagccctgg aatgatgatg ggcatgttca atatgttaag 2160

cactgtatta ggcgtgtcca tcctgaatct tggacaaaag agatacacca agactactta 2220

ctggtgggat ggtcttcaat cctctgacga ttttgctctg attgtgaatg cacccaatca 2280

tgaagggatt caagccggag tcgacaggtt ttatcgaacc tgtaagctac ttggaatcaa 2340

tatgagcaag aaaaagtctt acataaacag aacaggtaca tttgaattca caagtttttt 2400

ctatcgttat gggtttgttg ccaatttcag catggagctt cccagttttg gggtgtctgg 2460

gatcaacgag tcagcggaca tgagtattgg agttactgtc atcaaaaaca atatgataaa 2520

caatgatctt ggtccagcaa cagctcaaat ggcccttcag ttgttcatca aagattacag 2580

gtacacgtac cgatgccata gaggtgacac acaaatacaa acccgaagat catttgaaat 2640

aaagaaactg tgggagcaaa cccgttccaa agctggactg ctggtctccg acggaggccc 2700

aaatttatac aacattagaa atctccacat tcctgaagtc tgcctaaaat gggaattgat 2760

ggatgaggat taccaggggc gtttatgcaa cccactgaac ccatttgtca gccataaaga 2820

aattgaatca atgaacaatg cagtgatgat gccagcacat ggtccagcca aaaacatgga 2880

gtatgatgct gttgcaacaa cacactcctg gatccccaaa agaaatcgat ccatcttgaa 2940

tacaagtcaa agaggagtac ttgaggatga acaaatgtac caaaggtgct gcaatttatt 3000

tgaaaaattc ttccccagca gttcatacag aagaccagtc gggatatcca gtatggtgga 3060

ggctatggtt tccagagccc gaattgatgc acggattgat ttcgaatctg gaaggataaa 3120

gaaagaagag ttcactgaga tcatgaaaat ttgcagtaca atcgaggaac ttcggagaca 3180

gaagtagcag gtgcaacttg tggaaagcgg agggggagtg gtgcaaccag gaagatcact 3240

tagacttgat tgcaaagcat caggaataac attctcaaat tcaggaatgc attgggtgag 3300

acaagcacca ggaaaaggac ttgaatgggt ggcagtgata tggtatgatg gatcaaaaag 3360

atattatgca gattcagtga aaggaagatt cacaatatca agagataatt caaaaaatac 3420

acttttcctt caaatgaatt cacttagagc agaagataca gcagtgtatt attgcgcaac 3480

aaatgatgat tattggggac aaggaacact tgtgacagca tcatctccag agcccgaatt 3540

gatgcacgga ttgatttcga atctggaagg ataaagaaag aagagttcac tgagatcatg 3600

aagatctgtt ccaccattga agagctcaga cggcaaaaat agtgaattta gcttgtcctt 3660

catgaaaaaa tgccttgttt ctactaataa cccggcggcc caaaatgccg actcggagcg 3720

aaagatatac ctcccccggg gccgggaggt cgcgtcaccg accacgccgc cggcccaggc 3780

gacgcgcgac acggacacct gtccccaaaa acgccaccat cgcagccaca cacggagcgc 3840

ccggggccct ctggtcaacc ccaggacaca cgcgggagca gcgccgggcc ggggacgccc 3900

tcccggcggt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg 3960

ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 4020

cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 4080

tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 4140

gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctgcattaa 4200

tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 4260

ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 4320

gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4380

ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 4440

cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 4500

ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 4560

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 4620

catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 4680

gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 4740

tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 4800

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 4860

actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 4920

gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 4980

aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 5040

gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 5100

aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 5160

atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 5220

gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 5280

atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 5340

ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 5400

cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 5460

agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 5520

cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 5580

tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 5640

agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 5700

gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 5760

gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 5820

ccacatagca gaactttaaa 5840

<210> 3

<211> 5850

<212> DNA

<213>Artificial sequence

<400> 3

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 60

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 120

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 180

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 240

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 300

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 360

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 420

gacgtcgata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 480

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 540

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 600

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 660

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 720

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa 780

cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt 840

aacgctagca gttaaccgga gtactggtcg acctccgaag ttggggggga gcgaaagcag 900

gtactgatcc aaaatggaag attttgtgcg acaatgcttc aatccgatga ttgtcgagct 960

tgcggaaaaa acaatgaaag agtatgggga ggacctgaaa atcgaaacaa acaaatttgc 1020

agcaatatgc actcacttgg aagtatgctt catgtattca gattttcact tcatcaatga 1080

gcaaggcgag tcaataatcg tagaacttgg tgatccaaat gcacttttga agcacagatt 1140

tgaaataatc gagggaagag atcgcacaat ggcctggaca gtagtaaaca gtatttgcaa 1200

cactacaggg gctgagaaac caaagtttct accagatttg tatgattaca aggagaatag 1260

attcatcgaa attggagtaa caaggagaga agttcacata tactatctgg aaaaggccaa 1320

taaaattaaa tctgagaaaa cacacatcca cattttctcg ttcactgggg aagaaatggc 1380

cacaaaggca gactacactc tcgatgaaga aagcagggct aggatcaaaa ccagactatt 1440

caccataaga caagaaatgg ccagcagagg cctctgggat tcctttcgtc agtccgagag 1500

aggagaagag acaattgaag aaaggtttga aatcacagga acaatgcgca agcttgccga 1560

ccaaagtctc ccgccgaact tctccagcct tgaaaatttt agagcctatg tggatggatt 1620

cgaaccgaac ggctacattg agggcaagct gtctcaaatg tccaaagaag taaatgctag 1680

aattgaacct tttttgaaaa caacaccacg accacttaga cttccgaatg ggcctccctg 1740

ttctcagcgg tccaaattcc tactgatgga tgccttaaaa ttaagcattg aggacccaag 1800

tcatgaagga gagggaatac cgctatatga tgcaatcaaa tgcatgagaa cattctttgg 1860

atggaaggaa cccaatgttg ttaaaccaca cgaaaaggga ataaatccaa attatcttct 1920

gtcatggaag caagtactgg cagaactgca ggacattgag aatgaggaga aaattccaaa 1980

gactaaaaat atgaagaaaa caagtcagct aaagtgggca cttggtgaga acatggcacc 2040

agaaaaggta gactttgacg actgtaaaga tgtaggtgat ttgaagcaat atgatagtga 2100

tgaaccagaa ttgaggtcgc ttgcaagttg gattcagaat gagtttaaca aggcatgcga 2160

actgacagat tcaagctgga tagagctcga tgagattgga gaagatgtgg ctccaattga 2220

acacattgca agcatgagaa ggaattattt cacatcagag gtgtctcact gcagagccac 2280

agaatacata atgaaggggg tgtacatcaa tactgccttg cttaatgcat cttgtgcagc 2340

