CN101001869A - Carcinoembryonic antigen fusions and uses thereof - Google Patents

Abstract

Polynucleotides encoding carcinoembryonic antigen (CEA) fusion proteins are provided, the CEA fusion proteins comprising a CEA protein, or functional variant thereof, fused to a substantial portion of an immunoenhancing element. The polynucleotides of the present invention can elicit an immune response in a mammal, which, in preferred embodiments, is stronger than the immune response elicited by a wild-type CEA. The gene encoding CEA is commonly associated with the development of human carcinomas. The present invention provides compositions and methods to elicit or enhance immunity to the protein product expressed by the CEA tumor-associated antigen, wherein aberrant CEA expression is associated with a carcinoma or its development. This invention specifically provides adenoviral vector and plasmid constructs carrying polynucleotides encoding CEA fusion proteins and discloses their use in vaccines and pharmaceutical compositions for preventing and treating cancer.

Description

Carcinoembryonic antigen fusions and its purposes

Invention field

Relate generally to treatment for cancer of the present invention.More specifically, the present invention relates to the polynucleotide of encoding fusion protein, wherein said fusion rotein comprises at least a portion of tumor relative polypeptide carcinomebryonic antigen.The present invention also provides recombinant vectors and the host who comprises described polynucleotide, and the fusion rotein of purifying and use composition disclosed herein and molecule strengthen the immunoreactive method at CEA.

Background of invention

Immunoglobulin superfamily (IgSF) is by the proteic genomic constitution of the various functions of many codings, and one of function is intercellular adhesion.IgSF albumen contains at least one Ig dependency structure territory, and it is important for keeping suitable intermolecular binding interactions.Because this interaction is that IgSF member's various biological functions are necessary, the destruction of many IgSF adhesion molecules or unconventionality expression and many human diseasess are associated.

Carcinomebryonic antigen (CEA) belongs to the subtribe of Ig superfamily, and it is made up of the cell surface glycoprotein that is called CEA relevant cell adhesion molecule (CEACAM).Shown that CEACAM has served as adhesion molecule between homotype and heterocyst (Benchimol et al., Cell 57:327-334 (1989)).Except cell adhesion, CEA (being also referred to as CEACAM5) suppresses by cell from the caused necrocytosis of the separation of extracellular matrix, and can promote and some proto-oncogene, the for example Bcl2 cell transformation relevant with C-Myc (referring to, Berinstein, J.ClinOncol.20 (8): 2197-2207 (2002)).Sequence (U.S. Patent No. 5,274,087 of the human CEA that encodes have been cloned and have characterized; US Patent No 5,571,710; With US Patent No 5,843,761.Also referring to Beauchemin et al., Mol.Cell.Biol.7:3221-3230 (1987); Zimmerman et al., Proc.Natl.Acad.Sci.USA 84:920-924 (1987); Thompson et al.Proc.Natl.Acad.Sci.USA 84 (9): 2965-69 (1987)).

During the fetation and in grownup's mucous membrane of colon, detect the normal expression of CEA.In human colon's tumour, detected the overexpression (Goldand Freeman, J.Exp.Med.121:439-462 (1965)) of CEA before three more than ten years first, after this in nearly all colorectum tumour, all found.In addition, the CEA overexpression is detectable in the gland cancer of a high proportion of pancreas, liver, breast, ovary, uterine neck and lung.Because its popularity in these tumor types and limited normal tissue expression, CEA is considered to the target spot of autologous tumor related antigen and active and passive immunization therapy.Recently clinical data has determined that different vaccine strategies can produce human B and the T cell that is specific to CEA, provides other evidence proof CEA to be used for the treatment of the molecule of these cancer types and the target spot that immunology gets involved.

The methods of treatment of target CEA comprises uses anti-CEA antibody (referring to Chester et al., Cancer Chemother.Pharmacol.46 (Suppl): S8-S12 (2000)), and based on the vaccine (for summary, referring to Berinstein, the same) of CEA.Many developing vaccines and commercialization have been subjected to the obstruction of a difficult problem relevant with the high expression level that obtains foreign gene.Also be subjected in the individuality of treatment, can not producing the immunoreactive obstruction of q.s based on the success of the vaccine of DNA.Although developed the various proteic dna vaccinations of target, to compare with the vaccine of routine, the immune response of generation is weak relatively.

For the vaccine of developing in conjunction with the gene fusion strategy provides chance, antigen is connected with various immunostimulant elements the simplification of DNA operation in the gene fusion strategy.Be fused to heat shock protein (HSP) 70 (Liu et al., J.Virol.74:2888-94 (2000) by coding; Chenget al.J.Immunol.166:6218-26 (2001); Chen et al., Cancer Res.60:1035-42 (2000)), the Fc of IgG1 part (You et al., J.Immunol.165:4581-92 (2000)), lysosome related membrane protein (LAMP) (Su et al., CancerRes.62:5041-48 (2002)) with from general Th epi-position (Renardet al., the J.Immunol.171:1588-95 (2003) of tetanus toxin; King et al., Nature Med.4:1281-86 (1998); Lund et al., Cancer Gene Ther.10:365-76 (2003); Padua et al., Nature Med.9 (11): 1413-17 (2003); Savelyeva et al., NatureBiotechnol.19:760-64 (2001); Wahren et al., WO 2004/092216)) antigenic carrier, in animal model, proved immunoreactive enhancing to target antigen.In view of the limited immunogenicity of tumour antigen with need overcome tolerance and bring into play effective anticancer effect, the immune response that strengthens target antigen is relevant especially for cancer vaccine.

Therefore, although the aforesaid proteic wild-type nucleotide sequence of coding CEA of having identified, high expectations be to develop a kind of vaccine, when being delivered to Mammals, can cause enhanced CEA specific immune response with respect to wild type full-length CEA cDNA.Same expectation be that exploitation is used for the treatment of or prevent CEA dependency method for cancer, it has utilized safety and nucleic acid molecule or the albumen of strengthening the CEA specific immune response effectively.

Summary of the invention

The invention provides the polynucleotide of encoding fusion protein, wherein said fusion rotein comprises at least a portion of tumor relative polypeptide carcinomebryonic antigen, is fused to for example bacteriotoxic entity part of immunostimulant element (a substantial portion).In preferred embodiment, the CEA part of the CEA fusion rotein of coding is deleted its C-end anchors fixed structure territory.In preferred embodiment, the A of the heat labile enterotoxin that described immunostimulant element is E.coli or B subunit, or its entity part.Other preferred embodiment in, described immunostimulant element is the minimized structural domain (DOM) of tetanus toxin fragment C, or its entity part.The present invention also provides the recombinant vectors that comprises described polynucleotide, includes but not limited to adenovirus and plasmid vector and comprise the host cell of described recombinant vectors.Also provide fusion rotein at this by the purifying of polynucleotide encoding of the present invention.

The present invention further provides by using vaccine that comprises CEA fusions described here or CEA fusion rotein or the method that the pharmaceutical composition initiation suppresses in Mammals at the proteic immune response of CEA or preventing cancer develops.This method preferred embodiment in, with respect to the reaction that wild-type CEA vaccine causes, described immune response has been enhanced.

As at whole specification sheets with use in subsidiary claim, singulative " a ", " an " and " the " comprise plural content, unless indicated in addition significantly in the context.

As what in whole specification sheets and subsidiary claims, use, adopt to give a definition and to abridge:

Term " promotor " is meant on the DNA chain, RNA polymerase bonded recognition site with it.Promotor and RNA polymerase form initiation complex and start and drive transcriptional activity.By activation sequences that is called " enhanser " or the inhibition sequence that is called " silencer ", can modify described mixture.

Term " box " is meant will be from the Nucleotide or the gene order of vector expression, for example, and the Nucleotide or the gene order of coding hCEA-LTB fusions.Usually, box comprises the gene order that can be inserted in the carrier, and it provides the adjusting sequence that is used to express described Nucleotide or gene order in some embodiments.In other embodiments, described Nucleotide or gene order provide the adjusting that is used for its expression sequence.In further embodiment, described carrier provides some to regulate sequence, and described Nucleotide or gene order provide other to regulate sequence.For example, described carrier can be provided for transcribing the promotor of described Nucleotide or gene order, and described Nucleotide or gene order provide transcription termination sequence.Can include, but not limited to enhanser, transcription termination sequence, montage receptor and donor sequences, intron, ribosome binding sequence and poly (A) appended sequence by the adjusting sequence that carrier provides.

Term " carrier " is meant some instruments, dna fragmentation can be imported in host's organism or the host tissue by them.Exist the carrier of various types, comprise plasmid, virus (comprising adenovirus), phage and clay.

About the term " first-generation " that adenovirus carrier uses, the adenovirus carrier of replication defective has been described.First-generation adenovirus carrier generally has E1 gene region deletion or passivation, preferably has E3 gene region deletion or passivation.

Abbreviation " DOM " generally is meant the N-end structure territory of the fragment C of Toxoid,tetanus.

Abbreviation " LT " generally is meant the heat labile enterotoxin of E.coli." LT " can refer to complete enterotoxin, comprises the entity part of subunit A and B or subunit A or the entity part of subunit B.Abbreviation " LTA " is meant the A subunit of E.coli heat labile enterotoxin, or its entity part, be included in the terminal brachymemma of C-end or N-but kept the subunit of biologic activity, and contain inner aminoacid insertion, deletion or replacement but kept the subunit of biologic activity.Abbreviation " LTB " is meant the B subunit of E.coli heat labile enterotoxin, or its entity part, be included in the terminal brachymemma of C-end or N-but kept the subunit of biologic activity, and contain inner aminoacid insertion, deletion or replacement but kept the subunit of biologic activity.

Title " pV1J/hCEAopt " is meant plasmid construction body disclosed herein, comprise the CMV with intron A instant-codon optimized human CEA gene, Trobest deutero-polyadenylic acid and transcription termination sequence and the minimum pUC trunk (referring to embodiment 2) of early stage (IE) promotor, total length.Title " pV1J/hCEA " is meant basically construct as described above, and just this construct comprises human CEA gene of wild-type rather than codon optimized human CEA gene.

Title " pV1J/hCEA-LTB " is meant plasmid construction body disclosed herein, comprise the CMV with intron A instant-early stage (IE) promotor, lack the human CEA gene of its GPI grappling encoding sequence, terminal B subunit with the E.coli heat labile enterotoxin at its C-merges, Trobest deutero-polyadenylic acid and transcription termination sequence and minimum pUC trunk.

The construct as described above that title " pV1J/hCEAopt-LTB " is meant basically as is right after, just this construct comprises the codon optimized human CEA gene that lacks its GPI grappling encoding sequence rather than the corresponding section of the human CEA gene of wild-type.

The plasmid construction body as described above that title " pV1J/hCEAopt-LTBopt " is meant basically as is right after, just CEA sequence and LTB sequence are all codon optimized for high level expression in the human cell.

Title " pV1J/rhCEAopt-LTBopt " is meant basically construct as described above, is that the codon optimized CEA gene of the described mankind is replaced by the rhesus monkey CEA gene codon optimized for high level expression in the human cell.

Title " pV1J/hCEA-LTA " is meant plasmid construction body disclosed herein, comprise the CMV with intron A instant-early stage (IE) promotor, lack the human CEA gene of its GPI grappling encoding sequence, terminal A subunit with the E.coli heat labile enterotoxin at its C-merges, Trobest deutero-polyadenylic acid and transcription termination sequence and minimum pUC trunk.Being structured among the embodiment 2 of plasmid vector that comprises various CEA-LT fusions described.

Title " pV1J/hCEA-DOM " is meant plasmid construction body disclosed herein, comprise the CMV with intron A instant-early stage (IE) promotor, lack the human CEA gene of its GPI grappling encoding sequence, merge in its C-end and the N-end structure territory (DOM) of the fragment C of Toxoid,tetanus, Trobest deutero-polyadenylic acid and transcription termination sequence and minimum pUC trunk (embodiment 2).

Title " pV1J/rhCEAopt-DOMopt " is meant basically construct as described above, is that the codon optimized CEA gene of the described mankind is replaced by the rhesus monkey CEA gene codon optimized for high level expression in the human cell.

Title " pV1J/hCEA-FcIgG " is meant plasmid construction body disclosed herein, comprise the CMV with intron A instant-early stage (IE) promotor, lack the human CEA gene of its GPI grappling encoding sequence, merge in the heavy fragment of its C-end with the constant chain of immunoglobulin G 1, Trobest deutero-polyadenylic acid and transcription termination sequence and minimum pUC trunk.(embodiment 2).PV1J/hCEAopt-FcIgGopt is meant basically construct as described, and the nucleotide sequence of just encode CEA and FcIgG is codon optimized for high level expression in the human cell.

Title " Ad5/hCEAopt " and " Ad5/hCEA " are meant two kinds of constructs disclosed herein, and it comprises the Ad5 adenoviral gene group of having deleted E1 and E3 zone.In " Ad5/hCEAopt " construct, E1 is pressed in the zone the codon optimized human CEA gene of E1 parallel direction and is replaced, described codon optimized human CEA gene is a Trobest polyadenylic acid signal under the control of human cmv promoter that does not contain intron A afterwards." Ad5/hCEA " construct is that the genomic E1 of Ad5 zone is replaced by the human CEA sequence of wild-type basically as mentioned above.Title " Ad5/hCEAopt-LTB " is meant basically Ad5 construct as described above, is that described codon optimized human CEA sequence lacks GPI grappling encoding sequence, and terminal B subunit with the E.coli heat labile enterotoxin at its C-merges.The construction of recombinant adenovirus containing that comprises various CEA-LT fusions is described in embodiment 3.

" immunostimulant element " is meant the part of CEA fusion rotein of the present invention, and with respect to total length wild-type CEA, it can stimulate or strengthen the proteic immune response of CEA to linking to each other.Immunostimulant element of the present invention is selected from the group that constitutes by the heavy fragment (FcIgG) of the constant chain of the N-end structure territory (DOM) of fragment C (FrC), the FrC of heat shock protein (HSP) 70, lysosome related membrane protein (LAMP), Toxoid,tetanus, immunoglobulin G 1, vesicular stomatitis virus glycoprotein (VSV-G), from the heat labile enterotoxin (LT) of the Toxins,exo-, cholera (CT) of Vibrio cholerae (vibrio cholerae) and E.coli.Use interchangeably at this term " immunostimulant element " and term " adjuvant ".

As used herein, " fusion rotein " is meant the albumen with at least two covalently bound polypeptide, and one of them polypeptide is from a protein sequence or structural domain, and another polypeptide is from second protein sequence or structural domain.Fusion rotein of the present invention comprises CEA polypeptide or its fragment or variant and comprises second polypeptide of the entity part of immunostimulant element, and described immunostimulant element is being bacteriotoxin in some cases.Described CEA polypeptide, its fragment or variant can be human CEA or from the CEA homologue of other species.Preferably N-is terminal connects to C-is terminal for the polypeptide that constitutes fusion rotein.Described CEA polypeptide and toxin subunit can merge by any order.In some embodiments of the present invention, the C-end of CEA polypeptide is fused to the N-end of toxin subunit, as illustrative in accompanying drawing 1A.Yet the immunostimulant element fusion rotein that is fused to the N-end of CEA polypeptide is also considered therein.Term " CEA fusion rotein " intention is as generic term, and it is meant aforesaid fusions, and it comprises CEA polypeptide or its fragment or the variant that merges with polypeptide, and described previous polypeptide comprises the immunostimulant element.

Term " CEA-LT fusions " is meant a kind of nucleotide sequence, and wherein at least a portion of CEA gene is fused to the entity part of the LTA or the LTB subunit of E.coli heat labile enterotoxin.Term " CEA-LT fusion rotein " is meant by CEA-LT fusions encoded polypeptides as described.Term " CEA-LT fusions " and " CEA-LT fusion rotein " also are interpreted as those that are meant that its fragment, its homologue and its functional equivalent (collectively being called " variant "), for example one or more therein amino acid are inserted into, delete or replaced by other propylhomoserins.CEA-LT fusions of the present invention, can stimulate the immune response of helper cell or cytotoxic T cell, or stimulate production of antibodies for example during human administration to Mammals, and effect with " wild-type " CEA sequence is suitable at least for it.Of the present invention preferred embodiment in, CEA compares with wild-type, the CEA-LT fusions can enhancing immunity reaction.

Term " CEA-DOM fusions " is meant a kind of nucleotide sequence, and wherein the entity part of the minimized structural domain of at least a portion of CEA gene and tetanus toxin fragment C merges, unless indicated this term to be meant protein sequence in the context clearly.Term " CEA-DOM fusion rotein " is meant by CEA-DOM fusions encoded polypeptides as described.Term

" CEA-DOM fusions " and " CEA-DOM fusion rotein " also is interpreted as those that are meant that its fragment, its homologue and its functional equivalent (collectively being called " variant "), for example one or more therein amino acid are inserted into, delete or replaced by other propylhomoserins.CEA-DOM fusions of the present invention, can stimulate the immune response of helper cell or cytotoxic T cell, or stimulate production of antibodies for example during human administration to Mammals, and its effect is suitable with " wild-type " CEA sequence at least.Of the present invention preferred embodiment in, CEA compares with wild-type, the CEA-DOM fusions can enhancing immunity reaction.

Abbreviation " AD " is meant CEA gene or proteic anchoring structure territory.The anchoring structure territory of the human CEA of wild-type is positioned at about amino acid 679 of SEQ ID NO:20 to about amino acid 702.

Term " treatment " is meant the measure of therapeutic treatment and preventative or preventing property.Need the object of treatment to comprise that lacked of proper care and that those tend to lack of proper care or those need prevent the object of lacking of proper care.

" imbalance " is to benefit from any situation of the treatment of molecule of the present invention, and described molecule comprises nucleic acid molecule described here and by the fusion rotein of described nucleic acid molecule encoding.Term " imbalance " is included is chronic and acute imbalance or disease, comprises that those make Mammals tend to the pathological conditions of the imbalance discussed.Molecule intention of the present invention is the imbalance of feature or the treatment of situation with opposing with the abnormal cell proliferation, and described imbalance or situation include but not limited to mammary cancer, colorectal carcinoma and lung cancer.

Term " significant quantity " is meant and imports the polypeptide that enough vaccine compositions produce adequate level, thereby produces the immune response result.Those skilled in the art will know that this level can change.

" conservative amino acid replacement " is meant that an amino-acid residue is by another chemically similarly amino-acid residue replacement.The example that this conservative property is replaced comprises: replace another with a hydrophobic residue (Isoleucine, leucine, Xie Ansuan or methionine(Met)); Or (for example, arginine is replaced Methionin to replace another with a polar residues of identical charges; L-glutamic acid is replaced aspartic acid).

" hCEA " and " hCEAopt " refers to human carcinomas embryonal antigen and human codon optimized carcinomebryonic antigen respectively.

" rhCEA " and " rhCEAopt " refers to rhesus monkey carcinomebryonic antigen and the codon optimized carcinomebryonic antigen of rhesus monkey respectively.

" substantially similar " meaning be given nucleic acid or aminoacid sequence and reference sequences enjoy at least 75%, preferred 85%, preferred 90%, preferred again 95% identity.In the present invention, reference sequences can be the relevant portion of human CEA Nucleotide of wild-type or aminoacid sequence, or the wild-type Nucleotide of bacteriotoxin or its subunit or the relevant portion of aminoacid sequence, described bacteriotoxin or its subunit be the LTB or the LTA subunit of E.coli heat labile enterotoxin for example, and be indicated as the context of text.Reference sequences also can be, for example, and wild-type rhesus monkey CEA sequence.Thereby, with the human CEA albumen of wild-type or its fragment " substantially similar " CEA protein sequence, to on segmental length, enjoy at least 75% identity with the associated clip of the human CEA of wild-type, preferred 85% identity, preferred 90% identity, preferred again 95% identity.Whether the albumen of given CEA, LTB or LTA or nucleotide sequence and reference sequences are " substantially similar ", for example can be by using sequence analysis software as determining from GAP computer program, version 6.0 comparative sequences information that University of Wisconsin GeneticsComputer Group (UWGCG) obtains.The GAP program has been utilized comparison method that revised by Smith and Waterman (Adv.Appl.Math.2:482,1981), Needleman and Wunsch (J.Mol.Biol.48:443,1970).

" entity part " of gene, its variant, fragment or subunit be meant reference sequences at least 50%, preferred 75%, preferred 90%, preferred 95% part again.

" gene " is meant a kind of nucleic acid molecule, its nucleotide sequence coded polypeptide molecule.Gene can be the continuous sequence of Nucleotide, and perhaps they can comprise fragment such as intron, promoter region, splice site and the tumor-necrosis factor glycoproteins of insertion.Gene can be RNA or DNA.Preferred gene is the gene of coding peptide of the present invention.

Term " nucleic acid " or " nucleic acid molecule " mean Yeast Nucleic Acid (RNA) or thymus nucleic acid (DNA), probe, oligonucleotide, its fragment or part and primer.DNA can be complementary DNA (cDNA) or genomic dna, the gene of the CEA fusion rotein of for example encoding.

" wild-type CEA " or " wild-type protein " or " wt albumen " is meant albumen or its variant of the amino-acid sequence that comprises natural generation.The aminoacid sequence of the human CEA of wild-type is shown in the accompanying drawing 7E (SEQ ID NO:20).The aminoacid sequence (WO 2004/072287, referring to accompanying drawing 7A-7B) of wild-type rhesus monkey CEA has been described previously.

" wild-type CEA gene " is meant the gene of the proteic nucleotide sequence of CEA that comprises the natural generation of encoding, described albumen comprises the albumen of human origin or the albumen that obtains from other organisms, other organisms include but not limited to other Mammalss, for example rat, mouse and rhesus monkey.The nucleotide sequence of human CEA gene is this area obtainable (above-mentioned).Also referring to Beauchemin et al., Mol.Cell.Biol.7:3221-3230 (1987); Zimmermanet al., Proc.Natl.Acad.Sci.USA 84:920-924 (1987); With Thompson et al.Proc.Natl.Acad.Sci.USA 84 (9): 2965-69 (1987).The nucleotides sequence of wild-type rhesus monkey gene is listed in shown in the accompanying drawing 7C-7D.

Term " Mammals " is meant any Mammals, comprises the mankind.

Abbreviation " Ag " is meant antigen.

Abbreviation " Ab " and " mAb " refers to antibody and monoclonal antibody respectively.

Abbreviation " ORF " is meant the open reading frame of gene.

Brief description of drawings

Accompanying drawing 1 has shown the synoptic diagram of the carrier of developing in this research.Coding CEA-LTA and the plasmid of CEA-LTB fusions and the essential characteristic of Ad carrier have been shown.Also shown Ad5 genomic oppositely terminal repetition (ITR).

Accompanying drawing 2 has shown the Nucleotide (SEQ ID NO:7, picture A) and the aminoacid sequence (SEQ ID NO:8, picture B) of exemplary hCEA-LTA fusions.Shown the LTA nucleotide sequence with runic.