aatggatgat ttccaattaa ttccaatgat aagcaagtgt agaactaagg agggaaggcg 2400

aaagaccaac ttgtatggtt tcatcataaa aggaagatcc cacttaagga atgacaccga 2460

cgtggtaaac tttgtgagca tggagttttc tctcactgac ccaagacttg aaccacataa 2520

atgggagaag tactgtgttc ttgagatagg agatatgctt ataagaagtg ccataggcca 2580

ggtttcaagg cccatgttct tgtatgtgag aacaaatgga acctcaaaaa ttaaaatgaa 2640

atggggaatg gagatgaggc gttgcctcct ccagtcactt caacaaattg agagtatgat 2700

tgaagctgag tcctctgtca aagagaaaga catgaccaaa gagttctttg agaacaaatc 2760

agaaacatgg cccattggag agtcccccaa aggagtggag gaaagttcca ttgggaaggt 2820

ctgcaggact ttattagcaa agtcggtatt caacagcttg tatgcatctc cacaactaga 2880

aggattttca gctgaatcaa gaaaactgct tcttatcgtt caggctctta gggacaacct 2940

tgaacctggg acctttgatc ttggggggct atatgaagca attgaggagt gcctgattaa 3000

tgatccctgg gttttgctta acgccagctg gtttaattct tttttgacgc acgcgctatc 3060

atagggaagc ggggcaacaa acttctcttt actgaagcaa gctggtgatg tggaggagaa 3120

ccccggtcca atgtatcgta tgcaactgct gagctgcata gcactgtctt tagcacttgt 3180

gacaaacagc gaaattgtgc ttacacaatc accagcaaca ctttcacttt caccaggaga 3240

aagagcaaca ctttcatgca gagcatcaca atcagtgtca tcatatcttg catggtatca 3300

acaaaaacca ggacaagcac caagacttct tatatatgat gcatcaaata gagcaacagg 3360

aataccagca agattctcag gatcaggatc aggaacagat ttcacactta caatatcatc 3420

acttgaacca gaagatttcg cagtgtatta ttgccaacaa tcatcaaatt ggccaagaac 3480

attcggacaa ggaacaaaag tggaaattaa acttgaacct gggacctttg atcttggggg 3540

gctatatgaa gcaattgagg agtgcctgat taatgatccc tgggttttgc ttaatgcttc 3600

ttggttcaac tcattcctta cacatgcatt gagttagttg tggcagtgct actatttgct 3660

atccatactg tccaaaaaag taccttgttt ctactaataa cccggcggcc caaaatgccg 3720

actcggagcg aaagatatac ctcccccggg gccgggaggt cgcgtcaccg accacgccgc 3780

cggcccaggc gacgcgcgac acggacacct gtccccaaaa acgccaccat cgcagccaca 3840

cacggagcgc ccggggccct ctggtcaacc ccaggacaca cgcgggagca gcgccgggcc 3900

ggggacgccc tcccggcggt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc 3960

cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 4020

gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 4080

ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 4140

acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 4200

gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 4260

cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 4320

tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 4380

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 4440

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 4500

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4560

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4620

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 4680

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 4740

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 4800

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 4860

ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 4920

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 4980

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 5040

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 5100

gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 5160

aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 5220

acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 5280

gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 5340

cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 5400

cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 5460

tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 5520

cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 5580

gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 5640

cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 5700

ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 5760

gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 5820

taataccgcg ccacatagca gaactttaaa 5850

<210> 4

<211> 5871

<212> DNA

<213>Artificial sequence

<400> 4

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 60

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 120

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 180

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 240

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 300

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 360

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 420

gacgtcgata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 480

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 540

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 600

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 660

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 720

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa 780

cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt 840

aacgctagca gttaaccgga gtactggtcg acctccgaag ttggggggga gcgaaagcag 900

gcaaaccatt tgaatggatg tcaatccgac cttacttttc ttaaaagtgc cagcacaaaa 960

tgctataagc acaactttcc cttatactgg agaccctcct tacagccatg ggacaggaac 1020

aggatacacc atggatactg tcaacaggac acatcagtac tcagaaaagg gaagatggac 1080

aacaaacacc gaaactggag caccgcaact caacccgatt gatgggccac tgccagaaga 1140

caatgaacca agtggttatg cccaaacaga ttgtgtattg gaggcgatgg ctttccttga 1200

ggaatcccat cctggtattt ttgaaaactc gtgtattgaa acgatggagg ttgttcagca 1260

aacacgagta gacaagctga cacaaggccg acagacctat gactggactc taaatagaaa 1320

ccaacctgct gcaacagcat tggccaacac aatagaagtg ttcagatcaa atggcctcac 1380

ggccaatgag tctggaaggc tcatagactt ccttaaggat gtaatggagt caatgaacaa 1440

agaagaaatg gggatcacaa ctcattttca gagaaagagg cgggtgagag acaatatgac 1500

taagaaaatg ataacacaga gaacaatggg taaaaagaag cagagattga acaaaaggag 1560

ttatctaatt agagcattga ccctgaacac aatgaccaaa gatgctgaga gagggaagct 1620

aaaacggaga gcaattgcaa ccccagggat gcaaataagg gggtttgtat actttgttga 1680

gacactggca aggagtatat gtgagaaact tgaacaatca gggttgccag ttggaggcaa 1740

tgagaagaaa gcaaagttgg caaatgttgt aaggaagatg atgaccaatt ctcaggacac 1800

cgaactttct ttcaccatca ctggagataa caccaaatgg aacgaaaatc agaatcctcg 1860

gatgtttttg gccatgatca catatatgac cagaaatcag cccgaatggt tcagaaatgt 1920

tctaagtatt gctccaataa tgttctcaaa caaaatggcg agactgggaa aagggtatat 1980

gtttgagagc aagagtatga aacttagaac tcaaatacct gcagaaatgc tagcaagcat 2040

cgatttgaaa tatttcaatg attcaacaag aaagaagatt gaaaaaatcc gatcgctctt 2100

aatagagggg actgcatcat tgagccctgg aatgatgatg ggcatgttca atatgttaag 2160

cactgtatta ggcgtgtcca tcctgaatct tggacaaaag agatacacca agactactta 2220

ctggtgggat ggtcttcaat cctctgacga ttttgctctg attgtgaatg cacccaatca 2280

tgaagggatt caagccggag tcgacaggtt ttatcgaacc tgtaagctac ttggaatcaa 2340

tatgagcaag aaaaagtctt acataaacag aacaggtaca tttgaattca caagtttttt 2400

ctatcgttat gggtttgttg ccaatttcag catggagctt cccagttttg gggtgtctgg 2460

gatcaacgag tcagcggaca tgagtattgg agttactgtc atcaaaaaca atatgataaa 2520

caatgatctt ggtccagcaa cagctcaaat ggcccttcag ttgttcatca aagattacag 2580