Accompanying drawing 3 has shown the Nucleotide (SEQ ID NO:9, picture A) and the aminoacid sequence (SEQ ID NO:10, picture B) of exemplary hCEA-LTB fusions.Shown the LTB nucleotide sequence with runic.

Accompanying drawing 4 has shown the nucleotide sequence (SEQ IDNO:11) of exemplary hCEAopt-LTB fusions.Shown the LTB nucleotide sequence with runic.

Accompanying drawing 5 has shown Nucleotide (SEQ ID NO:12, picture A) and the aminoacid sequence (SEQ ID NO:13, picture B) that is called hCEAopt-LTBopt, the exemplary hCEA-LTB fusions of optimizing fully at this.LTB Nucleotide and aminoacid sequence have been shown with runic.The joint sequence that the clone's strategy that is adopted by fusion CEA and LTB sequence produces is a underscore.

Accompanying drawing 6 has shown Nucleotide (SEQ ID NO:14, picture A) and the aminoacid sequence (SEQ ID NO:15, picture B) that is called rhesus monkey CEA-LTB fusions rhCEAoptLTBopt, that optimize fully at this.LTB Nucleotide and aminoacid sequence have been shown with runic.The joint sequence that the clone's strategy that is adopted by fusion CEA and LTB sequence produces is a underscore.

Accompanying drawing 7 has shown nucleotide sequence (the picture A and the B of the wild type gene of coding rhesus monkey CEA, SEQ ID NO:16 and 17) and the aminoacid sequence of corresponding proteins (picture C and D, SEQ ID NO:18 and 19), as described (U.S.S.N.60/447,203) previously.Picture E has shown the aminoacid sequence (SEQ ID NO:20) of the human CEA of wild-type, its be previous the description (referring to, for example, U.S. Patent No. 5,274,087).

Accompanying drawing 8 has shown the comparison of CEA expression efficiency in different CEA construct cells transfected.Picture A has described with 3 μ g plasmids of the wild-type sequence that carries hCEA, hCEA-LTA and hCEA-LTB, together with the expression efficiency of 0.2 μ g plasmid pV1J/mEPO as the HeLa cell of tracer transfection.Picture B has shown the result who uses the similar transfection experiment of pV1J/hCEAopt and pV1J/hCEAopt-LTB.After the transfection three days by measuring the proteic quantity of CEA that occurs in the cell extract and, measuring expression efficiency by expressing this numerical standardization according to EPO.Data presented relates to the average CEA expression values of twice independent transfection.

Accompanying drawing 9 has shown the comparison of the expression efficiency of the different adenovirus recombination vectors of expressing CEA.Infect the HeLa cell with Ad/hCEAopt and Ad/hCEAopt-LTB with 100 and 1000 moi.Measure expression efficiency by the proteic quantity of CEA that the measurement infection discharged after three days in cell extract.Twice independent average CEA expression values that infects of data presented reflection.

Accompanying drawing 10 has shown the cell-mediated immunoreactive analysis that the different plasmid vectors by the human CEA of coding are caused.Organize the plasmid that Electricinjection 50 μ g indicate in the C57BL/6 mouse muscle (CEA, CEA-LTA fusions or CEA-LTB fusions) in 0 and 3 Wednesdays.The 4th group of mouse is with the mixture immunity of 25 μ g pV1J/hCEA-LTA and 25 μ g pV1J/hCEA-LTB.Picture A.Two weeks after the reinforcement comprise that by use the ELISPOT analysis is carried out to the splenocyte from single mouse (empty circles) in the peptide storehouse of intact proteins, measure the number of the IFN gamma-secretase T cell that is specific to CEA.Also indicated geometrical mean (solid diamond).Picture B has described to use peptide storehouse D that the splenocyte of concentrating from mice immunized is carried out painted result in the IFN gamma cells.Each group has been shown that non-specific IFN γ produces (DMSO).

Accompanying drawing 11 has shown the antibody titers with the plasmid DNA carrier mice immunized of coding CEA.By ELISA to the serum measurement of the single mouse of use by oneself plasmid pV1J/hCEA, pV1J/hCEA-LTA and pV1J/hCEA-LTB immunity at the proteic independent titre of the human CEA of purifying.Also shown mean value (solid diamond).

Accompanying drawing 12 has shown the cell-mediated immunoreactive analysis that the different plasmid vectors by coding CEA are caused.Group (accompanying drawing 4) with each 4 BALB/c mouse of as noted above the plasmid Electricinjection of indicating.Two weeks after the last injection comprise that by use the ELISPOT analysis is carried out to the splenocyte from single mouse (empty circles) in the peptide storehouse of intact proteins, measure the number of the IFN γ secretion T cell that is specific to CEA.Also indicated mean value (solid diamond).

Accompanying drawing 13 has shown the CEA specific C D8 that the different plasmid vectors by coding CEA are caused ⁺The analysis of t cell responses.With the aforesaid plasmid Electricinjection C57/DR4 mouse of indicating (referring to accompanying drawing 4).In last injection two weeks of back, use peptide storehouse D that the splenocyte of concentrating from mice immunized is carried out dyeing in the IFN gamma cells.Each group has been shown that non-specific IFN γ produces (DMSO).

Accompanying drawing 14 has shown the CEA specific C D8 that the different plasmid vectors by coding CEA are caused ⁺The analysis of t cell responses.With the aforesaid plasmid Electricinjection HHD mouse of indicating (referring to accompanying drawing 4).In last injection two weeks of back, use peptide storehouse B and D that the splenocyte of concentrating from mice immunized is carried out dyeing in the IFN gamma cells.Each group has been shown that non-specific IFN γ produces (DMSO).

Accompanying drawing 15 has shown with 5 cell-mediated and immune responses body fluid of the CEA transgenic mice (N=9) of Electricinjection immunity weekly of plasmid of indicating.The plasmid DNA of each vaccine inoculation i.m. injection total amount 50 μ g.Picture A.Two weeks after the last injection, by using peptide storehouse D the splenocyte from single mouse (circle) is carried out the cell intrinsic color, measure the number of the IFN γ secretion T cell that is specific to CEA.Also indicated the value (trilateral) of geometric mean.Picture B.By ELISA to each serum measurement of use by oneself plasmid pV1J/hCEAopt and pV1J/hCEA-LTB mice immunized at the proteic independent titre of the human CEA of purifying.The value (solid diamond) that has also shown geometric mean.These data show that the CEA-LTB fusions has destroyed the tolerance to CEA in transgenic mice.

Accompanying drawing 16 has shown the CEA specific C D8 that the different adenovirus carriers by coding CEA are caused ⁺The analysis of t cell responses.Ad/hCEAopt and Ad/CEAopt-LTB immunity CEA transgenic mice in 0 and 2 weeks with various dose.In two weeks after the last injection, use peptide storehouse D (solid circles) that the PBMC from each mice immunized is carried out IFN gamma cells intrinsic color.The value (solid diamond) that has also shown flat geometric mean.The non-specific IFN γ of the group of each injection produces (DMSO) and is less than or equal to 0.01%.

Accompanying drawing 17 has shown the result to the tumor protection research of the CEA transgenic mice of the immunity of attacking with the MC38-CEA cell.The group of each 10 CEA transgenic mice with the plasmid DNA of indicating (50 μ g/ injection) 5 times weekly Electricinjection carry out immunity.In two weeks after the last DNA injection, mouse is with 1 * 10 ¹⁰The corresponding Ad carrier single injection of vp is strengthened.After adenovirus is strengthened 14 days are with 5 * 10 ⁵Mouse is attacked in individual MC38-CEA cell subcutaneous injection.Picture A has shown the per-cent that does not have mice with tumor at the time point of indicating.Picture B has reported the mean tumour volume of the group of each immunity.These data show, have protected mouse to avoid tumor development with CEA-LTB immunity CEA transgenic mice.

Accompanying drawing 18. picture A have shown the synoptic diagram of the representative CEA fusion rotein that uses in this research.The carrier of expressing the CEA fusion rotein derives from the plasmid pV1Jns that describes among the embodiment 2.This construct comprises the CEA nucleotide sequence from nt1 to nt2037, has the clean deletion corresponding to 64 aa of GPI anchor series, and the aa1 of expression CEA is to aa679.Also indicated each the proteic sequence coordinate that is fused to CEA.Picture B has shown the expression of pV1J deutero-construct in cells transfected.As embodiment 5 describe with plasmid pV1J/CEA-VSV-G, pV1J/CEA-FcIgG, pV1J/CEA-DOM, pV1J/CEA-HSP70, pV1J/CEA-LAMP or pV1J/CEA transfection HeLa cell, handle and be used for the Western engram analysis.Indicated the specificity of the antibody that is used for the Western trace.Indicated CEA albumen (black arrow).The position that has also shown molecular size standard substance (kilodalton).

Accompanying drawing 19 has shown the comparison of the expression efficiency of CEA fusions construct.As the plasmid transfection HeLa cell that the usefulness of embodiment 8 descriptions is indicated, measure the CEA derived protein that in cellular lysate (A) and supernatant liquor (B), occurs by ELISA.The result who obtains is the representative of two independent experiments.

Accompanying drawing 20 has shown the immunogenic comparison of the different constructs of coding CEA fusion rotein.The plasmid intramuscular electroporation C57BL/6 mouse of indicating with 5 or 50 μ g/ agent.Injected at the 0th and 14 day.Picture A describes as embodiment 6 and 15, uses the storehouse of the peptide that covers aa497-703 (storehouse D) to measure the quantity that the IFN γ among the PBMC secretes the T cell in each independent mouse.The average quantity (solid circles) that has also shown IFN γ secretion T cell.The SFC value of pV1J/CEA-DOM and pV1J/CEA-FcIgG is different from the value of pV1J/CEA significantly.Picture B.Use the CEA of purifying to measure antibody titers by ELISA as substrate.Shown mean value with each group of the plasmid immunity of indicating of 50 μ g dosage.Mark with an asterisk with those significantly different titres of the mouse of injecting pV1J/CEA.

Accompanying drawing 21 has shown inducing of in CEA transgenic mice CEA specific immune response.The group of each 12 CEA transgenic mice plasmid DNA (50 μ g/ agent Electricinjection in musculus quadriceps) or the adenovirus carrier (10 of the cDNA of the codon utilization ratio optimization of carrying CEA, CEA-DOM or CEA-FcIgG ⁹The vp/ agent) carries out immunity.Carrying out IFN γ in the cell by the PBMC to each mice immunized dyes and measures the CEA specific C D8 that is caused by DNA/DNA (A) and Ad/Ad (C) immunization ways ⁺The T cell.The mean value (solid circles) that has also shown each group.CEA-DOM and CEA-FcIgG group with DNA/DNA and the immunity of Ad/Ad mode are different from the group that CEA inoculates significantly.Measure the CEA specific antibody titre of each the single mouse that inoculates with DNA/DNA (B) or Ad/Ad (D) immunization ways by ELISA.The titre that CEA-DOM and CEA-FcIgG carrier cause is different from the titre that CEA causes significantly.

Accompanying drawing 22 has shown the immunogenicity of DNA/Ad mode (regimen).The group of each 12 CEA transgenic mice plasmid DNA (50 μ g/ agent) and adenovirus carrier (10 ⁹The vp/ agent) carry out immunity, described plasmid DNA and adenovirus carrier carry the cDNA of the codon utilization ratio optimization of CEA, CEA-DOM or CEA-FcIgG.Carrying out in the cell IFN γ by the PBMC to each mice immunized dyes and measures CEA specific C D8 ⁺T cell (A).The mean value (solid circles) that has also shown each group.CEA-DOM and CEA-FcIgG group are different from the group of CEA inoculation significantly.Measure the CEA specific antibody titre (B) of each independent mouse by ELISA.The titre that CEA-DOM and CEA-FcIgG carrier cause is different from the titre that CEA causes significantly.Shown mean value (solid circles).

Accompanying drawing 23 has shown the CD4 that detects at tetanus toxoid protein ⁺T cell responses.As the usefulness pV1J/CEA-DOMopt immunity CEA transgenic mice of describing among the embodiment 16.With peptide p30 the PBMC that concentrates from mice immunized is carried out dyeing in the IFN gamma cells.Whole lymphocytic gatings (gating) have been shown and to CD8 ⁺(R3) and CD4 ⁺The gating of T cell (R4).

Accompanying drawing 24 has shown the antitumous effect that the carrier with the codon optimized cDNA that carries CEA, CEA-DOM or CEA-FcIgG carries out vaccine inoculation.As described in Example 18, use the cDNA that the codon utilization ratio carry CEA, CEA-DOM or CEA-FcIgG optimizes plasmid DNA and Ad carrier, with DNA/DNA (A), Ad/Ad (B) and DNA/Ad (C) vaccine inoculation mode the group of each 10 CEA transgenic mice is carried out immunity.Two weeks after the last injection are with 5 * 10 ⁵MC38-CEA tumour cell sc inoculates and attacks mouse.The per-cent of no mice with tumor in the group of weekly mensuration immunization is with comparing of untreated contrast.Mouse (DNA/Ad form) with CEA-DOM carrier immunization is different from control mice (log rank tests p＜0.05) significantly.

Accompanying drawing 25 has shown that CD4, CDS or NK exhaust the antitumous effect inductive influence by CEA-DOM DNA/Ad immune induction.Repeat to inject weekly with 50 μ g pV1J/CEA-DOMopt, use 1 * 10 subsequently ⁹The Ad-CEA-DOMopt of vp strengthens, and comes immune CEA transgenic mice (embodiment 19).After one week of last injection, do not exhaust or exhaust the CD4 of mouse ⁺T cell, CD8 ⁺T cell or NK cell.Two weeks after the last immunity are with 5 * 10 ⁵MC38-CEA tumour cell sc inoculates and attacks mouse.The per-cent of no mice with tumor compares with untreated contrast in the group of weekly mensuration immunization.Data show, the group that the per-cent of the no mice with tumor in inoculation group is different from untreated contrast significantly and exhausts.

Accompanying drawing 26 has shown the nucleotide sequence (SEQ ID NO:21) that is called hCEAoptDOMopt, the exemplary hCEA-DOM fusions of optimizing fully at this.The aminoacid sequence (SEQ ID NO:45) that has also shown encoded protein.The CEA part of the nucleotide sequence of this specific CEA fusions is made of Nucleotide 1 to 2037, and it is codon optimized for high level expression in human host cell.With the DOM part of runic demonstration nucleotide sequence, also codon optimized for high level expression in the human cell.The joint sequence that the clone's strategy that is adopted by fusion CEA and LTB sequence produces is a underscore.

Accompanying drawing 27 has shown at this and has been called exemplary nucleotide sequence hCEAoptFcIgGopt, the hCEA-FcIgGopt fusions (SEQ ID NO:25).The sequence (SEQ ID NO:46) that has also shown encoded protein.The CEA part of the nucleotide sequence of this specific CEA fusions is made of Nucleotide 1 to 2037, and it is codon optimized for high level expression in human host cell.With the FcIgG part of runic demonstration nucleotide sequence, it is codon optimized for high level expression in the human cell.The joint sequence that the clone's strategy that is adopted by fusion CEA and LTB sequence produces is a underscore.

Accompanying drawing 28 has shown nucleotide sequence (the SEQ ID NO:22 of the part of the human CEA cDNA of the human CEA cDNA of the wild-type from nt1 to nt2037 wild-type, picture A), the proteic part of the hCEA of coding from aa1 to aa679 (SEQ ID NO:23, picture B).

Accompanying drawing 29 has shown the unoptimizable nucleotide sequence that minimizes structural domain (DOM) cDNA (SEQ ID NO:47) of the tetanus toxin fragment C from nt1 to nt825, and encoding D OM albumen also shows (SEQ ID NO:48).

Accompanying drawing 30 has shown the unoptimizable nucleotide sequence (SEQ ID NO:49) of exemplary human CEA-DOM fusions.The CEA of the nucleotide sequence of this specific CEA fusions part is made up of to 2037 Nucleotide 1.The DOM part of this nucleotide sequence shows with runic.

Accompanying drawing 31 has shown at this and has been called exemplary nucleotide sequence rhCEA-DOMopt, rhesus monkey CEA-DOM fusions (SEQ ID NO:50).The sequence (SEQ ID NO:51) that has also shown the fusion rotein of coding.The CEA part of the nucleotide sequence of this specific CEA fusions is made up of Nucleotide 1 to 2037, and it is codon optimized for high level expression in human host cell.With the DOM part of runic demonstration nucleotide sequence, it is codon optimized for high level expression in the human cell.

Accompanying drawing 32 has shown at this and has been called exemplary nucleotide sequence rhCEA-CTBopt, rhesus monkey CEA-CTB fusions (SEQ ID NO:52).The sequence (SEQ ID NO:53) that has also shown the fusion rotein of coding.The CEA part of the nucleotide sequence of this specific CEA fusions is made up of Nucleotide 1 to 2037, and it is codon optimized for high level expression in human host cell.With the CTB part of runic demonstration nucleotide sequence, it is codon optimized for high level expression in the human cell.

Detailed description of the invention

Carcinomebryonic antigen (CEA) is relevant with the development of gland cancer usually.The present invention relates to composition and method, come to cause or strengthen immunity at expressed protein product by the CEA tumor associated antigen, wherein unusual CEA expression is relevant with cancer or its development.Unusually the CEA expression does not need CEA albumen all to express in tumor tissues at any time point of tumor development with the dependency of cancer, because expressing, unusual CEA may and can not detect in the appearance of tumour initial period in the late period of tumor development, otherwise or.

For this reason, the invention provides the polynucleotide, carrier, host cell and the encoded protein that comprise CEA sequence or its variant, be used for vaccine and pharmaceutical composition and be used for treatment for cancer and/or prevention.Polynucleotide of the present invention comprise the nucleotide sequence of coding CEA albumen or its variant, be fused to the nucleotide sequence of at least one subunit of coding immunostimulant element, described immunostimulant element is bacterial enterotoxin or its entity part for example, and it can assist a ruler in governing a country the immune response at relevant CEA effectively.

CEA nucleotide sequence of the present invention can be a human origin, maybe can be the CEA homologue from other species.Reported the human CEA nucleotide sequence of wild-type (referring to, for example, U.S. Patent No. 5,274,087; US Patent No 5,571,710; With US Patent No 5,843,761).Rhesus monkey CEA sequence (WO 2004/072287) has been described recently.The CEA part of CEA fusions can be a total length, or is enough to cause in Mammals any variant of CEA specific immune response.CEA variant of the present invention include, but are not limited to the terminal brachymemma of C-or N-sequence, have the sequence that conservative property replaces and have the inner deletion or the sequence of insertion.

Of the present invention preferred embodiment in, the CEA of CEA fusions partly is human CEA or its function equivalent.Other preferred embodiment in, described CEA partly is rhesus monkey CEA, or its function equivalent.

Therefore, the present invention relates to comprise the synthetic polyribonucleotides of the nucleotide sequence of coding CEA fusion rotein, described fusion rotein comprises CEA albumen or proteic biological active fragment of CEA or the mutant form that merges with immunostimulant element or its subunit, and described immunostimulant element or its subunit can strengthen the proteic immune response to CEA effectively.The proteic mutant form of described CEA includes but not limited to: conservative amino acid is replaced, N-terminal blocks, C-terminal blocks, delete or add.Any such biological active fragment and/or mutant are with proteins encoded or protein fragments, and it has simulated the proteic immunological properties of listed CEA among the SEQ ID NO:20 at least basically.Synthetic polyribonucleotides coding of the present invention is expressed the mRNA molecule of functional CEA fusion rotein, thereby is useful in the exploitation of therapeutic or preventative cancer vaccine.

Of the present invention preferred embodiment in, the CEA of the CEA fusion rotein of coding partly is human CEA (SEQ ID NO:20) or its function equivalent, for example, deleted the human CEA (SEQ ID NO:23) in its C-end anchors fixed structure territory (AD), described C-end anchors fixed structure territory is positioned at about amino acid 679 of human total length CEA to about amino acid 702.Without being limited by theory, the secretion of CEA fusion rotein has been improved in deletion anchoring structure territory, thereby has strengthened the immunoreactive cross-excitation of CEA-LTB (priming).Other preferred embodiment in, described CEA partly is rhesus monkey CEA (SEQ ID NO:18 and 19), or its function equivalent.

The immunostimulant componentry of CEA fusion rotein of the present invention can stimulate or strengthen the proteic immune response of CEA that links to each other, and is selected from by the heavy fragment (FcIgG) of the constant chain of the N-end structure territory (DOM) of fragment C (FrC), the FrC of heat shock protein (HSP) 70, lysosome related membrane protein (LAMP), Toxoid,tetanus, immunoglobulin G 1, vesicular stomatitis virus glycoprotein (VSV-G), from the group of heat labile enterotoxin (LT) formation of the Toxins,exo-, cholera (CT) of Vibrio cholerae and E.coli.Of the present invention preferred embodiment in, the adjuvant of CEA fusions partly comprises the N-end structure territory (DOM) of FrC, it has shown strengthens the immunogenicity of antigens of sending jointly consumingly.In further preferred embodiment, the adjuvant of CEA fusions partly comprises subunit or its entity part of LT.Further preferred embodiment in, the adjuvant of CEA fusions partly is the entity part of FcIgG.

The CEA fusions (Lund et al. Cancer Gene Therapy10:365-376 (2003)) of the single epi-position (Q830-L844) of human CEA that comprises brachymemma and the tetanus toxin that merges has with it been described.Do not resemble this single epi-position fusions, CEA fusions of the present invention comprises the entity part of immunostimulant element or its subunit, and as mentioned above, it can strengthen the immunogenicity of CEA albumen or its variant.The entity part that is used for the immunostimulant element of composition described here and method does not comprise 50% the part that is less than total length toxin subunit.Employing strategy as used herein guarantees that this strategy has utilized total length adjuvant subunit or its entity part at the better immune response of the CEA sequence that merges.Without being limited by theory, what believe is, comprises greater than an auxiliary epi-position if be elected to be the bacteriotoxin of adjuvant, the toxin sequence of fusion rotein is restricted to single epi-position will causes effect to the immunogenic reduction of target protein demonstrably.In addition, what believe is, if the interaction of adjuvant and specific cells acceptor is depended in the immune response enhancing of adjuvant mediation rather than based on general epi-position, then acceptor interaction may depend on specific structure conformation, this will need the entity part of immunostimulant element to bring into play adjuvant effect.In this case, the adjuvant sequence that comprises the weak point of single epi-position will be not enough to mediated immune reaction and improve.

Also considering to be used for of the present invention is the variant of coding immunostimulant element described here or the nucleotide sequence of mutant, including but not necessarily limited to: Nucleotide is replaced, deletes, is added N-terminal brachymemma and C-terminal brachymemma.In some cases, add to the nucleotide sequence of coding adjuvant or its subunit that specific point mutation reduces or the toxicity of eliminating encoded protein may be useful.In the illustrative embodiments in this aspect of the invention, the LT subunit is fused to the CEA sequence of CEA fusions, wherein the brachymemma of LT subunit its signal sequence.Without being limited by theory, the toxin signal sequence is the deletion of LTB signal sequence for example, has guaranteed that the translation post-treatment of CEA fusions is driven by the CEA signal sequence.