gtacacgtac cgatgccata gaggtgacac acaaatacaa acccgaagat catttgaaat 2640

aaagaaactg tgggagcaaa cccgttccaa agctggactg ctggtctccg acggaggccc 2700

aaatttatac aacattagaa atctccacat tcctgaagtc tgcctaaaat gggaattgat 2760

ggatgaggat taccaggggc gtttatgcaa cccactgaac ccatttgtca gccataaaga 2820

aattgaatca atgaacaatg cagtgatgat gccagcacat ggtccagcca aaaacatgga 2880

gtatgatgct gttgcaacaa cacactcctg gatccccaaa agaaatcgat ccatcttgaa 2940

tacaagtcaa agaggagtac ttgaggatga acaaatgtac caaaggtgct gcaatttatt 3000

tgaaaaattc ttccccagca gttcatacag aagaccagtc gggatatcca gtatggtgga 3060

ggctatggtt tccagagccc gaattgatgc acggattgat ttcgaatctg gaaggataaa 3120

gaaagaagag ttcactgaga tcatgaaaat ttgcagtaca atcgaggaac ttcggagaca 3180

gaagtagcaa gtgcaacttg tgcaatcagg agcagaagtg aaaaaaccag gatcatcagt 3240

gaaagtgtca tgcaaaacat caggagatac attctcaaca tatgcaatat catgggtgag 3300

acaagcacca ggacaaggac ttgaatggat gggaggaatt attccaattt tcggaaaagc 3360

acattatgca caaaaattcc aaggaagagt gacaataaca gcagatgaaa gcacatcaac 3420

agcatatatg gaactttcat cacttagatc agaagataca gcagtgacat tctgcgcaag 3480

aaaattccat ttcgtgtcag gatcaccatt cggaatggat gtgtggggac aaggaacaac 3540

agtgacagtg tcatcatcca gagcccgaat tgatgcacgg attgatttcg aatctggaag 3600

gataaagaaa gaagagttca ctgagatcat gaagatctgt tccaccattg aagagctcag 3660

acggcaaaaa tagtgaattt agcttgtcct tcatgaaaaa atgccttgtt tctactaata 3720

acccggcggc ccaaaatgcc gactcggagc gaaagatata cctcccccgg ggccgggagg 3780

tcgcgtcacc gaccacgccg ccggcccagg cgacgcgcga cacggacacc tgtccccaaa 3840

aacgccacca tcgcagccac acacggagcg cccggggccc tctggtcaac cccaggacac 3900

acgcgggagc agcgccgggc cggggacgcc ctcccggcgg tcacctaaat gctagagctc 3960

gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg 4020

tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 4080

ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 4140

gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg 4200

cttctgaggc ggaaagaacc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4260

tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4320

gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4380

ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4440

ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4500

acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4560

tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4620

ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4680

ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4740

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4800

actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4860

gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 4920

tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4980

caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 5040

atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 5100

acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5160

ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5220

ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5280

tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 5340

tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5400

gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 5460

tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 5520

tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5580

ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5640

tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5700

ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5760

gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 5820

ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa a 5871

<210> 5

<211> 5847

<212> DNA

<213>Artificial sequence

<400> 5

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 60

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 120

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 180

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 240

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 300

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 360

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 420

gacgtcgata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 480

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 540

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 600

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 660

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 720

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa 780

cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt 840

aacgctagca gttaaccgga gtactggtcg acctccgaag ttggggggga gcgaaagcag 900

gtactgatcc aaaatggaag attttgtgcg acaatgcttc aatccgatga ttgtcgagct 960

tgcggaaaaa acaatgaaag agtatgggga ggacctgaaa atcgaaacaa acaaatttgc 1020

agcaatatgc actcacttgg aagtatgctt catgtattca gattttcact tcatcaatga 1080

gcaaggcgag tcaataatcg tagaacttgg tgatccaaat gcacttttga agcacagatt 1140

tgaaataatc gagggaagag atcgcacaat ggcctggaca gtagtaaaca gtatttgcaa 1200

cactacaggg gctgagaaac caaagtttct accagatttg tatgattaca aggagaatag 1260

attcatcgaa attggagtaa caaggagaga agttcacata tactatctgg aaaaggccaa 1320

taaaattaaa tctgagaaaa cacacatcca cattttctcg ttcactgggg aagaaatggc 1380

cacaaaggca gactacactc tcgatgaaga aagcagggct aggatcaaaa ccagactatt 1440

caccataaga caagaaatgg ccagcagagg cctctgggat tcctttcgtc agtccgagag 1500

aggagaagag acaattgaag aaaggtttga aatcacagga acaatgcgca agcttgccga 1560

ccaaagtctc ccgccgaact tctccagcct tgaaaatttt agagcctatg tggatggatt 1620

cgaaccgaac ggctacattg agggcaagct gtctcaaatg tccaaagaag taaatgctag 1680

aattgaacct tttttgaaaa caacaccacg accacttaga cttccgaatg ggcctccctg 1740

ttctcagcgg tccaaattcc tactgatgga tgccttaaaa ttaagcattg aggacccaag 1800

tcatgaagga gagggaatac cgctatatga tgcaatcaaa tgcatgagaa cattctttgg 1860

atggaaggaa cccaatgttg ttaaaccaca cgaaaaggga ataaatccaa attatcttct 1920

gtcatggaag caagtactgg cagaactgca ggacattgag aatgaggaga aaattccaaa 1980

gactaaaaat atgaagaaaa caagtcagct aaagtgggca cttggtgaga acatggcacc 2040

agaaaaggta gactttgacg actgtaaaga tgtaggtgat ttgaagcaat atgatagtga 2100

tgaaccagaa ttgaggtcgc ttgcaagttg gattcagaat gagtttaaca aggcatgcga 2160

actgacagat tcaagctgga tagagctcga tgagattgga gaagatgtgg ctccaattga 2220

acacattgca agcatgagaa ggaattattt cacatcagag gtgtctcact gcagagccac 2280

agaatacata atgaaggggg tgtacatcaa tactgccttg cttaatgcat cttgtgcagc 2340

aatggatgat ttccaattaa ttccaatgat aagcaagtgt agaactaagg agggaaggcg 2400

aaagaccaac ttgtatggtt tcatcataaa aggaagatcc cacttaagga atgacaccga 2460

cgtggtaaac tttgtgagca tggagttttc tctcactgac ccaagacttg aaccacataa 2520

atgggagaag tactgtgttc ttgagatagg agatatgctt ataagaagtg ccataggcca 2580

ggtttcaagg cccatgttct tgtatgtgag aacaaatgga acctcaaaaa ttaaaatgaa 2640

atggggaatg gagatgaggc gttgcctcct ccagtcactt caacaaattg agagtatgat 2700

tgaagctgag tcctctgtca aagagaaaga catgaccaaa gagttctttg agaacaaatc 2760

agaaacatgg cccattggag agtcccccaa aggagtggag gaaagttcca ttgggaaggt 2820