Described immunostimulant element, subunit or its entity part can merge with the N-terminal or the C-terminal of CEA sequence.Further, immunostimulant element sequences and CEA sequence can merge to N-to N-end or N-end to C-end, C-end to N-end, C-end by the N-end terminally.Of the present invention preferred embodiment in, the terminal fusion of the terminal N-of the C-of CEA polypeptide with the immunostimulant element.

The present invention relates to comprise the synthetic nucleic acid molecule (polynucleotide) of the sequence of Nucleotide, the mRNA of new CEA fusion rotein is expressed in the sequence encoding of described Nucleotide; For example, the nucleotide sequence of listed fusion rotein among the coding SEQ ID NO:8,10,13,15,45,46,51 and 53.Nucleic acid molecule of the present invention is substantially free of other nucleic acid.

The invention still further relates to the recombinant vectors and the recombinant host cell of protokaryon and eucaryon, they contain disclosed nucleic acid molecule in whole specification sheets.Synthetic dna molecular of the present invention, relevant carrier and host are useful for the exploitation of cancer vaccine.

Exemplary core acid molecule of the present invention comprises the nucleotide sequence that is selected from by SEQ ID NO:7,9,11,12,14,21,25,49,50 and 52 groups that constitute, shown in accompanying drawing 2-6,26-27,30-32, its encode exemplary CEA-LTA of the present invention, CEA-LTB, CEA-DOM, CEA-FcIgG and CEA-CTB fusion rotein.

The present invention also comprises SEQ ID NO:7,9,11,12,14,21,25,49,50 and 52 biological active fragment or mutant, and its coding is expressed the mRNA of exemplary CEA fusion rotein.Any such biological active fragment and/or mutant are with proteins encoded or protein fragments, and it has simulated the proteic pharmacological property of hCEA at least basically, and described hCEA albumen includes but not limited to hCEA albumen listed among the SEQ ID NO:20.Any such polynucleotide are including but not necessarily limited to Nucleotide replacement, deletion, interpolation, N-terminal brachymemma and C-terminal brachymemma.The mRNA molecule of mutant code of the present invention expressive function CEA fusion rotein in eukaryotic cell, thus in the exploitation of cancer vaccine, be useful.

Also comprise within the scope of the invention be the sudden change that does not change basically in the dna sequence dna of final physical character of expressed proteins.For example, Xie Ansuan may not can cause the variation of the function aspects of polypeptide to the replacement of glutamine to Methionin or l-asparagine to leucine, arginine.

As mentioned above, the invention further relates to recombinant vectors, it comprises disclosed nucleic acid molecule in the whole specification sheets.These carriers can be made up of DNA or RNA.For great majority clone purpose, dna vector is preferred.Typical carrier comprises virus, baculovirus, phage, clay, the yeast artificial chromosome of plasmid, modification, the DNA of the other forms of free or integration of the CEA fusion rotein of maybe can encoding.Be in and in those skilled in the art's limit of power be, special genes is shifted or other purposes are determined suitable carriers.

The present invention also provides the CEA fusion rotein by the purifying of disclosed nucleic acid encoding in whole specification sheets.In the illustrative embodiments in this aspect of the invention, described CEA fusion rotein comprises the aminoacid sequence that is selected from by SEQ ID NO:8,10,13,15,45,46,51 and 53 groups that constitute.

Be included in the present invention be under stringent condition with the dna sequence dnas of SEQ ID NO:7,9,11,12,14,21,25,49,50 or 52 hybridization.For instance, but not restriction, use the process of high stringent condition as follows.The prehybridization that contains the filter membrane of DNA carries out about 2 hours to spending the night at about 65 ℃ in damping fluid, described damping fluid is by 6 * SSC, and the salmon sperm dna of 5 * Denhardt ' s solution and 100 μ g/ml sex change is formed.In salmon sperm dna that contains 100 μ g/ml sex change and 5-20 * 10 ⁶Cpm's ³²In the prehybridization mixture of the probe of P mark, made filter hybridization about 12 to 48 hours at 65 ℃.The washing of filter membrane was carried out under 37 ℃ in the solution that contains 2 * SSC, 0.1%SDS about 1 hour.In 0.1 * SSC, 0.1%SDS, washing 45 minutes before the radioautograph subsequently at 50 ℃.Use other processes of high stringent condition to comprise, carried out hybridization step about 12 to 48 hours at about 42 ℃, or in 0.2 * SSPE, 0.2%SDS, washed about 30 to 60 minutes at about 65 ℃ at 5 * SSC, 5 * Denhardt ' s solution, 50% methane amide.The reagent that being used to of mentioning in said process carried out high strict hybridization is well known in the art.Can be at Sambrook et al., Molecular Cloning:ALaboratory Manual 2 ^NdEdition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1989) or Sambrook and Russell, Molecular Cloning:A Laboratory Manual, 3 ^RdEdition.Cold SpringHarbor Laboratory Press, Plainview finds the details of the composition of these reagent among the NY (2001).Except that above-mentioned, operable other high stringent conditions also are well known in the art.

The expression vector that contains CEA fusion rotein coding nucleic acid molecule is used in high level expression CEA fusion rotein in the recombinant host cell.Expression vector can include, but not limited to the cloning vector of cloning vector, modification, custom-designed plasmid or virus.And if expectation, various bacterial expression vectors can be used in bacterial cell express recombinant CEA fusions sequence.In addition, various fungal cell's expression vectors can be used in fungal cell's express recombinant CEA fusions sequence.Further, various insect cell expression carriers can be used at the expressed in insect cells recombinant protein.

The invention still further relates to the carrier conversion that comprises nucleic acid molecule of the present invention or the host cell of transfection.Recombinant host cell can be protokaryon or eucaryon, includes but not limited to, bacterium is E.coli for example, and the fungal cell is yeast for example, and mammalian cell includes but not limited to, the clone in ox, pig, monkey and rodent source; And insect cell, include but not limited to fruit bat and silkworm deutero-clone.This recombinant host cell can be cultivated under the condition that is fit to produce CEA fusion rotein or biology equivalents.Of the present invention preferred embodiment in, host cell is human.As defined herein, the host cell in the transgenosis mankind's health, human foetus or the human embryos do not planned to be included in term " host cell ".

As mentioned above, the expression vector that contains the DNA of coding CEA fusion rotein is used in and expresses the CEA fusion rotein in the recombinant host cell.Therefore, another aspect of the present invention is to express the method for CEA fusion rotein in recombinant host cell, comprise: (a) in the human host cell that is fit to, import the carrier that comprises nucleic acid, described nucleic acid comprises the sequence of the Nucleotide of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, the entity part of itself and immunostimulant element or its subunit merges, and wherein said immunostimulant element or its subunit are selected from by heat shock protein (HSP) 70, lysosome related membrane protein (LAMP), the fragment C of Toxoid,tetanus (FrC), the N-end structure territory (DOM) of FrC, the heavy fragment (FcIgG) of the constant chain of immunoglobulin G 1, vesicular stomatitis virus glycoprotein (VSV-G), the group that constitutes from the heat labile enterotoxin (LT) of the Toxins,exo-, cholera (CT) of Vibrio cholerae and E.coli; Can in Mammals, produce immune response with wherein said fusion rotein; And, (b) under the condition that allows the described CEA fusion rotein of expression, cultivate described host cell.

The preferred immunostimulant element that is used for this aspect of the present invention is selected from the group that is made of LTB, LTA, DOM and FcIgG.

In the further preferred embodiment in this aspect of the invention, the nucleotide sequence of the immunostimulant componentry of the CEA of described fusions part and/or described fusions is codon optimized for high level expression in the human cell.

The present invention also provides the method for expressing the CEA-LT fusion rotein in recombinant host cell, comprise: (a) in the human host cell that is fit to, import the carrier that comprises nucleic acid, described nucleic acid comprises the sequence of the Nucleotide of coding CEA-LT fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, its entity part and wherein said fusion rotein that is fused to the LT subunit can produce immune response in Mammals; And, (b) under the condition that allows the described CEA-LT fusion rotein of expression, cultivate described host cell.

The method of expressing the CEA-LT fusion rotein as mentioned above preferred embodiment in, described LT subunit is the entity part of LTB, wherein said LTB sequence has been deleted its signal sequence.In other embodiments, described LT subunit is LTA, or its entity part.

The present invention also provides the method for expressing the CEA-DOM fusion rotein in recombinant host cell, comprise: (a) in the human host cell that is fit to, import the carrier that comprises nucleic acid, described nucleic acid comprises the sequence of the Nucleotide of coding CEA-DOM fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, its entity part and wherein said fusion rotein in N-end structure territory (DOM) that is fused to the fragment C of tetanus toxin can produce immune response in Mammals; And, (b) under the condition that allows the described CEA-DOM fusion rotein of expression, cultivate described host cell.

The method of expressing the CEA-DOM fusion rotein as mentioned above preferred embodiment in, described DOM is partly codon optimized for high level expression in the human cell.Other preferred embodiment in, the CEA of described CEA fusions is partly codon optimized for high level expression in the human cell.Further preferred embodiment in, CEA and DOM part is all codon optimized for high level expression in the human cell.

In host cell, express after the CEA fusions, can reclaim the CEA fusion rotein and provide CEA fusion rotein with activity form.Some protein purification processes are available and suitable.Various combinations that can be by salt classification, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography or independent should be used for purification of recombinant proteins from cellular lysate and extract.In addition, by using, recombinant C EA fusion rotein and other cell proteins can be separated by mono-clonal that is specific to CEA albumen or the proteic polypeptide fragment of CEA or immune affinity column that polyclonal antibody is made.

With respect to the full-length cDNA of coding CEA, the nucleic acid molecule of the CEA of comprising fusions of the present invention and coded fusion rotein are to be designed to strengthen the CEA specific immune response, are used for vaccine development.In order further to strengthen the immunogenic properties of CEA fusions sequence of the present invention, in some embodiment described here, the polynucleotide of coding CEA fusion rotein are included as in host cell further high level expression and the codon optimized, and are as described below.In these embodiments, at least a portion of the codon of design CEA fusions is with the codon of the host cell preference of application plan, and host cell is the human cell described in preferred embodiment.The CEA fusions of optimizing can be used to develop the dna vaccination based on recombinant adenovirus or plasmid, and it provides effective immunoprophylaxis at the CEA associated cancer by neutralizing antibody and cell-mediated immunity.Synthetic molecules can be used as immunogenic composition.The invention provides codon optimized CEA fusions polynucleotide, induce coded proteic expression in described animal when it is directly imported in the vertebrates body, described vertebrates comprises Mammals, for example the primates and the mankind.

As mentioned above, In some embodiments of the present invention, synthetic molecules comprises the sequence of Nucleotide, and some of them Nucleotide has been changed the codon with end user's class cell preference, thereby allows high-caliber expressing fusion protein in human host cell.Synthetic molecules for example can be used as the CEA fusion rotein, the source of CEA-LTB fusion rotein, and described fusion rotein can be used for cancer vaccine so that the effective immunoprophylaxis at the CEA associated cancer to be provided by neutralizing antibody and cell-mediated immunity.Nucleic acid molecule disclosed herein also can be used as the basis based on the cancer vaccine of DNA.

" triplet " codon of four kinds of possible nucleotide bases can exist and surpass 60 kinds of versions.Because these codons provide the only information of 20 kinds of different aminoacids (and transcription initiation and termination), some amino acid can be a kind of phenomenon that is called the codon redundancy by surpassing a codon coding.Because the reason that some is not exclusively understood, in the interior source DNA of dissimilar cells, selectable codon does not occur equably.In fact, in the cell of some type, as if there are variable natural grade (hierarchy) or " preference " for some codon.As an example, the amino acid leucine comprises CTA, CTC, CTG, CTT, TTA and TTG by any one appointment of six kinds of DNA codons.Exhaustive analysis to the genome codon frequency of microorganism has disclosed, and the interior source DNA of E.coli the most normally contains CTG leucine-designated pin, and the DNA of yeast and sline cungi the most normally comprises TTA leucine appointment-codon.In view of this grade, generally believe that the possibility that is rich in the high level expression of leucine polypeptide by E.coli host's acquisition will be somewhat dependent upon the frequency that codon uses.For example, possible is, the gene that is rich in the TTA codon will be expressed in E.coli indigently, and the gene that is rich in CTG may be expressed at this host's camber.Similarly, be TTA in yeast host cell, expressing the preferred codon that is rich in the leucine polypeptide.

Codon preference phenomenon is tangible to the meaning of recombinant DNA technology, this phenomenon can be used for explaining previous in successful host transformed organism, realize the foreign gene high level expression many failures-more the codon of " preference " may repeatedly not occur in the gene that inserts, the host cell mechanism that is used to express may not operate effectively.This phenomenon has been pointed out, and has been designed to comprise the synthetic gene of codon of the host cell preference of plan, and the exogenous genetic material of optimised form for the practice of recombinant DNA technology is provided.Thereby one aspect of the present invention is the CEA fusions gene for expressing codon optimized in the human cell.In the preferred embodiment of the present invention, have been found that the selectable codon of use coding same protein sequence can be eliminated express the restriction of external source CEA fusion rotein in the human cell.

According to some embodiment of the present invention, convert the nucleic acid molecule of coding CEA fusion rotein to have identical translation sequences but have alternative codon utilization polynucleotide sequence, as Lathe, " Synthetic Oligonucleotide Probes Deduced from Amino AcidSequence Data:Theoretical and Practical Considerations " J.Molec.Bio.183:1-12 (1985) is described, by reference it is incorporated in this.This method generally comprises in wild-type sequence to be identified and the common incoherent codon of Human genome of highly expressing, and be used in best codon replacement that human cell's camber expresses they.Check then in this new gene order by these codons replace the sequence of not expecting that produces (for example, " ATTTA " sequence, being not intended to of intron montage recognition site produces, unwanted restriction endonuclease sites, or the like).Eliminate the sequence of not expecting by replacing existing codon with the different codons of coding same amino acid.Test the expression of the segmental improvement of synthetic gene then.

Aforesaid method is used to produce the synthetic gene sequence of coding CEA fusion rotein, produces the gene of the codon that is included as high level expression and optimizes.Though above-mentioned process provides the general introduction of the method for the codon optimized gene that we are designed for cancer vaccine, what it will be understood by those skilled in the art that is that the expression of the raising of similar vaccine potency or gene can realize by the minor alteration in the process or by the minor alteration in the sequence.

Those skilled in the art will recognize that the high-level CEA fusions that the nucleic acid molecule that can make up other is provided among the human cell is expressed, wherein the only part of the codon of this dna molecular is codon optimized.For example, In some embodiments of the present invention, the codon that is contained in the CEA part of CEA fusions is optimized for high level expression in the human cell, and the codon that is contained in the adjuvant part of CEA fusions is substantially similar to the nucleotide sequence of wild-type coding adjuvant.In other embodiments of the present invention, the codon that is contained in the adjuvant part of CEA fusions is optimized for high level expression in the human cell, and the codon that is contained in the CEA part of CEA fusions is substantially similar to wild-type CEA gene.In other embodiment more of the present invention, the CEA of CEA fusions and adjuvant part are all codon optimized for high level expression in the human cell.Only at the CEA of CEA fusions and/or the optimised CEA fusions of subclass of the codon in the adjuvant part, also be that the present invention considers therein.

Nucleic acid of the present invention can be mounted in the expression cassette, and described expression cassette comprises the sequence that is designed to be provided at the effective protein expression among the human cell.Described box preferably contains CEA fusion rotein encoding gene, has with it can be operatively connected relevant to transcribe and translate control sequence, for example promotor and terminator sequence.In preferred embodiment, described promotor is the cytomegalovirus promoter (CMV) that does not have the intron A sequence, and person of skill in the art will appreciate that, can use any in for example strong immunoglobulin (Ig) of many other known promotors or other eukaryotic gene promotors.Preferred transcription terminator is the Trobest terminator, yet also can use other known transcription terminators.The combination of CMV-BGH terminator is particularly preferred.

According to the present invention, CEA fusions expression cassette is inserted in the carrier.Carrier is adenovirus or plasmid vector preferably, yet also can use the linear DNA that links to each other with promotor, or other carriers, for example the vaccinia virus of adeno-associated virus (AAV) or modification, retroviral or lentiviral vectors.

If the carrier of selecting is an adenovirus, preferably, this carrier is so-called first-generation adenovirus carrier.The feature of these adenovirus carriers is the adenovirus E 1 gene regions that have non-functional E1 gene region and preferably have deletion.At some embodiment, described expression cassette is inserted into the common residing position of adenovirus E 1 gene.In addition, these carriers are optional has E3 zone non-functional or deletion.Preferably, the adenoviral gene group of use is deleted E1 and E3 zone (Δ E1 Δ E3).This adenovirus can be bred in expressing the known clone of viral E1 gene, for example 293 cells or PERC.6 cell, or by of short duration or stable conversion with express extra proteic, derive from 293 or the clone of PERC.6 cell in breed.For example, when use has the construct of controlled genetic expression, for example during the adjustable promoter systems of tsiklomitsin, described clone can be expressed in the composition that relates in this regulation system.An example of this clone is T-Rex-293; Other be known in the art.

Operate adenovirus carrier for convenience, adenovirus can be the shuttle plasmid form.The invention still further relates to shuttle vector, it comprises plasmid part and adenovirus part, described adenovirus partly comprises the adenoviral gene group of E1 with deletion and the E3 deletion of choosing wantonly, and has the expression cassette of the insertion that comprises CEA fusion rotein coding nucleotide sequence.In preferred embodiment, there be the restriction site of flank, thereby can easily remove adenovirus carrier in the adenovirus part of plasmid.Shuttle plasmid can duplicate in prokaryotic cell prokaryocyte or eukaryotic cell.

Of the present invention preferred embodiment in, expression cassette is inserted into (referring to Emini et al., WO 02/22080, by reference it is incorporated in this) in the pMRKAd5-HV0 adenoviral plasmid.This plasmid comprises the Ad5 genome of having deleted E1 and E3 zone.Be considered to cause the enhanced virus amplification, and improved the design of pMRKAd5-HV0 plasmid with respect to previous adenovirus carrier by 5 ' cis acting packaging area being further expanded to mix in the E1 gene at important element aspect the viral packing of optimization.Advantageously, this enhanced adenovirus carrier can be kept genetic stability after high pass is for propagation.

The standard molecular biological technology of preparation and purify DNA construct allows preparation adenovirus of the present invention, shuttle plasmid and dna immunization former.

What determine according to the present invention is, CEA-LT fusion rotein coding molecule described here (promptly, SEQ ID NO:12), it comprises the entity part of the LTA or the LTB subunit of E.coli heat labile enterotoxin, compares with corresponding wild type CEA sequence with the efficient that is equal to and expresses (referring to embodiment 4).Shown also that at this plasmid pV1J/hCEA-LTA and pV1J/hCEA-LTB have caused the antibody response bigger than pV1J/hCEA, confirmed the adjuvant effect (referring to embodiment 11) of LT subunit the performance of CEA specific immune response.Thereby digital proof described here, the fusion of CEA encoding sequence and LTA or LTB cDNA has caused the raising of CEA specific immune response.Seem that LTB has brought into play better reinforced effects to immune response, mainly induce CD8 ⁺The T cell, and that the LTA initiation mainly is CD4 ⁺Reaction.

The immunogenic properties of the raising of CEA-LTB fusions also shown according to the present invention, owing to respect to total length wild-type CEA cDNA, can more effectively be destroyed the tolerance to the CEA self antigen.When the plasmid of complete codon optimized cDNA of CEA-LTB fusions is carried in injection, also observe the reinforced effects of LTB to the immunogenic properties of CEA.At last, use the adenovirus carrier that carries the CEA-LT fusions, result described here shows that the enhanced immunogenicity of CEA-LT fusions is not limited to plasmid DNA immunity (referring to embodiment 13).

What further show according to the present invention is, compares CEA, plasmid pV1J/hCEA-DOM and pV1J/hCEA-FcIgG caused better CEA specific cell mediation with immune response (referring to embodiment 15) body fluid.Also shown according to the present invention, because the immunogenic properties of the raising of CEA fusions,, can more effectively destroy tolerance the CEA self antigen with DOM described here and FcIgG CEA fusions with respect to total length wild-type CEA cDNA.With DNA or the immunity of Ad carrier the time, observe the enhanced immunogenic properties of these fusion roteins, show that the enhanced immunogenicity of CEA-LT fusions is not limited to plasmid DNA immunity (referring to embodiment 16).

Therefore, aforesaid carrier can be used for immunogenic composition and vaccine, is used to prevent to express with unusual CEA the development of relevant gland cancer, and/or is used for the treatment of existing cancer.By eliminating the difficult problem aspect the high expression level that in host transformed organism successfully, obtains external source CEA, with can cause the immunoreactive CEA fusion rotein of enhanced by providing when for example human when being administered to Mammals, carrier of the present invention has allowed vaccine development and commercialization.

For this reason, one aspect of the present invention is the method for prevention or treatment CEA associated cancer, comprise the vaccine carrier that comprises polynucleotide to administration, described polynucleotide comprise the nucleotide sequence of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from by heat shock protein (HSP) 70, lysosome related membrane protein (LAMP), the fragment C of Toxoid,tetanus (FrC), the N-end structure territory (DOM) of FrC, the heavy fragment (FcIgG) of the constant chain of immunoglobulin G 1, vesicular stomatitis virus glycoprotein (VSV-G), the group that constitutes from the heat labile enterotoxin (LT) of the Toxins,exo-, cholera (CT) of Vibrio cholerae and E.coli; Wherein said fusion rotein can produce immune response in Mammals.

Method described here preferred embodiment in, described immunostimulant element is selected from the group that is made of LTB, LTA, DOM and FcIgG.

According to aforesaid method, can use described vaccine carrier and be used for including but not limited to: lung cancer, mammary cancer and colorectal carcinoma in any Mammals treatment or preventing cancer.Of the present invention preferred embodiment in, described Mammals is human.

Further, those skilled in the art can select the carrier of any kind, is used for described treatment and prevention method.Preferably, described carrier is adenovirus carrier or plasmid vector.Of the present invention preferred embodiment in, described carrier is an adenovirus carrier, it comprises the deletion that has in the adenovirus E 1 zone and the adenoviral gene group of the inset in the adenovirus E 1 zone, wherein said inset comprises expression cassette, described expression cassette comprises: (a) sequence of the Nucleotide of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, and it is fused to immunostimulant element or its entity part; Wherein said immunostimulant element is selected from the group that constitutes by the heavy fragment (FcIgG) of the constant chain of the N-end structure territory (DOM) of fragment C (FrC), the FrC of heat shock protein (HSP) 70, lysosome related membrane protein (LAMP), Toxoid,tetanus, immunoglobulin G 1, vesicular stomatitis virus glycoprotein (VSV-G), from the heat labile enterotoxin (LT) of the Toxins,exo-, cholera (CT) of Vibrio cholerae and E.coli; Wherein said fusion rotein can produce immune response in Mammals; (b) promotor that can be operatively connected with described polynucleotide.

The invention further relates to the adenovirus vaccine carrier vector, it comprises the deletion that has in the E1 zone and the adenoviral gene group of the inset in the E1 zone, wherein said inset comprises expression cassette, described expression cassette comprises: (a) sequence of the Nucleotide of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, and it is fused to the entity part of immunostimulant element; Wherein said immunostimulant element is selected from the group that is made of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT, LTA and LTB; Wherein said fusion rotein can produce immune response in Mammals; (b) promotor that can be operatively connected with described polynucleotide.

In the preferred implementation in this aspect of the invention, described adenovirus carrier is the Ad5 carrier.

Of the present invention another preferred embodiment in, described adenovirus carrier is the Ad6 carrier.

Another preferred embodiment in, described adenovirus carrier is the Ad24 carrier.

Consider that also being used for of the present invention is the adenovirus vaccine carrier that comprises the adenoviral gene group, it infects natively and is different from human species, includes but not limited to the chimpanzee adenovirus carrier.This aspect of the present invention preferred embodiment be chimpanzee Ad3 vaccine carrier.

In yet another aspect, the present invention relates to comprise the vaccine plasmid of plasmid part and expression cassette part, described expression cassette partly comprises: (a) sequence of the Nucleotide of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to immunostimulant element or its entity part, and described immunostimulant element is selected from the group that is made of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT, LTA and LTB; Wherein said fusion rotein can produce immune response in Mammals; (b) promotor that can be operatively connected with described polynucleotide.

In some embodiments of the present invention, the polynucleotide vaccine based on recombinant adenovirus and plasmid disclosed herein is used to various exciting/strengthen (prime/boost) combination to induce the enhanced immune response.In this case, use two kinds of carriers in " excite and strengthen " mode.For example use the carrier one or many of the first kind, after preset time, for example, 2 weeks, 1 month, 2 months, six months or other proper spacing are used the carrier one or many of second type after the time.Preferably, described carrier carries the expression cassette of identical polynucleotide of coding or polynucleotide combination.In the embodiment that also uses plasmid DNA, preferably, this carrier contains by one or more promotors of Mammals or insect cell identification.In preferred embodiment, described plasmid will contain strong promoter, such as but not limited to, the CMV promotor.Synthetic CEA fusion gene or other genes of expressing will be connected with this promotor.The example of this plasmid can be described Mammals expression plasmid V1Jns (J.Shiver et.al.inDNA Vaccines, M.Liu et al.eds., N.Y.Acad.Sci., N.Y., 772:198-208 (1996) is incorporated in this with it by reference).

As mentioned above, adenovirus carrier vaccine and plasmid vaccine can be administered to vertebrates and come the induction of immunity reaction as the part of single therapy mode.For this reason, the present invention relates to protect Mammals to avoid the method for CEA associated cancer, comprise: (a) in Mammals, import first carrier, described first carrier comprises: the i) sequence of the Nucleotide of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT, LTA and LTB; Wherein said fusion rotein can produce immune response in Mammals; The (ii) promotor that can be operatively connected with described polynucleotide; (b) allow through preset time; (c) in Mammals, import second carrier, described second carrier comprises: (i) sequence of the Nucleotide of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT, LTA and LTB; Wherein said fusion rotein can produce immune response in Mammals; The (ii) promotor that can be operatively connected with described polynucleotide.

In an embodiment of aforesaid guard method, described first carrier is a plasmid, and second carrier is an adenovirus carrier.In selectable embodiment, first carrier is an adenovirus carrier, and second carrier is a plasmid.

In aforesaid method, the carrier that can use the first kind surpasses once, and each vector administration is preset time at interval.The series of this first kind carrier is applied in through after the preset time can follow using of one or many second type of carrier.Be similar to the treatment of using first kind carrier, second type of carrier also can give once after the preset time interval or surpass once.

The invention further relates to treatment and suffer from the mammiferous method of the relevant gland cancer of CEA, comprise: (a) in this Mammals, import first carrier, described first carrier comprises: the i) sequence of the Nucleotide of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT and LT; Wherein said fusion rotein can produce immune response in Mammals; The (ii) promotor that can be operatively connected with described polynucleotide; (b) allow through preset time; (c) in this Mammals, import second carrier, described second carrier comprises: (i) sequence of the Nucleotide of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of HSP70, LAMP, FrC, DOM, FcIgG, VSV-G, CT and LT; Wherein said fusion rotein can produce immune response in Mammals; The (ii) promotor that can be operatively connected with described polynucleotide.

In an embodiment of aforesaid methods of treatment, described first carrier is a plasmid, and second carrier is an adenovirus carrier.In selectable embodiment, first carrier is an adenovirus carrier, and second carrier is a plasmid.

Method as mentioned above preferred embodiment in, described carrier comprises the sequence of the Nucleotide of coding CEA-LT fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of LT subunit.Further preferred embodiment in, described carrier comprises the sequence of the Nucleotide of coding CEA-LTB fusion rotein.In the further preferred embodiment of method as mentioned above, described carrier comprises the sequence of the Nucleotide of coding CEA-DOM fusion rotein, and wherein said CEA fusion rotein comprises CEA albumen or its variant, is fused to the entity part of DOM subunit.Further preferred embodiment in, described carrier comprises the sequence of the Nucleotide of coding CEA-FcIgG fusion rotein.

The quantity of the RNA that imports vaccine recipient's effable DNA or transcribe partly depends on the immunogenicity of the gene product of the intensity of promotor of use and expression.Usually, with about 1ng to 100mg, preferred about 10 μ g to 300 μ g immunity or the plasmid vaccine carrier of prevention effective dose be applied directly in the muscle tissue.The effective dose of recombinant adenovirus is about 10 ⁶-10 ¹²Individual particle, preferred about 10 ⁷-10 ¹¹Individual particle.Subcutaneous injection, intracutaneous import, pressed skin and other mode of administration, and for example endoperitoneal, intravenous, intramuscular or suction is sent also and considered.

Of the present invention preferred embodiment in, by intramuscular injection vaccine carrier is imported the recipient.

Vaccine carrier of the present invention can expose, that is, do not associate with any albumen or immune other reagent of influencing the recipient.In this case, hope be that vaccine carrier is in the acceptable solution of physiology, such as but not limited to, Sterile Saline or aseptic buffer saline.Alternatively, what possibility was useful is to use the reagent of the cellular uptake that helps DNA, such as but not limited to calcium ion.These reagent are commonly referred to as transfection and promote reagent and pharmaceutically acceptable carrier.Those skilled in the art can determine specific reagent or pharmaceutically acceptable carrier and suitable time and the mode used.

Method and material in order to describe and openly may to interrelate and use with the present invention merge all publications referred in this by reference.This any content all can not be interpreted as admitting the present invention do not have qualification according to formerly invention on the date early than this open.

Be described with reference to the drawings preferred embodiment of the present invention, should be understood that to the invention is not restricted to these clear and definite embodiments, can carry out various changes and modification and do not deviate from as defined scope and spirit of the present invention in the subsidiary claim by those skilled in the art.

Following examples illustrate but do not limit the present invention.

Embodiment 1

The structure of CEA fusion rotein

In order to determine the immunogenicity of CEA fusion rotein, made up a series of carriers, its coding is fused to the proteic amino acid of human CEA (hereinafter claiming aa) 1 to 679 (referring to embodiment 2) of one group of selected polypeptide.Consider that the immunostimulant character that they are in the news selects these sequences, this has proved in various experimental systems.Be connected to allogenic polypeptide by the proteic cDNA of CEA that will delete the GPI anchor series and come through engineering approaches CEA fusions (in accompanying drawing 18A, having described exemplary construct).With tumour antigen be connected to HSP70, FcIgG or LAMP sequence determine the enhancing of antigen uptake or internally the target again (retargeting) of chamber, tagma whether cause the immune response of rising.Similarly, make up with the fragment C (FrC) of tetanus toxin or promote body fluid with the fusions that lacks the minimal structure territory (DOM is referring to accompanying drawing 29) (Rice et al.J.Immunol.169:3908-13 (2002)) that possible competitive MHCI class combines epi-position or CD4 ⁺T cell responses.Also CEA is connected to the VSV-G encoding sequence and determines whether the fusions with viral glycoprotein influences the immunogenic properties of CEA.

The encoding sequence of these CEA fusions is cloned into the control (embodiment 2) that is in human CMV/ intron A promotor and Trobest (BGH) polyadenylic acid signal among the carrier pV1Jns.Plasmid pV1J/CEA-FRC, pV1J/CEA-DOM, pV1J/CEA-FcIgG, pV1J/CEA-LAMP, pV1J/CEA-VSV-G and pV1J/CEA-HSP70 have carried the wild-type CEA cDNA of the encoding sequence that is fused to the allogenic polypeptide of indicating.The Exemplary core thuja acid of hCEA-DOM and hCEA-FcIgG fusions and aminoacid sequence are shown in accompanying drawing 26,27 and 30.

For the LTA that evaluates the E.coli heat labile enterotoxin and LTB subunit to the immunogenic influence of CEA, generate a series of other fusions constructs, its coding CEA Argine Monohydrochloride 1 to 679 also is fused to LTA (aa18 to 259) or LTB (aa21 to 125) encoding sequence.For the synoptic diagram of the structure of the exemplary CEA-LTA of these research and development and CEA-LTB fusions shown in Figure 1.The Exemplary core thuja acid of CEA-LT fusions and aminoacid sequence are shown in the accompanying drawing 2-6.

Be connected to the LT subunit of having removed signal coding sequence by the proteic cDNA of the CEA that will delete anchor series and come through engineering approaches CEA-LT fusions.CEA fusions encoding sequence is cloned into the control that is in human cytomegalovirus (CMV)/intron A promotor and Trobest (BGH) polyadenylic acid signal among the carrier pV1Jns.Plasmid pV1J/hCEA-LTA and pV1J/hCEA-LTB carry the wild-type CEA cDNA (referring to embodiment 2) of the encoding sequence that is fused to LTA and LTB respectively.

Produce all constructs that carry the CEA-LTB fusions by merging with the LTBcDNA fragment that comprises nt64 to 375 from the CEA cDNA of nt1 to 2037.By using sequence specific primers

LTB-S15 '-TATTCTAGATGCTCCCCAGACTATTACAGAA-3 ' (SEQID NO:1) and LTB-A15 '-TATGCGGCCGCCTAGTTTTCCATACTGATTGCCGC-3 ' (SEQ ID NO:2) pcr amplification E.coli genomic dna obtains the LTB encoding sequence.The DNA of amplification is imported to 3 ' end of CEA encoding sequence, produce plasmid.

Embodiment 2

The plasmid construction body

PV1J/CEAopt and pV1J/CEA: these two constructs carry respectively the codon utilization ratio that optimize with CEA cDNA wild-type.The CEA encoding sequence is between instant early promoter of the CMV/ of cytomegalovirus intron A and Trobest polyadenylic acid signal.In order to produce pV1J/hCEAopt, at 37 ℃ with EcoRI digested plasmid pCR-hCEAopt 1 hour.The 2156bp inset that purifying produces, and be cloned into plasmid pV1JnsB ((Montgomery etal.DNA Cell Biol 12 (9): in EcoRI site 777-83 (1993)).

In order to produce pV1J/hCEA, with EcoRI digested plasmid pCI/hCEA (Song et al.Regulation of T-helper-1 versus T-helper-2 activity and enhancement oftumour immunity by combined DNA-based vaccination and nonviralcytokine gene transfer.Gene Therapy 7:481-492 (2000)).The 2109bp inset that produces is cloned in the EcoRI site of plasmid pV1JnsA (Montgomery et al., the same).

PV1J/hCEA-LTB and pV1J/hCEA _Opt-LTB: by oligonucleotide assembling (Geneart GmbH, Regensburg, Germany) the codon optimized cDNA of synthetic LTB and be cloned into the pCR-script carrier (Stratagene, LA Jolla, CA) in.In order to produce pV1J/hCEA _Opt-LTB _Opt, use following PCR primer by pcr amplification LTB _Opt: LTBopt-5 ' XbaI (5 ' end) 5 '-GCTCTAGAGCCCCCCAGAGCATCACCGAGCTGTGC-3 ' (SEQ ID NO:3) and LTBopt-3 ' BglII(3 ' end) 5 '-GCTCTAGAACCCCTCAGAACATCACCGATCTGTGCGCC-3 ' (SEQ ID NO:4).Product with amplification is inserted into plasmid pV1J/hCEA then _OptThe XbaI/BglII site in.

PV1J/hCEA-LTA: by using sequence specific primers LTA-S15 '-TATTCTAGATAATGGCGACAAATTATACCG-3 ' (SEQID NO:5) and LTA-A15 '-TATGCGGCCGCTCATAATTCATCCCGAATTCTGTT-3 ' (SEQ ID NO:6).The E.coli genomic dna is carried out PCR to increase corresponding to the LTA encoding sequence of nt54 to 774 of coding aa18 to 259.With the DNA of suitable digestion with restriction enzyme amplification and be inserted among the plasmid pV1J/hCEA.

PV1J/rhCEAopt-LTB: by carry out the increase 3 ' fragment (nt1641 to 2026) of rhesus monkey CEA cDNA of PCR from pV1J-rhCEAopt, it is codon optimized for high level expression in the human cell.The cDNA of amplification lacks GPI deadman encoding sequence and has the XbaI/BglII restriction site.This fragment is inserted into the PstI site of pCR-blunt-rhCEAopt, thereby obtains intermediary pCR-blunt-rhCEAoptXbaI/BglII.As BglII/SalI snippet extraction rhCEAopt, be cloned into the same loci among the pV1J-nsB, thereby obtain pV1J-rhCEAopt XbaI/BglII., be cloned among the pV1J-rhCEAopt XbaI/BglII, thereby obtain from having added the pCR-script-LTBopt amplification LTBopt in XbaI and BglII site respectively by PCR at 5 ' end and 3 ' end PV1J- RhCEAopt-LTBopt

PV1J/CEA-FrC, pV1J/CEA-DOM, pV1J/CEA-FcIgG, DV1J/CEA-LAMP, pV1J/CEA-HSP70 and pV1J/CEA-VSV-G: by arriving aa679 (SEQ ID NO:23 corresponding to aa1, accompanying drawing 28B) nt1 of CEA cDNA is to nt2037 (SEQ ID NO:22, accompanying drawing 28A), with merge all constructs that produce coded reference CEA fusion rotein corresponding to following cDNA fragment: the fragment C (CEA-FrC of Toxoid,tetanus, SEQ ID NO:24), the N-end structure territory (CEA-DOM of FrC, SEQ ID NO:21 and 49), heavy fragment (the CEA-FcIgG of the constant chain of immunoglobulin G 1, SEQ ID NO:25), lysosome related membrane protein (CEA-LAMP, SEQ ID NO:26), heat shock protein 70 (CEA-HSP70, SEQID NO:27) or vesicular stomatitis virus glycoprotein (CEA-VSV-G, SEQ ID NO:28).

The pRep-TeT.C plasmid of describing from Rice et al. (J.Immunol.169:3908-13 (2002)) by pcr amplification obtains FrC and DOM encoding sequence.Obtain FcIgG from total RNA of human PBMC.VSV-G and HSP70 obtain from p-FAST-VSV-G and plasmid pY3111 respectively.LAMP1 obtains by the gene assembling.Use following primer to increase: FrC justice (5 '-TATTCTAGATTCAACACCAATTCCATTTTCTTATTC-3 ' and (SEQ ID NO:29) FrC antisense (5 '-GCGGCCGCTAGAATCATTTGTCCATCCTTCATC-3 ' (SEQ ID NO:30), DOM justice (5 '-TATTCTAGATTCAACACCAATTCCATTTTCTTATTC-3 ' and (SEQ ID NO:31) DOM antisense (5-TTAGCGGCCGCTAGTTCTGTATCATATCGTAAAGGG-3 ' (SEQ ID NO:32), FcIgG justice (5 '-TCTAGATAAAACTCACACATGCCCA-3 ' and (SEQ ID NO:33) FcIgG antisense (5 '-GCCGACTCATTTACCCGGAGACAGGGAG-3 ' (SEQ ID NO:34), LAMP justice (5-TCTAGATTTGATCCCCATTGCTGTGGGCGGTGCCCTG-3 ' and (SEQ ID NO:35) LAMP antisense (5 '-GGCGTGACTCCTCTTCCTGCCAATGAGGTAGGCAATGAG-3 ' (SEQ ID NO:36), VSV-G justice (5 '-ATATCTAGATTTCACCATAGTTTTTCCACACAACC-3 ' (SEQ ID NO:37) VSV-G antisense (5 '-GCGGCCGCCTTCCTTCCAAGTCGGTTCATCTCTATG-3 ' (SEQ ID NO:38), HSP70 justice (5 '-GCTCTAGATATGGCTCGTGCGGTCGGGATCGACC-3 ' (SEQ IDNO:39)) and HSP70 antisense (5 ' GCCGCGGCCGCTCACTTGGCCTCCCGGCCGTCGTCG-3 ' (SEQ ID NO:40).The DNA of amplification is imported 3 ' terminal plasmid pV1J/CEA-FrC, pV1J/CEA-DOM, pV1J/CEA-FcIgG, pV1J/CEA-LAMP, pV1J/CEA-HSP70 and the pV1J/CEA-VSV-G of producing of CEA encoding sequence.

PV1J/CEA-DOMopt and pV1J/CEA-FcIgGopt: by oligonucleotide assembling (Geneart GmbH, Regensburg, the Germany) cDNA that optimizes of the codon utilization ratio of synthetic DOM and FcIgG, and be cloned into the pCR-script carrier (Stratagene, LaJolla, CA) in.In order to produce pV1J/CEA-DOM _Opt, by following primer PCR amplification DOM _Opt: Dom _OptJustice (5 '-GTTATCTAGAAGCACCCCCATCCC-3 ' (SEQ ID NO:41)) and Dom _OptOppositely (5 '-TTAAGATCTCTAAGATCTGGTGTCGTATCTCAGGGG-3 ' (SEQ ID NO:42).Product with amplification is inserted in the XbaI/BglII site of plasmid pV1J/CEAopt then.In order to produce pV1J/CEA-FcIgGopt, by using following primer PCR amplification FcIgG _Opt: FcIgG _OptJustice (5 '-TTATCTAGAAAGACCCACACCTGCCCCCCTTGC-3 ' (SEQ IDNO:43)) and FcIgG _OptOppositely (5 '-TATAGATCTTAGGGTACCTTACTTGCCGGGG-3 ' (SEQ ID NO:44)), the product of amplification is inserted in the XbaI/BglII site of plasmid pV1J/CEAopt.

Embodiment 3

Adenovirus carrier

AdS/hCEA _Opt: with EcoRI digested plasmid pCR-hCEAopt.The 2156bp inset that purifying produces, and be cloned in the EcoRI site of polyMRK-Ad5 shuttle plasmid.

Ad5/CEA: the shuttle plasmid pMRK-hCEA that obtains to be used to produce the Ad5 carrier with SspI and EcoRV digested plasmid pDelta1sp1B/hCEA.9.52kb fragment is connected with the product that limit, the Klenow processing of the 1272bp BglII/BamHI from plasmid polyMRK then.To contain the expression cassette of hCEA and E1 flank Ad5 zone, in BJ5183 E.coli cell, recombinate among the linearizing plasmid pAd5 of ClaI from the PacI/StuI fragment of pMRK-hCEA and pMRK-hCEAopt.The plasmid that produces is respectively pAd5-hCEA and pAd5-hCEAopt.Cut two plasmids with the PacI enzyme and discharge Ad ITRs, and transfection is to the PerC-6 cell.Carry out the amplification of Ad5 carrier by continuous passage.Purify purifying MRKAd5/hCEA and MRKAd5/hCEAopt by standard C sCl gradient, relatively A105 damping fluid (5mMTris-Cl, pH8.0,0.1mM MgCl ₂, 75mM NaCl, 5% sucrose, 0.005 polysorbas20) and dialysis fully.

Ad5/hCEAopt-LTB: by cutting the polyMRK-Ad5 shuttle plasmid with the SwaI enzyme, with by being connected with linearized vector with 2300bp dna fragmentation, make up plasmid pMRK-hCEAopt-LTB, described dna fragmentation is from EcoRI, BglII restriction and the pV1J/hCEAopt-LTB that handles with Klenow.As noted above with in the pMRK-hCEAopt-LTB linearizing and the Ad genome of recombinating.

Ad5/CEA-DOMopt and Ad5/CEA-FcIgGopt: by cutting the polyMRK-Ad5 shuttle plasmid with the SwaI enzyme, with by being connected with the linearized vector that has from the 2.9kb dna fragmentation of pV1J/CEA-DOMopt, or be connected with linearized vector with 2700bp dna fragmentation, the pV1J/CEA-FcIgGlopt that described 2700bp dna fragmentation is used by oneself EcoRI, BglII restriction and handled with Klenow makes up plasmid pMRK-CEA-DOMopt and pMRK-CEA-FcIgGopt.As noted above with in pMRK-CEA-FcIgGopt and pMRK-CEA-DOMopt linearizing and the Ad genome of recombinating.

Embodiment 4

The comparative expression efficiency of various CEA-LT fusions constructs

Having shown that the gene vaccine inoculation that cDNA that the son that accesses to your password is optimized is used to resist virus disease has caused better immune response, is because the target protein that increases is expressed at least to a certain extent.In order to confirm whether the LTB encoding sequence also strengthens the immunogenic properties of CEA cDNA, described CEA cDNA is designed to insert the codon to mankind's preferences (humanization) of each amino-acid residue, has also made up plasmid pV1J/hCEAopt-LTB.At last, also use the codon optimized cDNA of synthetic property of LTB to make up the complete codon optimized version of CEA-LTB fusions, produce plasmid pV1J/hCEA-LTBopt.

Whether immunogenic influence is not limited to the plasmid DNA immunity to CEA in order to determine LTB, has also made up coding CEAopt-LTB fusions, flank is the adenovirus 5 type carriers of CMV/ intron A promotor and BGH polyadenylic acid signal.The molecular weight of the CEA fusion rotein of plasmid and Ad vector expression is not different to come the molecular weight (data not shown) of CEA fusion rotein of the respective carrier of own coding total length form A EA cDNA.

For the expression efficiency of the carrier of relatively encode CEA-LTA and CEA-LTB fusions, and the expression efficiency of the cDNA of total length CEA, with plasmid pV1J/hCEA-LTA and pV1J/hCEA-LTB transfection HeLa cell.The CEA of these constructs is expressed and the comparing of the corresponding plasmid pV1J/hCEA of the wt cDNA that carries CEA.Similarly, with the expression efficiency of plasmid pV1J/hCEAopt-LTB and comparing of pV1J/hCEAopt.The expression efficiency of these constructs is determined in transfection two days later by the proteic quantity of CEA in the supervision cell extract.