ctgcaggact ttattagcaa agtcggtatt caacagcttg tatgcatctc cacaactaga 2880

aggattttca gctgaatcaa gaaaactgct tcttatcgtt caggctctta gggacaacct 2940

tgaacctggg acctttgatc ttggggggct atatgaagca attgaggagt gcctgattaa 3000

tgatccctgg gttttgctta acgccagctg gtttaattct tttttgacgc acgcgctatc 3060

atagggaagc ggggcaacaa acttctcttt actgaagcaa gctggtgatg tggaggagaa 3120

ccccggtcca atgtatcgta tgcaactgct gagctgcata gcactgtctt tagcacttgt 3180

gacaaacagc gaaatagtgc ttacacaatc accagcaaca ctttcacttt caccaggaga 3240

aagagcaaca ctttcatgca gagcatcaca atcagtgtca tcatatcttg catggtatca 3300

acaaaaacca ggacaagcac caagacttct tatatatgat gcatcaaata gagcaacagg 3360

aataccagca agattctcag gatcaggatc aggaacagat ttcacactta caatttcatc 3420

acttgaacca gaagatttcg cagtgtatta ttgccaacaa agatcaaatt ggccaacatt 3480

cggacaagga acaaaagtgg aaataaaact tgaacctggg acctttgatc ttggggggct 3540

atatgaagca attgaggagt gcctgattaa tgatccctgg gttttgctta atgcttcttg 3600

gttcaactca ttccttacac atgcattgag ttagttgtgg cagtgctact atttgctatc 3660

catactgtcc aaaaaagtac cttgtttcta ctaataaccc ggcggcccaa aatgccgact 3720

cggagcgaaa gatatacctc ccccggggcc gggaggtcgc gtcaccgacc acgccgccgg 3780

cccaggcgac gcgcgacacg gacacctgtc cccaaaaacg ccaccatcgc agccacacac 3840

ggagcgcccg gggccctctg gtcaacccca ggacacacgc gggagcagcg ccgggccggg 3900

gacgccctcc cggcggtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt 3960

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 4020

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 4080

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 4140

atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 4200

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4260

ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4320

ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4380

agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4440

taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4500

cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4560

tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4620

gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4680

gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4740

tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4800

gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4860

cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4920

aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4980

tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 5040

ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5100

attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 5160

ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 5220

tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 5280

aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 5340

acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 5400

aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 5460

agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 5520

ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 5580

agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 5640

tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 5700

tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 5760

attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 5820

taccgcgcca catagcagaa ctttaaa 5847

<210> 6

<211> 5312

<212> DNA

<213>Artificial sequence

<400> 6

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 60

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 120

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 180

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 240

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 300

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 360

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 420

gacgtcgata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 480

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 540

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 600

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 660

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 720

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa 780

cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt 840

aacgctagca gttaaccgga gtactggtcg acctccgaag ttggggagca aaagcagggg 900

aaaataaaaa caaccaaaat gaaggcaaac ctactggtcc tgttatgtgc acttgcagct 960

gcagatgcag acacaatatg tataggctac catgcgaaca attcaaccga cactgttgac 1020

acagtactcg agaagaatgt gacagtgaca cactctgtta acctgctcga agacagccac 1080

aacggaaaac tatgtagatt aaaaggaata gccccactac aattggggaa atgtaacatc 1140

gccggatggc tcttgggaaa cccagaatgc gacccactgc ttccagtgag atcatggtcc 1200

tacattgtag aaacaccaaa ctctgagaat ggaatatgtt atccaggaga tttcatcgac 1260

tatgaggagc tgagggagca attgagctca gtgtcatcat tcgaaagatt cgaaatattt 1320

cccaaagaaa gctcatggcc caaccacaac acaaacggag taacggcagc atgctcccat 1380

gaggggaaaa gcagttttta cagaaatttg ctatggctga cggagaagga gggctcatac 1440

ccaaagctga aaaattctta tgtgaacaaa aaagggaaag aagtccttgt actgtggggt 1500

attcatcacc cgcctaacag taaggaacaa cagaatctct atcagaatga aaatgcttat 1560

gtctctgtag tgacttcaaa ttataacagg agatttaccc cggaaatagc agaaagaccc 1620

aaagtaagag atcaagctgg gaggatgaac tattactgga ccttgctaaa acccggagac 1680

acaataatat ttgaggcaaa tggaaatcta atagcaccaa tgtatgcttt cgcactgagt 1740

agaggctttg ggtccggcat catcacctca aacgcatcaa tgcatgagtg taacacgaag 1800

tgtcaaacac ccctgggagc tataaacagc agtctccctt accagaatat acacccagtc 1860

acaataggag agtgcccaaa atacgtcagg agtgccaaat tgaggatggt tacaggacta 1920

aggaacaatc cgtccattca atccagaggt ctatttggag ccattgccgg ttttattgaa 1980

gggggatgga ctggaatgat agatggatgg tatggttatc atcatcagaa tgaacaggga 2040

tcaggctatg cagcggatca aaaaagcaca caaaatgcca ttaacgggat tacaaacaag 2100

gtgaacactg ttatcgagaa aatgaacatt caattcacag ctgtgggtaa agaattcaac 2160

aaattagaaa aaaggatgga aaatttaaat aaaaaagttg atgatggatt tctggacatt 2220

tggacatata atgcagaatt gttagttcta ctggaaaatg aaaggactct ggatttccat 2280

gactcaaatg tgaagaatct gtatgagaaa gtaaaaagcc aattaaagaa taatgccaaa 2340

gaaatcggaa atggatgttt tgagttctac cacaagtgtg acaatgaatg catggaaagt 2400

gtaagaaatg ggacttatga ttatcccaaa tattcagaag agtcaaagtt gaacagggaa 2460

aaggtagatg gagtgaaatt ggaatcaatg gggatctatc agattctggc gatctactca 2520

actgtcgcca gttcactggt gcttttggtc tccctggggg caatcagttt ctggatgtgt 2580

tctaatggat ctttgcagtg cagaatatgc atctgaggaa gcggggcaac aaacttctct 2640

ttactgaagc aagctggtga tgtggaggag aaccccggtc caatgtatcg tatgcaactg 2700

ctgagctgca tagcactgtc tttagcactt gtgacaaaca gccaggtgca acttgtggaa 2760

agcggagggg gagtggtgca accaggaaga tcacttagac ttgattgcaa agcatcagga 2820

ataacattct caaattcagg aatgcattgg gtgagacaag caccaggaaa aggacttgaa 2880

tgggtggcag tgatatggta tgatggatca aaaagatatt atgcagattc agtgaaagga 2940

agattcacaa tatcaagaga taattcaaaa aatacacttt tccttcaaat gaattcactt 3000

agagcagaag atacagcagt gtattattgc gcaacaaatg atgattattg gggacaagga 3060

acacttgtga cagcatcatc actaatggat ctttgcagtg cagaatatgc atctgagatt 3120

agaatttcag agatatgagg aaaaacaccc ttgtttctac tacccggcgg cccaaaatgc 3180

cgactcggag cgaaagatat acctcccccg gggccgggag gtcgcgtcac cgaccacgcc 3240

gccggcccag gcgacgcgcg acacggacac ctgtccccaa aaacgccacc atcgcagcca 3300

cacacggagc gcccggggcc ctctggtcaa ccccaggaca cacgcgggag cagcgccggg 3360

ccggggacgc cctcccggcg gtcacctaaa tgctagagct cgctgatcag cctcgactgt 3420

gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 3480

aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 3540

taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 3600

agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 3660

cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 3720

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 3780

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3840

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3900

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3960

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 4020

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 4080

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 4140

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 4200

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 4260

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 4320

aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 4380

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 4440

ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 4500

atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 4560

atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 4620

tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 4680

gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 4740

tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 4800

gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 4860

cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 4920

gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 4980

atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 5040

aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 5100

atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 5160

aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 5220

aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 5280

gataataccg cgccacatag cagaacttta aa 5312

<210> 7

<211> 5026

<212> DNA

<213>Artificial sequence

<400> 7

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 60

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 120

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 180

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 240

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 300

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 360

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 420

gacgtcgata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 480

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 540

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 600

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 660

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 720

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa 780

cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt 840

aacgctagca gttaaccgga gtactggtcg acctccgaag ttggggagcg aaagcagggg 900

tttaaaatga atccaaatca gaaaataaca accattggat caatctgtct ggtagtcgga 960

ctaattagcc taatattgca aatagggaat ataatctcaa tatggattag ccattcaatt 1020

caaactggaa gtcaaaacca tactggaata tgcaaccaaa acatcattac ctataaaaat 1080

agcacctggg taaaggacac aacttcagtg atattaaccg gcaattcatc tctttgtccc 1140

atccgtgggt gggctatata cagcaaagac aatagcataa gaattggttc caaaggagac 1200

gtttttgtca taagagagcc ctttatttca tgttctcact tggaatgcag gacctttttt 1260

ctgacccaag gtgccttact gaatgacaag cattcaaatg ggactgttaa ggacagaagc 1320

ccttataggg ccttaatgag ctgccctgtc ggtgaagctc cgtccccgta caattcaaga 1380

tttgaatcgg ttgcttggtc agcaagtgca tgtcatgatg gcatgggctg gctaacaatc 1440

ggaatttcag gtccagataa tggagcagtg gctgtattaa aatacaacgg cataataact 1500

gaaaccataa aaagttggag gaagaaaata ttgaggacac aagagtctga atgtgcctgt 1560

gtaaatggtt catgttttac tataatgact gatggcccga gtgatgggct ggcctcgtac 1620

aaaattttca agatcgaaaa ggggaaggtt actaaatcaa tagagttgaa tgcacctaat 1680

tctcactatg aggaatgttc ctgttaccct gataccggca aagtgatgtg tgtgtgcaga 1740

gacaactggc atggttcgaa ccggccatgg gtgtctttcg atcaaaacct ggattatcaa 1800

ataggataca tctgcagtgg ggttttcggt gacaacccgc gtcccgaaga tggaacaggc 1860

agctgtggtc cagtgtatgt tgatggagca aacggagtaa agggattttc atataggtat 1920

ggtaatggtg tttggatagg aaggaccaaa agtcacagtt ccagacatgg gtttgagatg 1980

atttgggatc ctaatggatg gacagagact gatagtaagt tctctgttag gcaagatgtt 2040

gtggcaatga ctgattggtc agggtatagc ggaagtttcg ttcaacatcc tgagctaaca 2100

gggctagact gtatgaggcc gtgcttctgg gttgaattaa tcaggggacg acctaaagaa 2160

aaaacaatct ggactagtgc gagcagcatt tctttttgtg gcgtgaatag tgatactgta 2220

gattggtctt ggccagacgg tgctgagttg ccattcagca ttgacaagta gggaagcggg 2280

gcaacaaact tctctttact gaagcaagct ggtgatgtgg aggagaaccc cggtccaatg 2340

tatcgtatgc aactgctgag ctgcatagca ctgtctttag cacttgtgac aaacagcgaa 2400

attgtgctta cacaatcacc agcaacactt tcactttcac caggagaaag agcaacactt 2460

tcatgcagag catcacaatc agtgtcatca tatcttgcat ggtatcaaca aaaaccagga 2520

caagcaccaa gacttcttat atatgatgca tcaaatagag caacaggaat accagcaaga 2580

ttctcaggat caggatcagg aacagatttc acacttacaa tatcatcact tgaaccagaa 2640

gatttcgcag tgtattattg ccaacaatca tcaaattggc caagaacatt cggacaagga 2700

acaaaagtgg aaattaaaag gggacgacct aaagaaaaaa caatctggac tagtgcgagc 2760

agcatttctt tttgtggcgt gaatagtgat actgtagatt ggtcttggcc agacggtgct 2820

gagttgccat tcagcattga caagtagtct gttcaaaaaa ctccttgttt ctactacccg 2880

gcggcccaaa atgccgactc ggagcgaaag atatacctcc cccggggccg ggaggtcgcg 2940

tcaccgacca cgccgccggc ccaggcgacg cgcgacacgg acacctgtcc ccaaaaacgc 3000

caccatcgca gccacacacg gagcgcccgg ggccctctgg tcaaccccag gacacacgcg 3060

ggagcagcgc cgggccgggg acgccctccc ggcggtcacc taaatgctag agctcgctga 3120

tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 3180

tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 3240

tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 3300

ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct 3360

gaggcggaaa gaaccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 3420

gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 3480

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 3540

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 3600

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3660

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 3720

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 3780

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 3840

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3900

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3960

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 4020

tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 4080

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 4140

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 4200

aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 4260

aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 4320

aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 4380

gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 4440

gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 4500

caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 4560

ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 4620

attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 4680

ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 4740

gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 4800

ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 4860

tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 4920

gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 4980

cggcgtcaat acgggataat accgcgccac atagcagaac tttaaa 5026

<210> 8

<211> 5342

<212> DNA

<213>Artificial sequence

<400> 8

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 60

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 120

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 180

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 240

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 300

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 360

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 420

gacgtcgata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 480

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 540

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 600

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 660

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 720

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa 780

cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt 840

aacgctagca gttaaccgga gtactggtcg acctccgaag ttggggagca aaagcagggg 900

aaaataaaaa caaccaaaat gaaggcaaac ctactggtcc tgttatgtgc acttgcagct 960

gcagatgcag acacaatatg tataggctac catgcgaaca attcaaccga cactgttgac 1020

acagtactcg agaagaatgt gacagtgaca cactctgtta acctgctcga agacagccac 1080

aacggaaaac tatgtagatt aaaaggaata gccccactac aattggggaa atgtaacatc 1140

gccggatggc tcttgggaaa cccagaatgc gacccactgc ttccagtgag atcatggtcc 1200

tacattgtag aaacaccaaa ctctgagaat ggaatatgtt atccaggaga tttcatcgac 1260

tatgaggagc tgagggagca attgagctca gtgtcatcat tcgaaagatt cgaaatattt 1320

cccaaagaaa gctcatggcc caaccacaac acaaacggag taacggcagc atgctcccat 1380

gaggggaaaa gcagttttta cagaaatttg ctatggctga cggagaagga gggctcatac 1440

ccaaagctga aaaattctta tgtgaacaaa aaagggaaag aagtccttgt actgtggggt 1500

attcatcacc cgcctaacag taaggaacaa cagaatctct atcagaatga aaatgcttat 1560

gtctctgtag tgacttcaaa ttataacagg agatttaccc cggaaatagc agaaagaccc 1620

aaagtaagag atcaagctgg gaggatgaac tattactgga ccttgctaaa acccggagac 1680

acaataatat ttgaggcaaa tggaaatcta atagcaccaa tgtatgcttt cgcactgagt 1740

agaggctttg ggtccggcat catcacctca aacgcatcaa tgcatgagtg taacacgaag 1800

tgtcaaacac ccctgggagc tataaacagc agtctccctt accagaatat acacccagtc 1860

acaataggag agtgcccaaa atacgtcagg agtgccaaat tgaggatggt tacaggacta 1920

aggaacaatc cgtccattca atccagaggt ctatttggag ccattgccgg ttttattgaa 1980

gggggatgga ctggaatgat agatggatgg tatggttatc atcatcagaa tgaacaggga 2040

tcaggctatg cagcggatca aaaaagcaca caaaatgcca ttaacgggat tacaaacaag 2100

gtgaacactg ttatcgagaa aatgaacatt caattcacag ctgtgggtaa agaattcaac 2160

aaattagaaa aaaggatgga aaatttaaat aaaaaagttg atgatggatt tctggacatt 2220

tggacatata atgcagaatt gttagttcta ctggaaaatg aaaggactct ggatttccat 2280

gactcaaatg tgaagaatct gtatgagaaa gtaaaaagcc aattaaagaa taatgccaaa 2340

gaaatcggaa atggatgttt tgagttctac cacaagtgtg acaatgaatg catggaaagt 2400

gtaagaaatg ggacttatga ttatcccaaa tattcagaag agtcaaagtt gaacagggaa 2460

aaggtagatg gagtgaaatt ggaatcaatg gggatctatc agattctggc gatctactca 2520

actgtcgcca gttcactggt gcttttggtc tccctggggg caatcagttt ctggatgtgt 2580

tctaatggat ctttgcagtg cagaatatgc atctgaggaa gcggggcaac aaacttctct 2640

ttactgaagc aagctggtga tgtggaggag aaccccggtc caatgtatcg tatgcaactg 2700

ctgagctgca tagcactgtc tttagcactt gtgacaaaca gccaagtgca acttgtgcaa 2760

tcaggagcag aagtgaaaaa accaggatca tcagtgaaag tgtcatgcaa aacatcagga 2820

gatacattct caacatatgc aatatcatgg gtgagacaag caccaggaca aggacttgaa 2880

tggatgggag gaattattcc aattttcgga aaagcacatt atgcacaaaa attccaagga 2940

agagtgacaa taacagcaga tgaaagcaca tcaacagcat atatggaact ttcatcactt 3000

agatcagaag atacagcagt gacattctgc gcaagaaaat tccatttcgt gtcaggatca 3060

ccattcggaa tggatgtgtg gggacaagga acaacagtga cagtgtcatc actaatggat 3120

ctttgcagtg cagaatatgc atctgagatt agaatttcag agatatgagg aaaaacaccc 3180

ttgtttctac tacccggcgg cccaaaatgc cgactcggag cgaaagatat acctcccccg 3240

gggccgggag gtcgcgtcac cgaccacgcc gccggcccag gcgacgcgcg acacggacac 3300

ctgtccccaa aaacgccacc atcgcagcca cacacggagc gcccggggcc ctctggtcaa 3360

ccccaggaca cacgcgggag cagcgccggg ccggggacgc cctcccggcg gtcacctaaa 3420

tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 3480

cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 3540

aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 3600

ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 3660

gggctctatg gcttctgagg cggaaagaac cagctgcatt aatgaatcgg ccaacgcgcg 3720

gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 3780

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 3840

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 3900

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 3960

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 4020

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 4080

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 4140

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 4200

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 4260

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 4320

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 4380

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 4440

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 4500

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 4560

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 4620

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 4680

tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 4740

tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 4800

tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 4860

gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 4920

tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 4980

ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5040

gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 5100

aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 5160

ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 5220

tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 5280

ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 5340

aa 5342

<210> 9

<211> 5023

<212> DNA

<213>Artificial sequence

<400> 9

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 60

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 120

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 180

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 240

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 300

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 360

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 420

gacgtcgata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 480

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 540

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 600

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 660

caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 720

ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa 780

cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctgtt 840

aacgctagca gttaaccgga gtactggtcg acctccgaag ttggggagcg aaagcagggg 900

tttaaaatga atccaaatca gaaaataaca accattggat caatctgtct ggtagtcgga 960

ctaattagcc taatattgca aatagggaat ataatctcaa tatggattag ccattcaatt 1020

caaactggaa gtcaaaacca tactggaata tgcaaccaaa acatcattac ctataaaaat 1080

agcacctggg taaaggacac aacttcagtg atattaaccg gcaattcatc tctttgtccc 1140

atccgtgggt gggctatata cagcaaagac aatagcataa gaattggttc caaaggagac 1200

gtttttgtca taagagagcc ctttatttca tgttctcact tggaatgcag gacctttttt 1260

ctgacccaag gtgccttact gaatgacaag cattcaaatg ggactgttaa ggacagaagc 1320

ccttataggg ccttaatgag ctgccctgtc ggtgaagctc cgtccccgta caattcaaga 1380

tttgaatcgg ttgcttggtc agcaagtgca tgtcatgatg gcatgggctg gctaacaatc 1440

ggaatttcag gtccagataa tggagcagtg gctgtattaa aatacaacgg cataataact 1500

gaaaccataa aaagttggag gaagaaaata ttgaggacac aagagtctga atgtgcctgt 1560

gtaaatggtt catgttttac tataatgact gatggcccga gtgatgggct ggcctcgtac 1620

aaaattttca agatcgaaaa ggggaaggtt actaaatcaa tagagttgaa tgcacctaat 1680

tctcactatg aggaatgttc ctgttaccct gataccggca aagtgatgtg tgtgtgcaga 1740

gacaactggc atggttcgaa ccggccatgg gtgtctttcg atcaaaacct ggattatcaa 1800

ataggataca tctgcagtgg ggttttcggt gacaacccgc gtcccgaaga tggaacaggc 1860

agctgtggtc cagtgtatgt tgatggagca aacggagtaa agggattttc atataggtat 1920

ggtaatggtg tttggatagg aaggaccaaa agtcacagtt ccagacatgg gtttgagatg 1980

atttgggatc ctaatggatg gacagagact gatagtaagt tctctgttag gcaagatgtt 2040

gtggcaatga ctgattggtc agggtatagc ggaagtttcg ttcaacatcc tgagctaaca 2100

gggctagact gtatgaggcc gtgcttctgg gttgaattaa tcaggggacg acctaaagaa 2160

aaaacaatct ggactagtgc gagcagcatt tctttttgtg gcgtgaatag tgatactgta 2220

gattggtctt ggccagacgg tgctgagttg ccattcagca ttgacaagta gggaagcggg 2280

gcaacaaact tctctttact gaagcaagct ggtgatgtgg aggagaaccc cggtccaatg 2340

tatcgtatgc aactgctgag ctgcatagca ctgtctttag cacttgtgac aaacagcgaa 2400

atagtgctta cacaatcacc agcaacactt tcactttcac caggagaaag agcaacactt 2460

tcatgcagag catcacaatc agtgtcatca tatcttgcat ggtatcaaca aaaaccagga 2520

caagcaccaa gacttcttat atatgatgca tcaaatagag caacaggaat accagcaaga 2580

ttctcaggat caggatcagg aacagatttc acacttacaa tttcatcact tgaaccagaa 2640

gatttcgcag tgtattattg ccaacaaaga tcaaattggc caacattcgg acaaggaaca 2700

aaagtggaaa taaaaagggg acgacctaaa gaaaaaacaa tctggactag tgcgagcagc 2760

atttcttttt gtggcgtgaa tagtgatact gtagattggt cttggccaga cggtgctgag 2820

ttgccattca gcattgacaa gtagtctgtt caaaaaactc cttgtttcta ctacccggcg 2880

gcccaaaatg ccgactcgga gcgaaagata tacctccccc ggggccggga ggtcgcgtca 2940

ccgaccacgc cgccggccca ggcgacgcgc gacacggaca cctgtcccca aaaacgccac 3000

catcgcagcc acacacggag cgcccggggc cctctggtca accccaggac acacgcggga 3060

gcagcgccgg gccggggacg ccctcccggc ggtcacctaa atgctagagc tcgctgatca 3120

gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 3180

ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 3240

cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 3300

gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 3360

gcggaaagaa ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 3420

ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 3480

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 3540

caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 3600

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 3660

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 3720

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 3780

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 3840

tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 3900

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 3960

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 4020

agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 4080

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 4140

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 4200

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 4260

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 4320

gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 4380

taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 4440

tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 4500

tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 4560

gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 4620

gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 4680

ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 4740

cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 4800

tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 4860

cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 4920

agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 4980

cgtcaatacg ggataatacc gcgccacata gcagaacttt aaa 5023

<210>10

<211>15

<212>PRT

<213>Artificial sequence

<400>10

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>11

<211> 137

<212> PRT

<213>Artificial sequence

<400>11

Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val

1 5 10 15

Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys

20 25 30

Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn

35 40 45

Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala

50 55 60

Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His

65 70 75 80

Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser

85 90 95

Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe

100 105 110

Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser

115 120 125

Glu Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

Claims (10)

1. a kind of method of the recombinant virus for the antibody for preparing expression anti-tumor protein, includes the following steps:

Anti-tumor protein antibody or the encoding gene containing its fusion protein are passed through into virus rescue, obtain expressing antitumor egg The recombinant virus of white antibody-encoding genes.