Compare with plasmid pV1J/CEA (91 μ g/l), the transfection of plasmid pV1J/hCEA-LTA and pV1J/hCEA-LTB has produced the detected CEA albumen (being respectively 183 and 139 μ g/l, accompanying drawing 8A) that is about twice quantity in culture supernatant.Similarly, the expression efficiency of construct pV1J/hCEAopt and pV1J/hCEAopt-LTB also is comparablely (to be respectively 113 and 136 μ g/l; Accompanying drawing 8B).At last, by infecting the HeLa cell, also compared the expression efficiency of Ad/hCEAopt and Ad/hCEAopt-LTB with different moi.At moi1000, the CEA expression efficiency of these two carriers is that comparable (comparable) (is respectively 1790 and 1400 μ g/l, accompanying drawing 9), and at moi100, compare with Ad/hCEAopt, carrier A d/hCEAopt-LTB has produced the detected CEA albumen (being respectively 1500 and 390 μ g/l) of about 1/4th multiple amounts in culture supernatant.

Thereby these results show, compare with the proteic corresponding cDNA of coding total length CEA, and the cDNA of coding CEA-LTA and CEA-LTB fusion rotein expresses with the efficient that is equal to.In addition, the compared CEA of these cDNA expresses the influence of the kind of the transgenosis vehicle of sending (vehicle) that is not used for them.

Embodiment 5

The detection that CEA expresses

The CEA that monitors plasmid and Ad carrier by Western engram analysis and ELISA expresses.With Lipofectamine 2000 (Life Technologies) with plasmid transfection in the HeLa cell.37 ℃ of adenovirus infections of in serum free medium, carrying out the HeLa cell 30 minutes, add fresh culture then.Hatch after 48 hours, gather in the crops whole cellular lysate.Use the rabbit polyclonal antiserum(antisera) to detect the CEA albumen that exists in the cellular lysate by the Western engram analysis.Detect albumen as the 180-200kDa band.Use Direct Elisa CEA test kit (DBC-Diagnostics Biochem Canada Inc) to detect the quantity of the CEA that expresses in the cellular lysate.

Use is specific to the antibody of CEA, VSV-G, FcIgG, tetanus toxin or HSP70 and checks Expression of Fusion Protein in the cells transfected by the Western engram analysis.Infect the HeLa cell with the plasmid transfection of indicating or with selected Ad carrier.After hatching 48 hours, gather in the crops whole cellular lysate and culture supernatant.

Also use Direct Elisa CEA test kit (DBC-Diagnostics BiochemCanada Inc) to monitor that the CEA in cellular lysate or the supernatant liquor expresses.With the CEA albumen of the antibody test that is specific to fusion polypeptide in the lysate of cells transfected, and in the control sample of simulation transfection, do not observe the expression (accompanying drawing 18B) of target antigen.The molecular weight of fusion rotein significantly is not different from the molecular weight of CEA.Obviously lacking molecular weight difference between various CEA polypeptide may be because the high glycosylation of tumour antigen.

For the carrier of CEA fusions and the expression efficiency of pV1J/CEA of relatively encoding, with different plasmid transfection HeLa cells, the CEA expression of these constructs is measured in transfection two days later by ELISA.Plasmid pV1J/CEA-FrC, pV1J/CEA-DOM, pV1J/CEA-FcIgG, pV1J/CEA-LAMP, pV1J/CEA-VSV-G and pV1J/CEA-HSP70 are to express CEA (accompanying drawing 19A) with the comparable efficient of pV1J/CEA.Most of fusion roteins are excretory and detect in cell conditioned medium liquid; Yet CEA-LAMP does not discharge from cells transfected, may be because it has gone to the lysosome compartment again.(accompanying drawing 19B).Thereby these results show, compare with the proteic cDNA of coding total length CEA, and the cDNA of coding CEA-FrC, CEA-DoM, CEA-VSV-G, CEA-FcIgG, CEA-HSP70 and CEA-LAMP fusion rotein expresses with the efficient that is equal to.

Embodiment 6

Peptide

Freeze dried hCEA peptide is resuspended among the DMSO with 40mg/ml available from Bio-Synthesis.Assemble storehouse (the Facciabene etal.J.Virol.78:8663-72 (2004) of the peptide of overlapping 11 residues, 15aa length as described.Final concentration is as follows: storehouse A=1.2mg/ml, storehouse B0.89mg/ml, storehouse C 0.89mg/ml, storehouse D 0.8mg/ml.Peptide is kept at-80 ℃.Use Toxoid,tetanus peptide p30 (F ₉₄₇NNFTVSFWLRVPKVSASHLE ₉₆₇(SEQ ID NO:54)) monitor immune response (Rice et al.J.Immunol.167:1558-65 (2001)) to DOM.

Embodiment 7

Mouse immune and tumor challenge

All zooscopies all pass through animal treatment of IRBM mechanism and the approval of the use council.Female C57BL/6 mouse (H-2 ^b) available from Charles River (Lecco, Italy).(Institute Pasteur, Paris France) provides HLA-A2.1 mouse (HHD) favorably by F.Lemmonier.The C57BL/DR4 mouse available from Taconic (Germantown, NY).CEA.tg mouse (H-2 ^b) provide by J.Primus (Vanderbilt University), remain on standard conditions (Clarke et aL.Cancer Res.58:1469-77 (1998)).As (Rizzuto et al.Proc.Natl.Acad.Sci.U.S.A.96 (11): the 6417-22 (1999)) of previous description, with 50 microgram plasmid DNA with 50 μ l volume Electricinjections in the mouse musculus quadriceps.In the mouse musculus quadriceps, carry out the Ad injection with 50 μ l volumes.In time series analysis body fluid of indicating and cell-mediated immune response.

Carrying out electricity irritation subsequently as previous (Rizzuto et al. is the same) of describing twice DNA injection C57BL/6 mouse in musculus quadriceps.Inject at interval with three weeks.Make 5 plasmid DNA injection (50 μ g/ injection), 2 Ad vector injections (1 * 10 weekly of CEA transgenic mice experience ⁹Virus particle/injection) or 5 injections weekly and strengthening with Ad subsequently.After last two weeks of injection, analysing body fluid and cell-mediated immune response.With 5 * 10 ⁵Mouse is attacked in (s.c.) injection under individual MC38-CEA (Clarke et al., the same) cell skin.Once in a week, check the tumor growth of mouse.

Embodiment 8

Antibody test and titration

Obtain to be used for the serum of antigen titration by back eye socket bloodletting.Be used in bag as (the Facciabene et al., the same) of previous description and be cushioned liquid (50mM NaHCO ₃, pH9.4) the highly purified CEA albumen in 100ng/ hole (Fitzgerald) of middle dilution wraps by ELISA flat board (Nunc maxisorp), and at 4 ℃ of hatching O/N.Sealed dull and stereotyped 1 hour with the PBS that contains 5%BSA at 37 ℃ then.In PBS 5%BSA, dilute mice serum (extent of dilution 1/50 is assessed seroconversion rate, extent of dilution 1: 10 to 1: 31,2150 assess titre).Pre-immune serum is as background.At 4 ℃ of serum hatching O/N with dilution.Wash with PBS1%BSA, 0.05% polysorbas20.(goat anti-mouse, IgG peroxidase Sigma) dilute 1/2000 to secondary antibody in PBS, 5%BSA, hatched 2-3 hour at RT on wobbler.After the washing, with tmb substrate (Pierce Biotechnology, Inc., Rockford, IL) the cultivation flat board in 100 μ l/ holes.With 25 μ l/ hole 1M H ₂SO ₄The solution stopped reaction reads flat board at 450nm/620nm. and anti-CEA serum titer is calculated as the reciprocallimiting dilution of serum, and it is at least 3 times of absorbancys from body pre-immune serum absorbancy that described serum produces at identical extent of dilution.

Embodiment 9

IFN-γ ELISPOT analyzes

As (the Facciabene et al., the same) of previous description, use mouse boosting cell and CEA specific peptide to analyze.Be used in the rat anti-mouse IFN-γ that is diluted to purifying 2.5 μ g/ml, 100 μ l/ holes among the aseptic PBS (IgG1, clone R4-6A2, Pharmingen) bag by 96 hole MAIP flat boards (Millipore Corp., Billerica, MA).With after the PBS washing, under 37 ℃, carry out flat board with the R10 substratum in 200 μ l/ holes and sealed 2 hours.

Remove spleen with sterile manner from the euthanasia mouse, destroy spleen by rubbing on metallic sieve and obtain splenocyte.By in cell mass, adding 1ml 0.1X PBS and vortex about 15 seconds, come to remove red corpuscle by the infiltration cracking.Add 1ml 2 * PBS then, volume is transferred to 4ml with 1 * PBS.By at RT centrifugal 10 minutes of 1200rpm with the cell mass granulation, granule is resuspended in the 1ml R10 substratum.Use T ü rks dyeing to viable count.

With 5 * 10 ⁵With 2.5 * 10 ⁵Cells/well is hatched 20hs with 1 μ g/ml suspended substance of every kind of peptide at 37 ℃ in duplicate with the splenocyte bed board.Use concanavalin A (ConA) as positive internal contrast to each mouse with 5 μ g/ml.After the washing of PBS, 0.05% polysorbas20, (RatIgG1, clone XMG 1.2 PharMingen) is hatched O/N at 4 ℃ to the dull and stereotyped rat anti-mouse IFN γ that combines vitamin H with the 50 μ l/ holes that are diluted to 1: 2500 in analysis buffer.After washing fully, (obviously as seen Rockford IL) cultivates dull and stereotyped development up to spot for PierceBiotechnology, Inc. by adding 50 μ l/ hole NBT/B-C1P.Come stopped reaction by washing flat board up hill and dale with distilled water.Air-dry flat board uses automatization ELISPOT reader that spot is counted.

Embodiment 10

The dyeing of the cell within a cell factor

As (the Facciabene et al., the same) of previous description, at 37 ℃ and 5%CO ₂, one to 2,000,000 mouse boosting cell that will be in 1ml RPMI 10%FCS or the storehouse (every kind of peptide of the final concentration of 5-6 μ g/ml) and brefeldin A (the 1 μ g/ml of PBMC and peptide; BDPharmingen cat#555028/2300kk) hatching is 12-16 hour.(PBS1%FBS, 0.01%NaN3) washed cell were hatched 15 minutes 4 ℃ of anti-mouse CD16/CD32 Fc fragments (BD Pharmingen cat#553142) with purifying to use the FACS damping fluid then.Washed cell then, use surface antibody: the anti-mouse of CD4-PE bonded (BD Pharmingen, cat#553049), the anti-mouse of PercP CD8 bonded (BD Pharmingen cat#553036) and the anti-mouse CD3e of APC bonded (BD Pharmingen cat#553066) in the dark at room temperature dyeed 30 minutes.After the washing with cell fixation, in the dark at 4 ℃ with Cytofix-Cytoperm solution (BD Pharmingen cat#555028/2300kk) infiltrationization 20 minutes.After PermWash solution (BD Pharmingen cat#55028/2300kk) washing, cell and IFN γ-FITC antibody (BD Pharmingen) hatching.Washed cell is used 1% formaldehyde fixed among the PBS then, and (Becton Diclinson, San Jose CA) analyze on the FACS-Calibur flow cytometer to use CellQuest software.

Embodiment 11

The immunogenicity of CEA-LT fusions

In order to check the plasmid inductive immune response of coding CEA-LTA and CEA-LTB fusions, the group of each 9 C57BL/6 mouse is come immune with every kind of plasmid pV1J/hCEA, pV1J/hCEA-LTA and the twice i.m. injection of pV1J/hCEA-LTB of 50 μ g.In addition, for whether the coexpression that confirms CEA-LTA and CEA-LTB fusion rotein has the adduction effect for the proteic immunogenicity of CEA, come immune one group of mouse by every kind of plasmid pV1J/hCEA-LTA and the pV1J/hCEA-LTB that injects 25 μ g altogether.Be separated by and three weeks carried out immunity.Consider enhanced transduction and immunogenicity (the Zucchelliet al.J.Virol.74:11598-11607 (2000) relevant with this particular procedure; Widera et al.J.Immunol.164:4635-4640 (2000)), with plasmid DNA routinely Electricinjection in mice skeletal.

After last 2 weeks of injection, analyze the cellular immunization of measuring different plasmids initiations by ELISPOT.The antigen-specific IFN γ secretion from the splenocyte of irriate is measured in four storehouses of using overlapping 11 aa and containing the 15mer peptide of complete CEA glycoprotein.Storehouse A covers aa1 to 147, and storehouse B covers aa137 to 237, and storehouse C covers aa317 to 507 and storehouse D covers aa497 to 703.As negative control, also use stimulate splenocyte with the DMSO that dissolves the employed same concentrations of CEA peptide after, measured the cytokine generation.

Proteic C-stub area mainly is partial in the immune response that dna vaccination inoculation causes in the C57BL/6 mouse, because with the background (accompanying drawing 10) of a little higher than all constructs of SFC value of peptide storehouse A detection.Higher geometrical mean by the SFC that detects with peptide storehouse B, C and D is shown, and pV1J/hCEA-LTB vaccine inoculation mode is better than the (pV1J/hCEA-LTB: be respectively 482,1436 and 2054 SFC/10 that pV1J/hCEA causes ⁶Individual splenocyte; PV1J/hCEA: be respectively 45,350 and 264 SFC/10 ⁶Individual splenocyte).Similarly, compare with pV1J/hCEA, plasmid pV1J/hCEA-LTA also has reinforced effects for the CEA specific immune response.Yet, only observed immunoreactive increase and (be respectively 925 and 528 SFC/10 with peptide storehouse C and D ⁶Individual splenocyte), and with the immune response that peptide storehouse B measures is low (15 SFC/10 ⁶Individual splenocyte).In addition, compare with the immune response of measuring in the group that pV1J/hCEA-LTB handles, the common injection of plasmid pV1J/hCEA-LTA and pV1J/hCEA-LTB does not have significant synergy for the immune response of CEA, but it has caused that the reduction of the SFC value that detects with peptide storehouse B and D (is respectively 210 and 528 SFC/10 ⁶Individual splenocyte).

The T cell-specific that causes in order to determine with different CEA construct vaccine inoculation carries out dyeing in the IFN gamma cells to the concentrated splenocyte from the mouse of using peptide storehouse D injection.In mouse, detect CD8 with the pV1J/hCEA-LTB injection ⁺Specific reaction (4.5%) is better than (being respectively 0.14% and 0.8%, accompanying drawing 10B) detected with pV1J/hCEA-LTA and pV1J/hCEA.On the contrary, pV1J/CEA-LTA has caused intensive CD4 ⁺Specific reaction (1.21%) is greater than with pV1J/hCEA-LTB and pV1J/hCEA observed (being respectively 0.55% and 0.58%).

Check induce (accompanying drawing 11) by measuring antigen-specific antibodies at the humoral immune reaction of CEA.Plasmid pV1J/hCEA-LTA and pV1J/hCEA-LTB have caused the antibody response bigger than pV1J/hCEA, have confirmed the adjuvant effect of LT subunit to the performance of CEA specific immune response.Thereby these digital proofs, the fusion of CEA encoding sequence and LTA or LTBcDNA has caused the raising of CEA specific immune response.Yet LTB seems that immune response is had better reinforced effects, and main inductive is CD8 ⁺The T cell, and that the LTA initiation mainly is CD4 ⁺Reaction.

Embodiment 12

The immunogenicity of CEA-LTB fusions in different mouse species

In order to determine that whether the LT subunit is not limited to single mouse genetic background to the reinforced effects of CEA specific immune response, carries out the immunity based on DNA in BALB/c, C57/DR4 and HLA-A2.1 (HHD) mouse.Consider that their immunocompetence selects BALB/c mouse, it is that immunization ways to various kinds has extremely reactive mouse species.The HHD transgenic mice is expressed human MHCI genoid.Similarly, the C57/DR4 transgenic mice carries human MHC II genoid.Thereby these two transgenic mice strains can provide the immunoreactive information about CEA-LT fusions in the environment of human MHC I and II class haplotype.

At first by the CEA specific immune response in the ELISPOT analyzing evaluation BALB/c mouse.After plasmid pV1J/hCEA-LTB immunity, detected the enhancing (pV1J/hCEA-LTB: be respectively 166,1353,796,899 SFC/10 of antigen specific immune reaction with peptide storehouse A, B, C, D ⁶Individual splenocyte; PV1J/hCEA: be respectively 57,312,327,318 SFC/10 ⁶Individual splenocyte, accompanying drawing 12).As in the C57BL/6 mouse, observing, compare with other parts of tumour antigen, it seems that the proteic N-stub area of CEA be minimum immunogenic.Compare with pV1J/hCEA, the pV1J/hCEA-LTA immunity has also produced the raising of antigen specific immune reaction.Detect immunoreactive raising with peptide storehouse B, C and D and (be respectively 936,727 and 650 SFC/10 ⁶Individual splenocyte).In addition, the common injection of two kinds of plasmid pV1J/hCEA-LTA and pV1J/hCEA-LTB has produced significant Overlay, and it mainly is (to be respectively 1783 and 2141 SFC/10 with what peptide storehouse C and D detected ⁶Individual splenocyte).

The immunity of pV1J/hCEA-LTB has strengthened the CEA specific immune response significantly in the C57/DR4 mouse, and it is that peptide storehouse D detected (accompanying drawing 13) is only arranged.Dyeing shows in the IFN gamma cells that the PBMC that concentrates from the mouse of injection is carried out, in the pV1J/hCEA-LTB mice immunized to the CD8 of CEA ⁺Reaction is the highest (15.32%), and very faint (0.5%) .pV1J/CEA-LTA immunity has only improved to moderate antigen specific immune reaction (0.43%) in the group that pV1J/hCEA handles, and does not further strengthen CEA immunogenicity (13.44%) when injecting altogether with the construct of coding CEA-LTB fusions.Enjoyably, in mice immunized, do not detect significant CD4 ⁺T cell responses (data not shown).

In the HHD mouse, evaluated the immune response that different CEA coding plasmids causes by the PBMC that concentrates being carried out dye in the IFN gamma cells.Only detect immune response with peptide storehouse B and D, shown in Figure 14, at CD8 at target antigen ⁺In the reaction, produced with the pV1J/hCEA-LTB immunity and to have surpassed 10 times raising.Opposite is, uses pV1J/hCEA-LTA separately or when injecting altogether with pV1J/hCEA-LTB, does not detect immunoreactive raising.In mice immunized, do not detect CD4 ⁺T cell responses (data not shown).

Integrate, these data acknowledgements, the fusion of LTB encoding sequence and CEA has caused the considerable increase of antigen specific immune reaction.Enjoyably, this reaction mainly is CD8 ⁺Specific, can in different mouse species, observe, thereby show that the reinforced effects of LT subunit performance is not that genotype is restrictive.

Embodiment 13

In the transgenic mice to the tolerance of human CEA

For whether the enhanced immunogenic properties of determining the hCEA-LTB fusions more effectively destroys tolerance to human CEA, with the immune hCEA transgenic mice of the carrier of the complete codon optimized cDNA that carries hCEA or CEA-LTB.These transgenic mices carry complete human CEA gene and flanking sequence, and express hCEA albumen in intestines, mainly are in caecum and colon.Thereby this mouse system is that research is at the security of the immunotherapy strategy of this tumour self antigen and the useful model (Clarke et al., Cancer Research58:1469-1477 (1998)) of effectiveness.

Undertaken by the PBMC of mouse that dyeing measures in the IFN gamma cells, caused significantly improve (the accompanying drawing 15A) of CEA specific immune response with the pV1J/hCEA-LTBopt immunity injection.With the enhancing of peptide storehouse D detection t cell responses, mainly be CD8 ⁺In addition, the reaction of CEA specificity humoral has also increased in the mouse that CEA-LTB handles, (the accompanying drawing 15B) as shown in increasing as 47 times of the geometrical mean aspect of the Ab titre of comparing with the group that pV1J/hCEAopt handles.

In order to determine whether LTB also can observe the reinforced effects of CEA specific immune response performance when carrying out immunity with the carrier that is different from plasmid DNA, with 1 * 10 ⁷, 1 * 10 ⁸With 1 * 10 ⁹The dosage of the vp group of Ad5/hCEAopt-LTB and each 12 CEA tg mouse of Ad/hCEAopt immunity.Mouse experiences the double injection of two weekly intervals, and injection two weeks of back are measured immune response by PBMC being carried out dye in the IFN gamma cells the last time.Use peptide storehouse D evaluation immune response.Ad/hCEAopt-LTB is stronger than Ad/hCEAopt immunogenicity, because with 1 * 10 ⁸The dosage of vp can detect the significant immune response to CEA, and 1 * 10 ⁹The Ad hCEAopt of vp destroys the tolerance of target antigen necessary (accompanying drawing 16).In any mice immunized, all do not detect CD4 ⁺Reaction (data not shown).

These data acknowledgements are because the immunogenic properties of the raising of CEA-LTB fusions can more effectively be destroyed the tolerance to this self antigen.In addition, carry the plasmid of complete codon optimized cDNA of CEA-LTB fusions in injection after, also observe the reinforced effects of LTB to the immunogenic properties of CEA.At last, these results show that the enhanced immunogenicity of CEA-LTB is not limited to the plasmid DNA immunity.

Embodiment 14

Tumor growth kinetics in the CEA transgenic mice of using the immunity of CEA-LTB fusions

Whether the immunogenicity of determining the raising of CEA-LTB fusions also produces the enhanced result of treatment that can disturb tumor development, deemed appropriate.For this purpose, 5 plasmid pV1J/hCEAopt or pV1J/CEAopt-LTB injections weekly of the group of each 10 CEA-tg mouse experience are with 1 * 10 subsequently ¹⁰The corresponding Ad carrier of vp is strengthened at last.Consider report recently, its shown by utilize plasmid DNA excite-form that Ad strengthens can induce the high-level cellular immunization at virus and bacterial antigens, adopted identical immunization protocol in this research.Two weeks after the last immunity are with 5 * 10 ⁵CEA tg mouse is attacked in the subcutaneous injection of individual MC38-CEA tumour cell.This homogenic clone derives from the colorectal carcinoma of chemical induction and expresses CEA.Attack the tumor development in (mock treated) mouse that detects simulation process after 22 days, the mouse of all processing no longer is (the accompanying drawing 17A) of first tumour.In addition, attack and existing the increase of following aspect the mean size of the tumor mass that reaches remarkable volume in back 34 days.The vaccinated mouse of carrier with coding pV1J/hCEAopt has shown partial resistance to tumor development, because 2 in the mouse of 10 processing were still tumor free in back 34 days in attack.The mean size of the tumour of this group is less than observed in the group of simulation process.Caused remarkable protection effect with the carrier immunity of coding CEAopt-LTB fusions to tumor development.It is tumor free that in the mouse of attack 10 processing in back 34 days 5 are still, and the mean size of the tumor mass in this group is significantly less than observed in the mouse that simulation process or pV1J/hAopt handle.Thereby these results show, with the carrier related enhanced CEA specific immune response of coding CEA-LTB fusions, and cause that the remarkable antitumous effect at the growth kinetics of the part protection of tumor growth and the tumor mass that reduces is relevant.

Embodiment 15

CEA-DOM and CEA-FcIgG fusions strengthen the proteic immunogenicity of CEA

In order to check the plasmid inductive immune response of coding CEA-FRC, CEA-DOM, CEA-VSV-G, CEA-FcIgG, CEA-HSP70 and CEA-LAMP fusions, the group of each 9 C57BL/6 mouse is injected with twice i.m. of every kind of plasmid of 50 or 5 μ g and is carried out immunity.Be separated by and three weeks carried out immunity.Consider enhanced transduction and immunogenicity (Zucchelli et al.J.Virology 74:11598 (2000) for the electroporation report, Widera et al., J.Immunol.164:4635 (2000)), with plasmid DNA routinely electroporation (DNA-EP) in mice skeletal.

After last 2 weeks of injection, analyze the immune response of measuring different plasmids initiations by IFN γ ELISPOT.Use overlapping 11aa and contain the storehouse (storehouse D, aa 497-703) of 15mer peptide of the C-stub area of CEA, measure from the splenocyte excretory antigen-specific IFN γ (Zucchelli et al., the same) that is stimulated.Carry out the immunoreactive analysis of CEA with peptide storehouse D, because in the C57BL/6 mouse, mainly be partial to this proteic C-stub area (Zuccelli et al., the same) at the cell immune response of CEA.As negative control, use stimulate splenocyte with the DMSO that dissolves the employed same concentrations of CEA peptide after, measured the cytokine generation.

Compare with pV1J/CEA, injection pV1J/CEA-DOM or pV1J/CEA-Fc have caused the bigger immune response at CEA.The bigger immunogenicity of these two fusion roteins has caused per 10 ⁶The spot of individual splenocyte forms the higher geometrical mean (accompanying drawing 20A) of cell (SFC).Plasmid pV1J/CEA-DOM and pV1J/CEA-FcIgG have similar immunogenic properties, (are respectively pV1J/CEA-DOM:590 and 1098SFC/10 in the increase of having brought into play 3 to 4 times aspect the CEA specific immune response when the plasmid DNA of injection 5 or 50 μ g ⁶Individual splenocyte, pV1J/CEA-FcIgG:510 and 1160, pV1J/CEA:146 and 264 SFC/10 ⁶Individual splenocyte).Between the SFC value that causes by pV1J/CEA-FrC, pV1J/CEA-LAMP, pV1J/CEA-HSP70 and pV1J/CEA, do not notice significant difference.In the negative control sample, do not detect the CEA specific immune response.

In order to determine the CEA fusions to the influence at the humoral response of CEA, the CEA albumen that uses purifying is tested serum (accompanying drawing 20B) from mice immunized as substrate in ELISA.Behind injection 50 μ g plasmid pV1J/CEA-DOM, pV1J/CEA-FcIgG, pV1J/CEA-FrC and pV1J/CEA-HSP70, observe the increase of CEA specific antibody titre.On the contrary, injection pV1J/CEA-LAMP and pV1J/EA-VSV-G have produced and have been similar to the observed CEA specific antibody reaction of pV1J/CEA immunity.Integrate, these digital proofs, the fusion of CEA encoding sequence and DOM or FcIgG cDNA caused the CEA specific cell mediation with increase humoral immune reaction.

Embodiment 16

CEA-DOM and CEA-FcIgG fusions destroy the tolerance to target antigen in the CEA transgenic mice

To the tolerance of target antigen is one of cancer vaccine obstacle that must overcome, to cause immune response and performance effective antitumour effect.Thereby whether the enhanced immunogenic properties of determining CEA-DOM and CEA-FcIgG fusions more effectively destroys tolerance to CEA than CEA albumen, deemed appropriate.For this reason, use comparing property of CEA transgenic mice immune Research.These transgenic mices carry complete human CEA gene and flanking sequence, and express CEA albumen in intestines, mainly are in caecum and colon.This mouse system is that research is at the security of the immunotherapy strategy of this tumour self antigen and the useful model (Clarke et al., the same) of effectiveness.

Consider the enhanced immunogenic properties of the carrier of the cDNA (cDNAopt) that the codon utilization ratio of carrying CEA is optimized, plasmid and adenovirus carrier are all carried the cDNAopt of CEA-DOM (CEA-DOMopt) or CEA-FcIgG (CEA-FcIgGopt) fusions by through engineering approaches.As observed, shown that CEA-DOMopt and CEA-FcIgGopt cDNAs express corresponding wild type cDNA with higher efficient, cause the enhanced immune response (data not shown) at CEA to CEA.

By based on using the plasmid DNA use alone or in combination and a series of immune Research of Ad carrier, with the immunogenicity of these two fusion roteins and comparing of CEA.The group of CEA transgenic mice carries out immunity with following variety of way: i) inject 50 μ g plasmid DNA (DNA/DNA) for 5 times once in a week, ii) with 1 * 10 ⁷To 1 * 10 ⁹The dosage of the adenovirus of individual virus particle (vp) 2 times is two injects adenovirus (Ad/Ad) allly, or iii) to inject plasmid DNA for 5 times weekly be 1 * 10 subsequently ⁹The final injection (DNA/Ad) of the adenovirus of vp.Dye and analyze immune response by using storehouse D peptide that the PBMC of each mice immunized or splenocyte are carried out in the cell IFN γ.In addition, monitor inducing of CEA specific antibody by ELISA.

The DNA/DNA immunity of CEA transgenic mice has disclosed, and CEA-DOMopt and CEA-FcIgGopt carrier have produced the measurable CD8 at target antigen ⁺T cell responses (accompanying drawing 21A).Thereby two kinds of constructs can both destroy the tolerance to CEA in these mouse.Indicate that as painted mean value in the IFN gamma cells antigen-specific reaction that CEA-DOM and CEA-FcIgG fusion rotein cause is comparable (being respectively 0.22 and 0.34%).However, the immune response that causes by these two kinds of constructs observed immune response (0.07%) when inoculating with pV1J/CEAopt.Similarly, anti-CEA humoral response is also when inoculating with amalgamation protein vaccine.All detect CEA specific antibody titre in all mouse with pV1J/CEA-DOMopt and pV1J/CEA-FcIgGopt immunity, the mean number of antibody titers is respectively 56,136 and 24,725.On the contrary, the group of pV1J/CEAopt immunity has shown 1/1 CEA specific antibody average titer (318) (accompanying drawing 21B).

The CEA transgenic mice of handling with Ad/Ad vaccine inoculation mode also shown, destroying aspect the tolerance of CEA, better efficient arranged when comparing with Ad-CEAopt with CEA-DOMopt and the inoculation of CEA-FcIgGopt Ad carrier bacterin the time.A spot of in injection as 10 ⁷In the mouse of inoculation, can observe CEA specific C D8 when the Ad-CEA-DOM of vp or Ad-CEA-PcIgG ⁺T cell responses, the CEA specific reaction can compare between two kinds of antigen, in injection 10 ⁹Improve during vp (being respectively 1.55% and 1.15%).On the contrary, 10 ⁹The Ad-CEAopt of vp causes significant CD8 ⁺T cell precursors frequency (2.1%) necessary (accompanying drawing 21C).In all mouse, detect the CEA specific antibody with Ad-CEA-DOMopt and Ad-CEA-FcIgGopt immunity.The mean number of antibody titers is respectively 19,600 and 33,000.Only in the mouse of 2 processing, injection Ad-CEAopt has produced measurable CEA specific reaction, antibody titers significantly lower (Zucchelli et al., the same) (accompanying drawing 21D).Enjoyably, at the CD8 that causes by CEA, CEA-DOM and CEA-FcIgG carrier ⁺T cell precursors frequency aspect, DNA/Ad immunity have shown the difference (accompanying drawing 22A) that reduces.Yet, the mean number of CEA specific antibody titre with the carrier inoculation of expressing CEA-DOM and CEA-FcIgG the time than CEA bigger (being respectively 31,200,26,120 and 412) (accompanying drawing 22B).

Enjoyably, do not consider antigen, in three kinds of vaccine inoculation modes any, do not detect significantly CD4 at CEA ⁺Cell Thl reacts (data not shown).Yet, after DNA/DNA vaccine inoculation, detect significant CD4 at auxiliary epi-position p30 ⁺Cell Thl reacts (0.4%), and p30 appears at DOM sequence inside (Rice et al.J.Immunol.167:1558-65 (2001)) (accompanying drawing 23).

Thereby these data show, CEA-DOM and CEA-FcIgG fusion rotein can be in transgenic mice destroy tolerance to CEA with the effectiveness bigger than CEA albumen.With DNA or the immunity of Ad carrier the time, can observe the enhanced immunogenic properties of these fusion roteins.Yet these two fusion roteins are at the CD8 that causes at CEA ⁺The bigger ability of T cell aspect can overcome by DNA/Ad vaccine inoculation mode at least in part.

Embodiment 17

The research of T cell depleting

(Perricone et al., J.Immunother.27 (4): 273-81 (2004) as described; Yoon et al., J.Ethnopharmacol.93 (2-3): 247-53 (2004)) injects anti-CD4 (GK1.5 hybridoma), anti-CD8 (Lyt2.2 hybridoma) or anti-Asialo GM1 (Wako Chemicals by i.p., Richmond VA) exhausts the CD4 of animal of immunity ⁺T cell, CD8 ⁺T cell, NK cell.In the-7 days injection of antibodies (ascites/dosage of 100 μ l dilution), in injection 5 * 10 with respect to tumor challenge ⁵Injected weekly for 3 weeks after the individual MC38-CEA cell.(PharMingen, San Diego CA) verify the situation of exhausting by wandering cells analysis of accounts peripheral blood in conjunction with the anti-CD4 of the MAbs of phycoerythrin, anti-CD8 and anti-NK by using; 99% cells involved subclass is depleted, and every other subclass remains on normal level.

Embodiment 18

The CEA-DOM immunity is brought into play antitumous effect in the CEA transgenic mice

Next we determined whether the immunogenicity of the raising of CEA-DOM or CEA-FcIgG fusions also produces the enhanced result of treatment that can disturb tumor development.For this purpose, the group of each 10 CEA transgenic mice experiences DNA/DNA, Ad/Ad or DNA/Ad immunization ways with CEA-DOM, CEA-FcIgG or CEA carrier.Last immunity two weeks of back are with 5 * 10 ⁵The CEA transgenic mice is attacked in the subcutaneous injection of individual MC38-CEA cell, and the MC38-CEA cell is the homogenic tumor cell line (Clarke et al., the same) of expressing CEA.Do not produce any significant antitumous effect with DNA/DNA or the immunity of Ad/Ad mode, what (accompanying drawing 24) the expressed albumen of the carrier of no matter injecting is.On the contrary, the DNA-EP/Ad immunity of the carrier of coding CEA-DOM fusion rotein has produced significant antitumous effect, and in the mouse of 10 processing 7 kept no tumour in back 34 days in attack.Thereby these results show that enhanced CEA specific immune response is relevant with CEA-DOMopt cDNA, and DNA/Ad vaccine inoculation mode is relevant with significant antitumous effect in the CEA transgenic mice.

Embodiment 19

The CEA-DOM antitumous effect depends on CD4 ⁺T cell, CD8 ⁺T cell and NK cell.

Characterized the effector cell who when the carrier with coding CEA-DOM fusions carries out DNA-EP and Ad immunity, relates in the observed antitumous effect.After the DNA/Ad immunity, but before tumor challenge, exhaust the CD4 of mouse by MAbs ⁺, CD8 ⁺T cell or NK cell.In the process of tumor challenge, give antibody to guarantee exhausting relevant NK and T cell subclass (subset) constantly.The antibody that use is specific to cell surface marker carries out exhaust (data not shown) that the wandering cells analysis of accounts monitors all three kinds of cell types.Exhaust CD4 ⁺, CD8 ⁺T cell or NK cell have side effect for the survival of mice immunized, compare a large amount of minimizings (accompanying drawing 25) that cause no mice with tumor with the group of inoculation.Thereby these data show, NK, CD4 ⁺And CD8 ⁺The T cell plays an important role in the antitumous effect of CEA-DOM vaccine inoculation performance.

Embodiment 20

Statistical study

In the place of indicating, check analytical results by log rank or two tailed Student t.It is significant that p value＜0.05 is considered to.

Sequence table

<110>Istituto?Di?Ricerche?Di?Biologia?Molecolare?P.Angeletti?S.P.A.

＜120〉carcinoembryonic antigen fusions and its purposes

<130>ITR0073Y?PCT

<150>60/635,791

<151>2004-12-14

<150>60/543,649

<151>2004-02-11

<160>54

<170>FastSEQ?for?Windows?Version?4.0

<210>1

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>1

tattctagat?gctccccaga?ctattacaga?a 31

<210>2

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>2

tatgcggccg?cctagttttc?catactgatt?gccgc 35

<210>3

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>3

gctctagagc?cccccagagc?atcaccgagc?tgtgc 35

<210>4

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>4

gctctagaac?ccctcagaac?atcaccgatc?tgtgcgcc 38

<210>5

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>5

tattctagat?aatggcgaca?aattataccg 30

<210>6

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>6

tatgcggccg?ctcataattc?atcccgaatt?ctgtt 35

<210>7

<211>2766

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEA-LTA fusions

<400>7

atggagtctc?cctcggcccc?tccccacaga?tggtgcatcc?cctggcagag?gctcctgctc 60

acagcctcac?ttctaacctt?ctggaacccg?cccaccactg?ccaagctcac?tattgaatcc 120

acgccgttca?atgtcgcaga?ggggaaggag?gtgcttctac?ttgtccacaa?tctgccccag 180

catctttttg?gctacagctg?gtacaaaggt?gaaagagtgg?atggcaaccg?tcaaattata 240

ggatatgtaa?taggaactca?acaagctacc?ccagggcccg?catacagtgg?tcgagagata 300

atatacccca?atgcatccct?gctgatccag?aacatcatcc?agaatgacac?aggattctac 360

accctacacg?tcataaagtc?agatcttgtg?aatgaagaag?caactggcca?gttccgggta 420

tacccggagc?tgcccaagcc?ctccatctcc?agcaacaact?ccaaacccgt?ggaggacaag 480

gatgctgtgg?ccttcacctg?tgaacctgag?actcaggacg?caacctacct?gtggtgggta 540

aacaatcaga?gcctcccggt?cagtcccagg?ctgcagctgt?ccaatggcaa?caggaccctc 600

actctattca?atgtcacaag?aaatgacaca?gcaagctaca?aatgtgaaac?ccagaaccca 660

gtgagtgcca?ggcgcagtga?ttcagtcatc?ctgaatgtcc?tctatggccc?ggatgccccc 720

accatttccc?ctctaaacac?atcttacaga?tcaggggaaa?atctgaacct?ctcctgccac 780

gcagcctcta?acccacctgc?acagtactct?tggtttgtca?atgggacttt?ccagcaatcc 840

acccaagagc?tctttatccc?caacatcact?gtgaataata?gtggatccta?tacgtgccaa 900

gcccataact?cagacactgg?cctcaatagg?accacagtca?cgacgatcac?agtctatgca 960

gagccaccca?aacccttcat?caccagcaac?aactccaacc?ccgtggagga?tgaggatgct 1020

gtagccttaa?cctgtgaacc?tgagattcag?aacacaacct?acctgtggtg?ggtaaataat 1080

cagagcctcc?cggtcagtcc?caggctgcag?ctgtccaatg?acaacaggac?cctcactcta 1140

ctcagtgtca?caaggaatga?tgtaggaccc?tatgagtgtg?gaatccagaa?cgaattaagt 1200

gttgaccaca?gcgacccagt?catcctgaat?gtcctctatg?gcccagacga?ccccaccatt 1260

tccccctcat?acacctatta?ccgtccaggg?gtgaacctca?gcctctcctg?ccatgcagcc 1320

tctaacccac?ctgcacagta?ttcttggctg?attgatggga?acatccagca?acacacacaa 1380

gagctcttta?tctccaacat?cactgagaag?aacagcggac?tctatacctg?ccaggccaat 1440

aactcagcca?gtggccacag?caggactaca?gtcaagacaa?tcacagtctc?tgcggagctg 1500

cccaagccct?ccatctccag?caacaactcc?aaacccgtgg?aggacaagga?tgctgtggcc 1560

ttcacctgtg?aacctgaggc?tcagaacaca?acctacctgt?ggtgggtaaa?tggtcagagc 1620

ctcccagtca?gtcccaggct?gcagctgtcc?aatggcaaca?ggaccctcac?tctattcaat 1680

gtcacaagaa?atgacgcaag?agcctatgta?tgtggaatcc?agaactcagt?gagtgcaaac 1740

cgcagtgacc?cagtcaccct?ggatgtcctc?tatgggccgg?acacccccat?catttccccc 1800

ccagactcgt?cttacctttc?gggagcgaac?ctcaacctct?cctgccactc?ggcctctaac 1860

ccatccccgc?agtattcttg?gcgtatcaat?gggataccgc?agcaacacac?acaagttctc 1920

tttatcgcca?aaatcacgcc?aaataataac?gggacctatg?cctgttttgt?ctctaacttg 1980

gctactggcc?gcaataattc?catagtcaag?agcatcacag?tctctgcatc?tggaactcta 2040

gttaatggcg?acaaattata?ccgtgctgac?tctagacccc?cagatgaaat?aaaacgttcc 2100

ggaggtctta?tgcccagagg?gcataatgag?tacttcgata?gaggaactca?aatgaatatt 2160

aatctttatg?atcacgcgag?aggaacacaa?accggctttg?tcagatatga?tgacggatat 2220

gtttccactt?ctcttagttt?gagaagtgct?cacttagcag?gacagtctat?attatcagga 2280

tattccactt?actatatata?tgttatagcg?acagcaccaa?atatgtttaa?tgttaatgat?2340

gtattaggcg?tatacagccc?tcacccatat?gaacaggagg?tttctgcgtt?aggtggaata?2400

ccatattctc?agatatatgg?atggtatcgt?gttaattttg?gtgtaattga?tgaacgatta?2460

catcgtaaca?gggaatatag?agaccggtat?tacagaaatc?tgaatatagc?tccggcagag?2520

gatggttaca?gattagcagg?tttcccaccg?gatcaccaag?cttggagaga?agaaccctgg?2580

attcatcatg?caccacaagg?ttgtggaaat?tcatcaagaa?caattacaga?tgatacttgt?2640

aatgaggaga?cccagaatct?gagcacaata?tatctcagga?aatatcaatc?aaaagttaag?2700

aggcagatat?tttcagacta?tcagtcagag?gttgacatat?ataacagaat?tcgggatgaa?2760

ttatga 2766

<210>8

<211>921

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CEA-LTB fusions

<400>8

Met?Glu?Ser?Pro?Ser?Ala?Pro?Pro?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Arg?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Lys?Leu?Thr?Ile?Glu?Ser?Thr?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Val?His?Asn?Leu?Pro?Gln?His?Leu?Phe?Gly

50 55 60

Tyr?Ser?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Gly?Asn?Arg?Gln?Ile?Ile

65 70 75 80

Gly?Tyr?Val?Ile?Gly?Thr?Gln?Gln?Ala?Thr?Pro?Gly?Pro?Ala?Tyr?Ser

85 90 95

Gly?Arg?Glu?Ile?Ile?Tyr?Pro?Asn?Ala?Ser?Leu?Leu?Ile?Gln?Asn?Ile

100 105 110

Ile?Gln?Asn?Asp?Thr?Gly?Phe?Tyr?Thr?Leu?His?Val?Ile?Lys?Ser?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Ala?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Gln

180 185 190

Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn?Val?Thr?Arg?Asn

195 200 205

Asp?Thr?Ala?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Ala?Arg

210 215 220

Arg?Ser?Asp?Ser?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Ser?Tyr?Arg?Ser?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Thr?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Asp?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Thr?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Pro?Pro?Lys?Pro?Phe?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu

325 330 335

Asp?Glu?Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Ile?Gln?Asn?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg

355 360 365

Leu?Gln?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Leu?Leu?Ser?Val?Thr

370 375 380

Arg?Asn?Asp?Val?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Glu?Leu?Ser

385 390 395 400

Val?Asp?His?Ser?Asp?Pro?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Asp?Pro?Thr?Ile?Ser?Pro?Ser?Tyr?Thr?Tyr?Tyr?Arg?Pro?Gly?Val?Asn

420 425 430

Leu?Ser?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser

435 440 445

Trp?Leu?Ile?Asp?Gly?Asn?Ile?Gln?Gln?His?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Ser?Asn?Ile?Thr?Glu?Lys?Asn?Ser?Gly?Leu?Tyr?Thr?Cys?Gln?Ala?Asn

465 470 475 480

Asn?Ser?Ala?Ser?Gly?His?Ser?Arg?Thr?Thr?Val?Lys?Thr?Ile?Thr?Val

485 490 495

Ser?Ala?Glu?Leu?Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro

500 505 510

Val?Glu?Asp?Lys?Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Ala?Gln

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Gly?Gln?Ser?Leu?Pro?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn

545 550 555 560

Val?Thr?Arg?Asn?Asp?Ala?Arg?Ala?Tyr?Val?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Val?Ser?Ala?Asn?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asp?Val?Leu?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Ser?Ser?Tyr?Leu?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Ala?Ser?Asn?Pro?Ser?Pro?Gln

6l0 615 620

Tyr?Ser?Trp?Arg?Ile?Asn?Gly?Ile?Pro?Gln?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ala?Lys?Ile?Thr?Pro?Asn?Asn?Asn?Gly?Thr?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Ser?Ile