2. according to the method described in claim 1, it is characterized by:

The anti-tumor protein antibody fusion protein include the anti-tumor protein antibody and other transforming growth factor receptors or The extracellular domain of this receptor；

And/or the anti-tumor protein antibody is immune globulin antibody or single-chain antibody or chimeric antibody；

And/or the virus is influenza virus or adenovirus or poxvirus or other oncolytic virus carriers.

3. according to the method described in claim 2, it is characterized by:

The virus rescue is to be coupled the anti-tumor protein antibody or the fusion protein with encoding gene in viral genome Encoding gene, then with viral genome remove for coupling gene other than remaining encoding gene be transferred in cell jointly, Packaging obtains recombinant virus.

4. according to the method described in claim 3, it is characterized by:

The anti-tumor protein antibody is anti-tumor protein single-chain antibody, and described method includes following steps: will be containing described anti- The fusion third of 1 encoding gene in the encoding gene of oncoprotein single-chain antibody and the viral genome and the disease It removes remaining encoding gene other than the gene for fusion in virus gene group to be transferred in cell jointly, packaging obtains recombination disease Poison, the expression of recombinant virus anti-tumor protein single-chain antibody；

Or, the anti-tumor protein antibody is immune globulin antibody, described method includes following steps: will contain immune globulin The fusion first of 1 encoding gene in the heavy chain encoding gene and the viral genome of Bai Kangti is immunized containing described The light chain encoding gene of globulin antibody and the fusion second of another encoding gene in the viral genome, and it is described It removes remaining encoding gene other than the gene for fusion in viral genome to be transferred in cell jointly, packaging obtains recombination disease Poison, the expression of recombinant virus immune globulin antibody；

Or, the anti-tumor protein antibody fusion protein includes the thin of the immune globulin antibody and transforming growth factor receptor Extracellular domain；Described method includes following steps: by the heavy chain fusion gene containing immune globulin antibody and the virus The fusion first of 1 encoding gene in genome, the light chain encoding gene containing the immune globulin antibody and described The fusion second of another encoding gene in viral genome, with gene of the removing for merging in the viral genome Remaining encoding gene in addition is transferred in cell jointly, and packaging obtains recombinant virus, and the expression of recombinant virus immunoglobulin is anti- The extracellular domain of body and transforming growth factor receptor；The heavy chain fusion gene of the immune globulin antibody includes immune ball The ligand binding domain coded sequence of the extracellular domain of the heavy chain encoding gene and transforming growth factor receptor of protein antibodies.

5. according to the method described in claim 4, it is characterized by:

The virus is influenza virus,

Encoding gene is PB2 protein coding gene, PB1 protein coding gene, PA encoding histone in the influenza virus gene group Gene, HA protein coding gene, NP protein coding gene, NA protein coding gene, M protein coding gene and NS encoding histone base Cause；

Or, the fusion or remaining described encoding gene pass through recombinant vector and import cell；

Or, the fusion first is from successively including HA protein coding gene in the viral genome the end 5' and described exempt from The heavy chain encoding gene of epidemic disease globulin antibody or the heavy chain fusion gene of the immune globulin antibody；

And the fusion second is from successively including NA protein coding gene in the viral genome the end 5' and described anti- The light chain encoding gene of the immune globulin antibody of oncoprotein；

And remaining described encoding gene is the PB2 protein coding gene, the PB1 protein coding gene, PA albumen volume Code gene, the NP protein coding gene, the M protein coding gene and the NS protein coding gene；

Or,

The fusion first is from successively including PB1 protein coding gene in the viral genome the end 5' and described immune The heavy chain encoding gene of globulin antibody or the heavy chain fusion gene of the immune globulin antibody；

And the fusion second is from successively including PA protein coding gene in the viral genome the end 5' and described anti- The light chain encoding gene of the immune globulin antibody of oncoprotein；

And remaining described encoding gene is the PB2 protein coding gene, the HA protein coding gene, NP albumen volume Code gene, the NA protein coding gene, the M protein coding gene and the NS protein coding gene.

6. according to the method any in claim 3-5, it is characterised in that:

The immune globulin antibody is immune detection point antibody；

And/or the immune detection point antibody is PD1 antibody or PDL1 antibody or CCR4 antibody or CTLA4；

And/or the immune detection point antibody is people's source protein antibody or source of mouse protein antibodies；

And/or the extracellular domain of the transforming growth factor receptor is the extracellular knot of transform growth factor-beta receptor II The ligand binding domain in structure domain.

7. by the recombinant virus of any the method preparation of claim 1-6.

Or, the antibody of the anti-tumor protein prepared by the recombinant virus；

Or, a kind of method for the antibody for preparing anti-tumor protein obtains anti-tumor protein for animal is immunized in the recombinant virus Antibody.

8. the antibody of recombinant virus as claimed in claim 7 or anti-tumor protein is resisting or is treating or inhibiting the application in tumour；

Or the antibody of recombinant virus as claimed in claim 7 or anti-tumor protein is inhibiting the application in tumor cell proliferation；

Or the antibody of recombinant virus as claimed in claim 7 or anti-tumor protein is anti-or treat or inhibit in tumour product in preparation Application；

Or the antibody of recombinant virus as claimed in claim 7 or anti-tumor protein inhibits in tumor cell proliferation product in preparation Using；

Or, a kind of antitumor product, active constituent is the antibody of recombinant virus as claimed in claim 7 or anti-tumor protein.

9. according to claim 1 described in -6 any the methods or claim 7 or application or described according to any one of claims 8 Product, it is characterised in that: the tumour is melanoma, liver cancer, lung cancer or other tumours.

10. prepared by the recombinant vector of gene or expressing said gene in viral genome or the virus of expressing said gene Application in antineoplastic genetic engineering drug.