660 665 670

Thr?Val?Ser?Ala?Ser?Gly?Thr?Leu?Val?Asn?Gly?Asp?Lys?Leu?Tyr?Arg

675 680 685

Ala?Asp?Ser?Arg?Pro?Pro?Asp?Glu?Ile?Lys?Arg?Ser?Gly?Gly?Leu?Met

690 695 700

Pro?Arg?Gly?His?Asn?Glu?Tyr?Phe?Asp?Arg?Gly?Thr?Gln?Met?Asn?Ile

705 7l0 715 720

Asn?Leu?Tyr?Asp?His?Ala?Arg?Gly?Thr?Gln?Thr?Gly?Phe?Val?Arg?Tyr

725 730 735

Asp?Asp?Gly?Tyr?Val?Ser?Thr?Ser?Leu?Ser?Leu?Arg?Ser?Ala?His?Leu

740 745 750

Ala?Gly?Gln?Ser?Ile?Leu?Ser?Gly?Tyr?Ser?Thr?Tyr?Tyr?Ile?Tyr?Val

755 760 765

Ile?Ala?Thr?Ala?Pro?Asn?Met?Phe?Asn?Val?Asn?Asp?Val?Leu?Gly?Val

770 775 780

Tyr?Ser?Pro?His?Pro?Tyr?Glu?Gln?Glu?Val?Ser?Ala?Leu?Gly?Gly?Ile

785 790 795 800

Pro?Tyr?Ser?Gln?Ile?Tyr?Gly?Trp?Tyr?Arg?Val?Asn?Phe?Gly?Val?Ile

805 810 815

Asp?Glu?Arg?Leu?His?Arg?Asn?Arg?Glu?Tyr?Arg?Asp?Arg?Tyr?Tyr?Arg

820 825 830

Asn?Leu?Asn?Ile?Ala?Pro?Ala?Glu?Asp?Gly?Tyr?Arg?Leu?Ala?Gly?Phe

835 840 845

Pro?Pro?Asp?His?Gln?Ala?Trp?Arg?Glu?Glu?Pro?Trp?Ile?His?His?Ala

850 855 860

Pro?Gln?Gly?Cys?Gly?Asn?Ser?Ser?Arg?Thr?Ile?Thr?Asp?Asp?Thr?Cys

865 870 875 880

Asn?Glu?Glu?Thr?Gln?Asn?Leu?Ser?Thr?Ile?Tyr?Leu?Arg?Lys?Tyr?Gln

885 890 895

Ser?Lys?Val?Lys?Arg?Gln?Ile?Phe?Ser?Asp?Tyr?Gln?Ser?Glu?Val?Asp

900 905 910

Ile?Tyr?Asn?Arg?Ile?Arg?Asp?Glu?Leu

915 920

<210>9

<211>2355

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEA-LTB fusions

<400>9

atggagtctc?cctcggcccc?tccccacaga?tggtgcatcc?cctggcagag?gctcctgctc?60

acagcctcac?ttctaacctt?ctggaacccg?cccaccactg?ccaagctcac?tattgaatcc?120

acgccgttca?atgtcgcaga?ggggaaggag?gtgcttctac?ttgtccacaa?tctgccccag?180

catctttttg?gctacagctg?gtacaaaggt?gaaagagtgg?atggcaaccg?tcaaattata?240

ggatatgtaa?taggaactca?acaagctacc?ccagggcccg?catacagtgg?tcgagagata?300

atatacccca?atgcatccct?gctgatccag?aacatcatcc?agaatgacac?aggattctac?360

accctacacg?tcataaagtc?agatcttgtg?aatgaagaag?caactggcca?gttccgggta?420

tacccggagc?tgcccaagcc?ctccatctcc?agcaacaact?ccaaacccgt?ggaggacaag?480

gatgctgtgg?ccttcacctg?tgaacctgag?actcaggacg?caacctacct?gtggtgggta?540

aacaatcaga?gcctcccggt?cagtcccagg?ctgcagctgt?ccaatggcaa?caggaccctc?600

actctattca?atgtcacaag?aaatgacaca?gcaagctaca?aatgtgaaac?ccagaaccca?660

gtgagtgcca?ggcgcagtga?ttcagtcatc?ctgaatgtcc?tctatggccc?ggatgccccc?720

accatttccc?ctctaaacac?atcttacaga?tcaggggaaa?atctgaacct?ctcctgccac?780

gcagcctcta?acccacctgc?acagtactct?tggtttgtca?atgggacttt?ccagcaatcc?840

acccaagagc?tctttatccc?caacatcact?gtgaataata?gtggatccta?tacgtgccaa?900

gcccataact?cagacactgg?cctcaatagg?accacagtca?cgacgatcac?agtctatgca?960

gagccaccca?aacccttcat?caccagcaac?aactccaacc?ccgtggagga?tgaggatgct?1020

gtagccttaa?cctgtgaacc?tgagattcag?aacacaacct?acctgtggtg?ggtaaataat?1080

cagagcctcc?cggtcagtcc?caggctgcag?ctgtccaatg?acaacaggac?cctcactcta?1140

ctcagtgtca?caaggaatga?tgtaggaccc?tatgagtgtg?gaatccagaa?cgaattaagt?1200

gttgaccaca?gcgacccagt?catcctgaat?gtcctctatg?gcccagacga?ccccaccatt?1260

tccccctcat?acacctatta?ccgtccaggg?gtgaacctca?gcctctcctg?ccatgcagcc?1320

tctaacccac?ctgcacagta?ttcttggctg?attgatggga?acatccagca?acacacacaa?1380

gagctcttta?tctccaacat?cactgagaag?aacagcggac?tctatacctg?ccaggccaat?1440

aactcagcca?gtggccacag?caggactaca?gtcaagacaa?tcacagtctc?tgcggagctg?1500

cccaagccct?ccatctccag?caacaactcc?aaacccgtgg?aggacaagga?tgctgtggcc?1560

ttcacctgtg?aacctgaggc?tcagaacaca?acctacctgt?ggtgggtaaa?tggtcagagc?1620

ctcccagtca?gtcccaggct?gcagctgtcc?aatggcaaca?ggaccctcac?tctattcaat?1680

gtcacaagaa?atgacgcaag?agcctatgta?tgtggaatcc?agaactcagt?gagtgcaaac?1740

cgcagtgacc?cagtcaccct?ggatgtcctc?tatgggccgg?acacccccat?catttccccc?1800

ccagactcgt?cttacctttc?gggagcgaac?ctcaacctct?cctgccactc?ggcctctaac?1860

ccatccccgc?agtattcttg?gcgtatcaat?gggataccgc?agcaacacac?acaagttctc?1920

tttatcgcca?aaatcacgcc?aaataataac?gggacctatg?cctgttttgt?ctctaacttg?1980

gctactggcc?gcaataattc?catagtcaag?agcatcacag?tctctgcatc?tggaactcta?2040

gatgctcccc?agtctattac?agaactatgt?tcggaatatc?gcaacacaca?aatatatacg?2100

ataaatgaca?agatactatc?atatacggaa?tcgatggcag?gtaaaagaga?aatggttatc?2160

attacattta?agagcggcgc?aacatttcag?gtcgaagtcc?cgggcagtca?acatatagac?2220

tcccaaaaaa?aagccattga?aaggatgaag?gacacattaa?gaatcacata?tctgaccgag?2280

accaaaattg?ataaattatg?tgtatggaat?aataaaaccc?ccaattcaat?tgcggcaatc?2340

agtatggaaa?actag 2355

<210>10

<211>784

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CEA-LTB fusions

<400>10

Met?Glu?Ser?Pro?Ser?Ala?Pro?Pro?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Arg?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Lys?Leu?Thr?Ile?Glu?Ser?Thr?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Val?His?Asn?Leu?Pro?Gln?His?Leu?Phe?Gly

50 55 60

Tyr?Ser?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Gly?Asn?Arg?Gln?Ile?Ile

65 70 75 80

Gly?Tyr?Val?Ile?Gly?Thr?Gln?Gln?Ala?Thr?Pro?Gly?Pro?Ala?Tyr?Ser

85 90 95

Gly?Arg?Glu?Ile?Ile?Tyr?Pro?Asn?Ala?Ser?Leu?Leu?Ile?Gln?Asn?Ile

100 105 110

Ile?Gln?Asn?Asp?Thr?Gly?Phe?Tyr?Thr?Leu?His?Val?Ile?Lys?Ser?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Ala?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Gln

180 185 190

Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn?Val?Thr?Arg?Asn

195 200 205

Asp?Thr?Ala?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Ala?Arg

210 215 220

Arg?Ser?Asp?Ser?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Ser?Tyr?Arg?Ser?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Thr?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Asp?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Thr?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Pro?Pro?Lys?Pro?Phe?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu

325 330 335

Asp?Glu?Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Ile?Gln?Asn?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg

355 360 365

Leu?Gln?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Leu?Leu?Ser?Val?Thr

370 375 380

Arg?Asn?Asp?Val?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Glu?Leu?Ser

385 390 395 400

Val?Asp?His?Ser?Asp?Pro?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Asp?Pro?Thr?Ile?Ser?Pro?Ser?Tyr?Thr?Tyr?Tyr?Arg?Pro?Gly?Val?Asn

420 425 430

Leu?Ser?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser

435 440 445

Trp?Leu?Ile?Asp?Gly?Asn?Ile?Gln?Gln?His?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Ser?Asn?Ile?Thr?Glu?Lys?Asn?Ser?Gly?Leu?Tyr?Thr?Cys?Gln?Ala?Asn

465 470 475 480

Asn?Ser?Ala?Ser?Gly?His?Ser?Arg?Thr?Thr?Val?Lys?Thr?Ile?Thr?Val

485 490 495

Ser?Ala?Glu?Leu?Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro

500 505 510

Val?Glu?Asp?Lys?Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Ala?Gln

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Gly?Gln?Ser?Leu?Pro?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn

545 550 555 560

Val?Thr?Arg?Asn?Asp?Ala?Arg?Ala?Tyr?Val?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Val?Ser?Ala?Asn?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asp?Val?Leu?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Ser?Ser?Tyr?Leu?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Ala?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Arg?Ile?Asn?Gly?Ile?Pro?Gln?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ala?Lys?Ile?Thr?Pro?Asn?Asn?Asn?Gly?Thr?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Ash?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Ser?Ile

660 665 670

Thr?Val?Ser?Ala?Ser?Gly?Thr?Leu?Asp?Ala?Pro?Gln?Ser?Ile?Thr?Glu

675 680 685

Leu?Cys?Ser?Glu?Tyr?Arg?Asn?Thr?Gln?Ile?Tyr?Thr?Ile?Asn?Asp?Lys

690 695 700

Ile?Leu?Ser?Tyr?Thr?Glu?Ser?Met?Ala?Gly?Lys?Arg?Glu?Met?Val?Ile

705 710 715 720

Ile?Thr?Phe?Lys?Ser?Gly?Ala?Thr?Phe?Gln?Val?Glu?Val?Pro?Gly?Ser

725 730 735

Gln?His?Ile?Asp?Ser?Gln?Lys?Lys?Ala?Ile?Glu?Arg?Met?Lys?Asp?Thr

740 745 750

Leu?Arg?Ile?Thr?Tyr?Leu?Thr?Glu?Thr?Lys?Ile?Asp?Lys?Leu?Cys?Val

755 760 765

Trp?Asn?Asn?Lys?Thr?Pro?Asn?Ser?Ile?Ala?Ala?Ile?Ser?Met?Glu?Asn

770 775 780

<210>11

<211>2355

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEAopt-LTB fusions

<400>11

atggagagcc?ccagcgcccc?cccccaccgc?tggtgcatcc?cctggcagcg?cctgctgctg?60

accgccagcc?tgctgacctt?ctggaacccc?cccaccaccg?ccaagctgac?catcgagagc?120

acccccttca?acgtggccga?gggcaaggag?gtgctgctgc?tggtgcacaa?cctgccccag?180

cacctgttcg?gctacagctg?gtacaagggc?gagcgcgtgg?acggcaaccg?ccagatcatc?240

ggctacgtga?tcggcaccca?gcaggccacc?cccggccccg?cctacagcgg?ccgcgagatc?300

atctacccca?acgccagcct?gctgatccag?aacatcatcc?agaacgacac?cggcttctac?360

accctgcacg?tgatcaagag?cgacctggtg?aacgaggagg?ccaccggcca?gttccgcgtg?420

taccccgagc?tgcccaagcc?cagcatcagc?agcaacaaca?gcaagcccgt?ggaggacaag?480

gacgccgtgg?ccttcacctg?cgagcccgag?acccaggacg?ccacctacct?gtggtgggtg?540

aacaaccaga?gcctgcccgt?gagcccccgc?ctgcagctga?gcaacggcaa?ccgcaccctg?600

accctgttca?acgtgacccg?caacgacacc?gccagctaca?agtgcgagac?ccagaacccc?660

gtgagcgccc?gccgcagcga?cagcgtgatc?ctgaacgtgc?tgtacggccc?cgacgccccc?720

accatcagcc?ccctgaacac?cagctaccgc?agcggcgaga?acctgaacct?gagctgccac?780

gccgccagca?acccccccgc?ccagtacagc?tggttcgtga?acggcacctt?ccagcagagc?840

acccaggagc?tgttcatccc?caacatcacc?gtgaacaaca?gcggcagcta?cacctgccag?900

gcccacaaca?gcgacaccgg?cctgaaccgc?accaccgtga?ccaccatcac?cgtgtacgcc?960

gagcccccca?agcccttcat?caccagcaac?aacagcaacc?ccgtggagga?cgaggacgcc?1020

gtggccctga?cctgcgagcc?cgagatccag?aacaccacct?acctgtggtg?ggtgaacaac?1080

cagagcctgc?ccgtgagccc?ccgcctgcag?ctgagcaacg?acaaccgcac?cctgaccctg?1140

ctgagcgtga?cccgcaacga?cgtgggcccc?tacgagtgcg?gcatccagaa?cgagctgagc?1200

gtggaccaca?gcgaccccgt?gatcctgaac?gtgctgtacg?gccccgacga?ccccaccatc?1260

agccccagct?acacctacta?ccgccccggc?gtgaacctga?gcctgagctg?ccacgccgcc?1320

agcaaccccc?ccgcccagta?cagctggctg?atcgacggca?acatccagca?gcacacccag?1380

gagctgttca?tcagcaacat?caccgagaag?aacagcggcc?tgtacacctg?ccaggccaac?1440

aacagcgcca?gcggccacag?ccgcaccacc?gtgaagacca?tcaccgtgag?cgccgagctg?1500

cccaagccca?gcatcagcag?caacaacagc?aagcccgtgg?aggacaagga?cgccgtggcc?1560

ttcacctgcg?agcccgaggc?ccagaacacc?acctacctgt?ggtgggtgaa?cggccagagc?1620

ctgcccgtga?gcccccgcct?gcagctgagc?aacggcaacc?gcaccctgac?cctgttcaac?1680

gtgacccgca?acgacgcccg?cgcctacgtg?tgcggcatcc?agaacagcgt?gagcgccaac?1740

cgcagcgacc?ccgtgaccct?ggacgtgctg?tacggccccg?acacccccat?catcagcccc?1800

cccgacagca?gctacctgag?cggcgccaac?ctgaacctga?gctgccacag?cgccagcaac?1860

cccagccccc?agtacagctg?gcgcatcaac?ggcatccccc?agcagcacac?ccaggtgctg?1920

ttcatcgcca?agatcacccc?caacaacaac?ggcacctacg?cctgcttcgt?gagcaacctg?1980

gccaccggcc?gcaacaacag?catcgtgaag?agcatcaccg?tgagcgccag?cggcacctct?2040

agagctcccc?agactattac?agaactatgt?tcggaatatc?gcaacacaca?aatatatacg?2100

ataaatgaca?agatactatc?atatacggaa?tcgatggcag?gcaaaagaga?aatggttatc?2160

attacattta?agagcggcga?aacatttcag?gtcgaagtcc?cgggcagtca?acatatagac?2220

tcccagaaaa?aagccattga?aaggatgaag?gacacattaa?gaatcacata?tctgaccgag?2280

accaaaattg?ataaattatg?tgtatggaat?aataaaaccc?ccaattcaat?tgcggcaatc?2340

agtatggaaa?actag 2355

<210>12

<211>2358

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEAopt-LTBopt fusions

<400>12

atggagagcc?ccagcgcccc?cccccaccgc?tggtgcatcc?cctggcagcg?cctgctgctg?60

accgccagcc?tgctgacctt?ctggaacccc?cccaccaccg?ccaagctgac?catcgagagc?120

acccccttca?acgtggccga?gggcaaggag?gtgctgctgc?tggtgcacaa?cctgccccag?180

cacctgttcg?gctacagctg?gtacaagggc?gagcgcgtgg?acggcaaccg?ccagatcatc?240

ggctacgtga?tcggcaccca?gcaggccacc?cccggccccg?cctacagcgg?ccgcgagatc?300

atctacccca?acgccagcct?gctgatccag?aacatcatcc?agaacgacac?cggcttctac?360

accctgcacg?tgatcaagag?cgacctggtg?aacgaggagg?ccaccggcca?gttccgcgtg?420

taccccgagc?tgcccaagcc?cagcatcagc?agcaacaaca?gcaagcccgt?ggaggacaag?480

gacgccgtgg?ccttcacctg?cgagcccgag?acccaggacg?ccacctacct?gtggtgggtg?540

aacaaccaga?gcctgcccgt?gagcccccgc?ctgcagctga?gcaacggcaa?ccgcaccctg?600

accctgttca?acgtgacccg?caacgacacc?gccagctaca?agtgcgagac?ccagaacccc?660

gtgagcgccc?gccgcagcga?cagcgtgatc?ctgaacgtgc?tgtacggccc?cgacgccccc?720

accatcagcc?ccctgaacac?cagctaccgc?agcggcgaga?acctgaacct?gagctgccac?780

gccgccagca?acccccccgc?ccagtacagc?tggttcgtga?acggcacctt?ccagcagagc?840

acccaggagc?tgttcatccc?caacatcacc?gtgaacaaca?gcggcagcta?cacctgccag?900

gcccacaaca?gcgacaccgg?cctgaaccgc?accaccgtga?ccaccatcac?cgtgtacgcc?960

gagcccccca?agcccttcat?caccagcaac?aacagcaacc?ccgtggagga?cgaggacgcc?1020

gtggccctga?cctgcgagcc?cgagatccag?aacaccacct?acctgtggtg?ggtgaacaac?1080

cagagcctgc?ccgtgagccc?ccgcctgcag?ctgagcaacg?acaaccgcac?cctgaccctg?1140

ctgagcgtga?cccgcaacga?cgtgggcccc?tacgagtgcg?gcatccagaa?cgagctgagc?1200

gtggaccaca?gcgaccccgt?gatcctgaac?gtgctgtacg?gccccgacga?ccccaccatc?1260

agccccagct?acacctacta?ccgccccggc?gtgaacctga?gcctgagctg?ccacgccgcc?1320

agcaaccccc?ccgcccagta?cagctggctg?atcgacggca?acatccagca?gcacacccag?1380

gagctgttca?tcagcaacat?caccgagaag?aacagcggcc?tgtacacctg?ccaggccaac?1440

aacagcgcca?gcggccacag?ccgcaccacc?gtgaagacca?tcaccgtgag?cgccgagctg?1500

cccaagccca?gcatcagcag?caacaacagc?aagcccgtgg?aggacaagga?cgccgtggcc?1560

ttcacctgcg?agcccgaggc?ccagaacacc?acctacctgt?ggtgggtgaa?cggccagagc?1620

ctgcccgtga?gcccccgcct?gcagctgagc?aacggcaacc?gcaccctgac?cctgttcaac?1680

gtgacccgca?acgacgcccg?cgcctacgtg?tgcggcatcc?agaacagcgt?gagcgccaac?1740

cgcagcgacc?ccgtgaccct?ggacgtgctg?tacggccccg?acacccccat?catcagcccc?1800

cccgacagca?gctacctgag?cggcgccaac?ctgaacctga?gctgccacag?cgccagcaac?1860

cccagccccc?agtacagctg?gcgcatcaac?ggcatccccc?agcagcacac?ccaggtgctg?1920

ttcatcgcca?agatcacccc?caacaacaac?ggcacctacg?cctgcttcgt?gagcaacctg?1980

gccaccggcc?gcaacaacag?catcgtgaag?agcatcaccg?tgagcgccag?cggcacctct?2040

agagcccccc?agagcatcac?cgagctgtgc?agcgagtacc?ggaacaccca?gatctacacc?2100

atcaacgaca?agatcctgag?ctacaccgag?agcatggccg?gcaagaggga?gatggtgatc?2160

atcaccttca?agagcggcgc?caccttccag?gtggaggtgc?ccggcagcca?gcacatcgac?2220

agccagaaga?aggccatcga?gcggatgaag?gacaccctgc?ggatcaccta?cctcaccgag?2280

accaagatcg?acaagctgtg?cgtgtggaac?aacaagaccc?ccaacagcat?cgccgccatc?2340

agcatggaga?attgataa 2358

<210>13

<211>784

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CEAopt-LTBopt fusions

<400>13

Met?Glu?Ser?Pro?Ser?Ala?Pro?Pro?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Arg?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Lys?Leu?Thr?Ile?Glu?Ser?Thr?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Val?His?Asn?Leu?Pro?Gln?His?Leu?Phe?Gly

50 55 60

Tyr?Ser?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Gly?Asn?Arg?Gln?Ile?Ile

65 70 75 80

Gly?Tyr?Val?Ile?Gly?Thr?Gln?Gln?Ala?Thr?Pro?Gly?Pro?Ala?Tyr?Ser

85 90 95

Gly?Arg?Glu?Ile?Ile?Tyr?Pro?Asn?Ala?Ser?Leu?Leu?Ile?Gln?Asn?Ile

100 105 110

Ile?Gln?Asn?Asp?Thr?Gly?Phe?Tyr?Thr?Leu?His?Val?Ile?Lys?Ser?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Ala?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Gln

180 185 190

Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn?Val?Thr?Arg?Asn

195 200 205

Asp?Thr?Ala?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Ala?Arg

2l0 215 220

Arg?Ser?Asp?Ser?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Ser?Tyr?Arg?Ser?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Thr?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Asp?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Thr?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Pro?Pro?Lys?Pro?Phe?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu

325 330 335

Asp?Glu?Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Ile?Gln?Asn?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg

355 360 365

Leu?Gln?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Leu?Leu?Ser?Val?Thr

370 375 380

Arg?Asn?Asp?Val?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Glu?Leu?Ser

385 390 395 400

Val?Asp?His?Ser?Asp?Pro?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Asp?Pro?Thr?Ile?Ser?Pro?Ser?Tyr?Thr?Tyr?Tyr?Arg?Pro?Gly?Val?Asn

420 425 430

Leu?Ser?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser

435 440 445

Trp?Leu?Ile?Asp?Gly?Asn?Ile?Gln?Gln?His?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Ser?Asn?Ile?Thr?Glu?Lys?Asn?Ser?Gly?Leu?Tyr?Thr?Cys?Gln?Ala?Asn

465 470 475 480

Asn?Ser?Ala?Ser?Gly?His?Ser?Arg?Thr?Thr?Val?Lys?Thr?Ile?Thr?Val

485 490 495

Ser?Ala?Glu?Leu?Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro

500 505 510

Val?Glu?Asp?Lys?Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Ala?Gln

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Gly?Gln?Ser?Leu?Pro?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn

545 550 555 560

Val?Thr?Arg?Asn?Asp?Ala?Arg?Ala?Tyr?Val?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Val?Ser?Ala?Asn?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asp?Val?Leu?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Ser?Ser?Tyr?Leu?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Ala?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Arg?Ile?Asn?Gly?IIe?Pro?Gln?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ala?Lys?Ile?Thr?Pro?Asn?Asn?Asn?Gly?Thr?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Ser?Ile

660 665 670

Thr?Val?Ser?Ala?Ser?Gly?Thr?Ser?Arg?Ala?Pro?Gln?Ser?IIe?Thr?Glu

675 680 685

Leu?Cys?Ser?Glu?Tyr?Arg?Asn?Thr?Gln?Ile?Tyr?Thr?Ile?Asn?Asp?Lys

690 695 700

Ile?Leu?Ser?Tyr?Thr?Glu?Ser?Met?Ala?Gly?Lys?Arg?Glu?Met?Val?Ile

705 710 715 720

Ile?Thr?Phe?Lys?Ser?Gly?Ala?Thr?Phe?Gln?Val?Glu?Val?Pro?Gly?Ser

725 730 735

Gln?His?Ile?Asp?Ser?Gln?Lys?Lys?Ala?Ile?Glu?Arg?Met?Lys?Asp?Thr

740 745 750

Leu?Arg?Ile?Thr?Tyr?Leu?Thr?Glu?Thr?Lys?Ile?Asp?Lys?Leu?Cys?Val

755 760 765

Trp?Asn?Asn?Lys?Thr?Pro?Asn?Ser?Ile?Ala?Ala?Ile?Ser?Met?Glu?Asn

770 775 780

<210>14

<211>2358

<212>DNA

＜213〉artificial sequence

<220>

＜223〉rhCEAopt-LTBopt fusions

<400>14

atgggcagcc?ccagcgcccc?cctgcaccgc?tggtgcatcc?cctggcagac?cctgctgctg?60

accgccagcc?tgctgacctt?ctggaacccc?cccaccaccg?cccagctgac?catcgagagc?120

cgccccttca?acgtggccga?gggcaaggag?gtgctgctgc?tggcccacaa?cgtgagccag?180

aacctgttcg?gctacatctg?gtacaagggc?gagcgcgtgg?acgccagccg?ccgcatcggc?240

agctgcgtga?tccgcaccca?gcagatcacc?cccggccccg?cccacagcgg?ccgcgagacc?300

atcgacttca?acgccagcct?gctgatccac?aacgtgaccc?agagcgacac?cggcagctac?360

accatccagg?tgatcaagga?ggacctggtg?aacgaggagg?ccaccggcca?gttccgcgtg?420

taccccgagc?tgcccaagcc?ctacatcagc?agcaacaaca?gcaaccccgt?ggaggacaag?480

gacgccgtgg?ccctgacctg?cgagcccgag?acccaggaca?ccacctacct?gtggtgggtg?540

aacaaccaga?gcctgcccgt?gagcccccgc?ctggagctga?gcagcgacaa?ccgcaccctg?600

accgtgttca?acatcccccg?caacgacacc?accagctaca?agtgcgagac?ccagaacccc?660

gtgagcgtgc?gccgcagcga?ccccgtgacc?ctgaacgtgc?tgtacggccc?cgacgccccc?720

accatcagcc?ccctgaacac?cccctaccgc?gccggcgaga?acctgaacct?gacctgccac?780

gccgccagca?accccaccgc?ccagtacttc?tggttcgtga?acggcacctt?ccagcagagc?840

acccaggagc?tgttcatccc?caacatcacc?gtgaacaaca?gcggcagcta?catgtgccag?900

gcccacaaca?gcgccaccgg?cctgaaccgc?accaccgtga?ccgccatcac?cgtgtacgcc?960

gagctgccca?agccctacat?caccagcaac?aacagcaacc?ccatcgagga?caaggacgcc?1020

gtgaccctga?cctgcgagcc?cgagacccag?gacaccacct?acctgtggtg?ggtgaacaac?1080

cagagcctga?gcgtgagcag?ccgcctggag?ctgagcaacg?acaaccgcac?cctgaccgtg?l140

ttcaacatcc?cccgcaacga?caccaccttc?tacgagtgcg?agacccagaa?ccccgtgagc?1200

gtgcgccgca?gcgaccccgt?gaccctgaac?gtgctgtacg?gccccgacgc?ccccaccatc?1260

agccccctga?acacccccta?ccgcgccggc?gagaacctga?acctgagctg?ccacgccgcc?1320

agcaaccccg?ccgcccagta?cagctggttc?gtgaacggca?ccttccagca?gagcacccag?1380

gagctgttca?tccccaacat?caccgtgaac?aacagcggca?gctacatgtg?ccaggcccac?1440

aacagcgcca?ccggcctgaa?ccgcaccacc?gtgaccgcca?tcaccgtgta?cgtggagctg?1500

cccaagccct?acatcagcag?caacaacagc?aaccccatcg?aggacaagga?cgccgtgacc?1560

ctgacctgcg?agcccgtggc?cgagaacacc?acctacctgt?ggtgggtgaa?caaccagagc?1620

ctgagcgtga?gcccccgcct?gcagctgagc?aacggcaacc?gcatcctgac?cctgctgagc?1680

gtgacccgca?acgacaccgg?cccctacgag?tgcggcatcc?agaacagcga?gagcgccaag?1740

cgcagcgacc?ccgtgaccct?gaacgtgacc?tacggccccg?acacccccat?catcagcccc?1800

cccgacctga?gctaccgcag?cggcgccaac?ctgaacctga?gctgccacag?cgacagcaac?1860

cccagccccc?agtacagctg?gctgatcaac?ggcaccctgc?gccagcacac?ccaggtgctg?1920

ttcatcagca?agatcaccag?caacaacagc?ggcgcctacg?cctgcttcgt?gagcaacctg?1980

gccaccggcc?gcaacaacag?catcgtgaag?aacatcagcg?tgagcagcgg?cgacagctct?2040

agagcccccc?agagcatcac?cgagctgtgc?agcgagtacc?ggaacaccca?gatctacacc?2100

atcaacgaca?agatcctgag?ctacaccgag?agcatggccg?gcaagaggga?gatggtgatc?2160

atcaccttca?agagcggcgc?caccttccag?gtggaggtgc?ccggcagcca?gcacatcgac?2220

agccagaaga?aggccatcga?gcggatgaag?gacaccctgc?ggatcaccta?cctcaccgag?2280

accaagatcg?acaagctgtg?cgtgtggaac?aacaagaccc?ccaacagcat?cgccgccatc?2340

agcatggaga?attgataa 2358

<210>15

<211>784

<212>PRT

＜213〉artificial sequence

<220>

＜223〉rhCEAopt-LTBopt fusions

<400>15

Met?Gly?Ser?Pro?Ser?Ala?Pro?Leu?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Thr?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Gln?Leu?Thr?Ile?Glu?Ser?Arg?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Ala?His?Asn?Val?Ser?Gln?Asn?Leu?Phe?Gly

50 55 60

Tyr?Ile?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Ala?Ser?Arg?Arg?Ile?Gly

65 70 75 80

Ser?Cys?Val?Ile?Arg?Thr?Gln?Gln?Ile?Thr?Pro?Gly?Pro?Ala?His?Ser

85 90 95

Gly?Arg?Glu?Thr?Ile?Asp?Phe?Asn?Ala?Ser?Leu?Leu?Ile?His?Asn?Val

100 105 110

Thr?Gln?Ser?Asp?Thr?Gly?Ser?Tyr?Thr?Ile?Gln?Val?Ile?Lys?Glu?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Glu

180 185 190

Leu?Ser?Ser?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro?Arg?Asn

195 200 205

Asp?Thr?Thr?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Val?Arg

210 215 220

Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Thr?Cys?His?Ala?Ala?Ser?Asn?Pro?Thr?Ala?Gln?Tyr?Phe?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Ile?Glu

325 330 335

Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser?Ser?Arg

355 360 365

Leu?Glu?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro

370 375 380

Arg?Asn?Asp?Thr?Thr?Phe?Tyr?Glu?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser

385 390 395 400

Val?Arg?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Ala?Pro?Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn

420 425 430

Leu?Asn?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Ala?Ala?Gln?Tyr?Ser

435 440 445

Trp?Phe?Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Pro?Asn?Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His

465 470 475 480

Asn?Ser?Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val

485 490 495

Tyr?Val?Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro

500 505 510

Ile?Glu?Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Val?Ala?Glu

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Ile?Leu?Thr?Leu?Leu?Ser

545 550 555 560

Val?Thr?Arg?Asn?Asp?Thr?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Glu?Ser?Ala?Lys?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Thr?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Leu?Ser?Tyr?Arg?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Asp?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Leu?Ile?Asn?Gly?Thr?Leu?Arg?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ser?Lys?Ile?Thr?Ser?Asn?Asn?Ser?Gly?Ala?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Asn?Ile

660 665 670

Ser?Val?Ser?Ser?Gly?Asp?Ser?Ser?Arg?Ala?Pro?Gln?Ser?Ile?Thr?Glu

675 680 685

Leu?Cys?Ser?Glu?Tyr?Arg?Asn?Thr?Gln?Ile?Tyr?Thr?Ile?Asn?Asp?Lys

690 695 700

Ile?Leu?Ser?Tyr?Thr?Glu?Ser?Met?Ala?Gly?Lys?Arg?Glu?Met?Val?Ile

705 710 715 720

Ile?Thr?Phe?Lys?Ser?Gly?Ala?Thr?Phe?Gln?Val?Glu?Val?Pro?Gly?Ser

725 730 735

Gln?His?Ile?Asp?Ser?Gln?Lys?Lys?Ala?Ile?Glu?Arg?Met?Lys?Asp?Thr

740 745 750

Leu?Arg?Ile?Thr?Tyr?Leu?Thr?Glu?Thr?Lys?Ile?Asp?Lys?Leu?Cys?Val

755 760 765

Trp?Asn?Asn?Lys?Thr?Pro?Asn?Ser?Ile?Ala?Ala?Ile?Ser?Met?Glu?Asn

770 775 780

<210>16

<211>2118

<212>DNA

<213>Macaca?mulatta

<400>16

atggggtctc?cctcagcccc?tcttcacaga?tggtgcatcc?cctggcagac?gctcctgctc?60

acagcctcac?ttctaacctt?ctggaacccg?cccaccactg?cccagctcac?tattgaatcc?120

aggccgttca?atgttgcaga?ggggaaggag?gttcttctac?ttgcccacaa?tgtgtcccag?180

aatctttttg?gctacatttg?gtacaaggga?gaaagagtgg?atgccagccg?tcgaattgga?240

tcatgtgtaa?taagaactca?acaaattacc?ccagggcccg?cacacagcgg?tcgagagaca?300

atagacttca?atgcatccct?gctgatccac?aatgtcaccc?agagtgacac?aggatcctac?360

accatacaag?tcataaagga?agatcttgtg?aatgaagaag?caactggcca?gttccgggta?420

tacccggagc?tgcccaagcc?ctacatctcc?agcaacaact?ccaaccccgt?ggaggacaag?480

gatgctgtgg?ccttaacctg?tgaacctgag?actcaggaca?caacctacct?gtggtgggta?540

aacaatcaga?gcctcccggt?cagtcccagg?ctggagctgt?ccagtgacaa?caggaccctc?600

actgtattca?atattccaag?aaatgacaca?acatcctaca?aatgtgaaac?ccagaaccca?660

gtgagtgtca?gacgcagcga?cccagtcacc?ctgaacgtcc?tctatggccc?ggatgcgccc?720

accatttccc?ctctaaacac?accttacaga?gcaggggaaa?atctgaacct?cacctgccac?780

gcagcctcta?acccaactgc?acagtacttt?tggtttgtca?atgggacgtt?ccagcaatcc?840

acacaagagc?tctttatacc?caacatcacc?gtgaataata?gcggatccta?tatgtgccaa?900

gcccataact?cagccactgg?cctcaatagg?accacagtca?cggcgatcac?agtctacgcg?960

gagctgccca?agccctacat?caccagcaac?aactccaacc?ccatagagga?caaggatgct?1020

gtgaccttaa?cctgtgaacc?tgagactcag?gacacaacct?acctgtggtg?ggtaaacaat?1080

cagagcctct?cggtcagttc?caggctggag?ctgtccaatg?acaacaggac?cctcactgta?1140

ttcaatattc?caagaaacga?cacaacgttc?tacgaatgtg?agacccagaa?cccagtgagt?1200

gtcagacgca?gcgacccagt?caccctgaat?gtcctctatg?gcccggatgc?gcccaccatt?1260

tcccctctaa?acacacctta?cagagcaggg?gaaaatctga?acctctcctg?ccacgcagcc?1320

tctaacccag?ctgcacagta?ctcttggttt?gtcaatggga?cgttccagca?atccacacaa?1380

gagctcttta?tacccaacat?caccgtgaat?aatagcggat?cctatatgtg?ccaagcccat?1440

aactcagcca?ctggcctcaa?taggaccaca?gtcacggcga?tcacagtcta?tgtggagctg?1500

cccaagccct?acatctccag?caacaactcc?aaccccatag?aggacaagga?tgctgtgacc?1560

ttaacctgtg?aacctgtggc?tgagaacaca?acctacctgt?ggtgggtaaa?caatcagagc?1620

ctctcggtca?gtcccaggct?gcagctctcc?aatggcaaca?ggatcctcac?tctactcagt?1680

gtcacacgga?atgacacagg?accctatgaa?tgtggaatcc?agaactcaga?gagtgcaaaa?1740

cgcagtgacc?cagtcaccct?gaatgtcacc?tatggcccag?acacccccat?catatccccc?1800

ccagacttgt?cttaccgttc?gggagcaaac?ctcaacctct?cctgccactc?ggactctaac?1860

ccatccccgc?agtattcttg?gcttatcaat?gggacactgc?ggcaacacac?acaagttctc?1920

tttatctcca?aaatcacatc?aaacaatagc?ggggcctatg?cctgttttgt?ctctaacttg?1980

gctaccggtc?gcaataactc?catagtcaag?aacatctcag?tctcctctgg?cgattcagca?2040

cctggaagtt?ctggtctctc?agctagggct?actgtcggca?tcataattgg?aatgctggtt?2100

ggggttgctc?tgatgtag 2118

<210>17

<211>2118

<212>DNA

<213>Macaca?mulatta

<400>17

atggggtctc?cctcagcccc?tcttcacaga?tggtgcatcc?cctggcagac?gctcctgctc?60

acagcctcac?ttctaacctt?ctggaacccg?cccaccactg?cccagctcac?tattgaatcc?120

aggccgttca?atgttgcaga?ggggaaggag?gttcttctac?ttgcccacaa?tgtgtcccag?180

aatctttttg?gctacatttg?gtacaaggga?gaaagagtgg?atgccagccg?tcgaattgga?240

tcatgtgtaa?taagaactca?acaaattacc?ccagggcccg?cacacagcgg?tcgagagaca?300

atagacttca?atgcatccct?gctgatccac?aatgtcaccc?agagtgacac?aggatcctac?360

accatacaag?tcataaagga?agatcttgtg?aatgaagaag?caactggcca?gttccgggta?420

tacccggagc?tgcccaagcc?ctacatctcc?agcaacaact?ccaaccccgt?ggaggacaag?480

gatgctgtgg?ccttaacctg?tgaacctgag?actcaggaca?caacctacct?gtggtgggta?540

aacaatcaga?gcctcccggt?cagtcccagg?ctggagctgt?ccagtgacaa?caggaccctc?600

actgtattca?atattccaag?aaatgacaca?acatcctaca?aatgtgaaac?ccagaaccca?660

gtgagtgtca?gacgcagcga?cccagtcacc?ctgaacgtcc?tctatggccc?ggatgcgccc?720

accatttccc?ctctaaacac?accttacaga?gcaggggaaa?atctgaacct?cacctgccac?780

gcagcctcta?acccaactgc?acagtacttt?tggtttgtca?atgggacgtt?ccagcaatcc?840

acacaagagc?tctttatacc?caacatcacc?gtgaataata?gcggatccta?tatgtgccaa?900

gcccataact?cagccactgg?cctcaatagg?accacagtca?cggcgatcac?agtctacgcg?960

gagctgccca?agccctacat?caccagcaac?aactccaacc?ccatagagga?caaggatgct?1020

gtgaccttaa?cctgtgaacc?tgagactcag?gacacaacct?acctgtggtg?ggtaaacaat?1080

cagagcctct?cggtcagttc?caggctggag?ctgtccaatg?acaacaggac?cctcactgta?1140

ttcaatattc?caagaaacga?cacaacgttc?tacgaatgtg?agacccagaa?cccagtgagt?1200

gtcagacgca?gcgacccagt?caccctgaat?gtcctctatg?gcccggatgc?gcccaccatt?1260

tcccctctaa?acacacctta?cagagcaggg?gaaaatctga?acctctcctg?ccacgcagcc?1320

tctaacccag?ctgcacagta?cttttggttt?gtcaatggga?cgttccagca?atccacacaa?1380

gagctcttta?tacccaacat?caccgtgaat?aatagcggat?cctatatgtg?ccaagcccat?1440

aactcagcca?ctggcctcaa?taggaccaca?gtcacggcga?tcacagtcta?tgtggagctg?1500

cccaagccct?acatctccag?caacaactcc?aaccccatag?aggacaagga?tgctgtgacc?1560

ttaacctgtg?aacctgtggc?tgagaacaca?acctacctgt?ggtgggtaaa?caatcagagc?1620

ctctcggtca?gtcccaggct?gcagctctcc?aatggcaaca?ggatcctcac?tctactcagt?1680

gtcacacgga?atgacacagg?accctatgaa?tgtggaatcc?agaactcaga?gagtgcaaaa?1740

cgcagtgacc?cagtcaccct?gaatgtcacc?tatggcccag?acacccccat?catatccccc?1800

ccagacttgt?cttaccgttc?gggagcaaac?ctcaacctct?cctgccactc?ggactctaac?1860

ccatccccgc?agtattcttg?gcttatcaat?gggacactgc?ggcaacacac?acaagttctc?1920

tttatctcca?aaatcacatc?aaacaataac?ggggcctatg?cctgttttgt?ctctaacttg?1980

gctaccggtc?gcaataactc?catagtcaag?aacatctcag?tctcctctgg?cgattcagca?2040

cctggaagtt?ctggtctctc?agctagggct?actgtcggca?tcataattgg?aatgctggtt?2100

ggggttgctc?tgatgtag 2118

<210>18

<211>705

<212>PRT

<213>Macaca?mulatta

<400>18

Met?Gly?Ser?Pro?Ser?Ala?Pro?Leu?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Thr?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Gln?Leu?Thr?Ile?Glu?Ser?Arg?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Ala?His?Asn?Val?Ser?Gln?Asn?Leu?Phe?Gly

50 55 60

Tyr?Ile?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Ala?Ser?Arg?Arg?Ile?Gly

65 70 75 80

Ser?Cys?Val?Ile?Arg?Thr?Gln?Gln?Ile?Thr?Pro?Gly?Pro?Ala?His?Ser

85 90 95

Gly?Arg?Glu?Thr?Ile?Asp?Phe?Asn?Ala?Ser?Leu?Leu?Ile?His?Asn?Val

100 105 110

Thr?Gln?Ser?Asp?Thr?Gly?Ser?Tyr?Thr?Ile?Gln?Val?Ile?Lys?Glu?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Glu

180 185 190

Leu?Ser?Ser?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro?Arg?Asn

195 200 205

Asp?Thr?Thr?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Val?Arg

210 215 220

Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Thr?Cys?His?Ala?Ala?Ser?Asn?Pro?Thr?Ala?Gln?Tyr?Phe?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Ile?Glu

325 330 335

Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser?Ser?Arg

355 360 365

Leu?Glu?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro

370 375 380

Arg?Asn?Asp?Thr?Thr?Phe?Tyr?Glu?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser

385 390 395 400

Val?Arg?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Ala?Pro?Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn

420 425 430

Leu?Asn?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Ala?Ala?Gln?Tyr?Ser

435 440 445

Trp?Phe?Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Pro?Asn?Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His

465 470 475 480

Asn?Ser?Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val

485 490 495

Tyr?Val?Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro

500 505 510

Ile?Glu?Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Val?Ala?Glu

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Ile?Leu?Thr?Leu?Leu?Ser

545 550 555 560

Val?Thr?Arg?Asn?Asp?Thr?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Glu?Ser?Ala?Lys?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Thr?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Leu?Ser?Tyr?Arg?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Asp?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Leu?Ile?Asn?Gly?Thr?Leu?Arg?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ser?Lys?Ile?Thr?Ser?Asn?Asn?Ser?Gly?Ala?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Asn?Ile

660 665 670

Ser?Val?Ser?Ser?Gly?Asp?Ser?Ala?Pro?Gly?Ser?Ser?Gly?Leu?Ser?Ala

675 680 685

Arg?Ala?Thr?Val?Gly?Ile?Ile?Ile?Gly?Met?Leu?Val?Gly?Val?Ala?Leu

690 695 700

Met

705

<210>19

<211>705

<212>PRT

<213>Macaca?mulatta

<400>19

Met?Gly?Ser?Pro?Ser?Ala?Pro?Leu?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Thr?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Gln?Leu?Thr?Ile?Glu?Ser?Arg?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Ala?His?Asn?Val?Ser?Gln?Asn?Leu?Phe?Gly

50 55 60

Tyr?Ile?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Ala?Ser?Arg?Arg?Ile?Gly

65 70 75 80

Ser?Cys?Val?Ile?Arg?Thr?Gln?Gln?Ile?Thr?Pro?Gly?Pro?Ala?His?Ser

85 90 95

Gly?Arg?Glu?Thr?Ile?Asp?Phe?Asn?Ala?Ser?Leu?Leu?Ile?His?Asn?Val

100 105 110

Thr?Gln?Ser?Asp?Thr?Gly?Ser?Tyr?Thr?Ile?Gln?Val?Ile?Lys?Glu?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Glu

180 185 190

Leu?Ser?Ser?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro?Arg?Asn

195 200 205

Asp?Thr?Thr?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Val?Arg

210 215 220

Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Thr?Cys?His?Ala?Ala?Ser?Asn?Pro?Thr?Ala?Gln?Tyr?Phe?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Ile?Glu

325 330 335

Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser?Ser?Arg

355 360 365

Leu?Glu?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro

370 375 380

Arg?Asn?Asp?Thr?Thr?Phe?Tyr?Glu?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser

385 390 395 400

Val?Arg?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 4l0 415

Ala?Pro?Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn

420 425 430

Leu?Asn?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Ala?Ala?Gln?Tyr?Phe

435 440 445

Trp?Phe?Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Pro?Asn?Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His

465 470 475 480

Asn?Ser?Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val

485 490 495

Tyr?Val?Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro

500 505 510

Ile?Glu?Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Val?Ala?Glu

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Ile?Leu?Thr?Leu?Leu?Ser

545 550 555 560

Val?Thr?Arg?Asn?Asp?Thr?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Glu?Ser?Ala?Lys?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Thr?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Leu?Ser?Tyr?Arg?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Asp?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Leu?Ile?Asn?Gly?Thr?Leu?Arg?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ser?Lys?Ile?Thr?Ser?Asn?Asn?Asn?Gly?Ala?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Asn?Ile

660 665 670

Ser?Val?Ser?Ser?Gly?Asp?Ser?Ala?Pro?Gly?Ser?Ser?Gly?Leu?Ser?Ala

675 680 685

Arg?Ala?Thr?Val?Gly?Ile?Ile?Ile?Gly?Met?Leu?Val?Gly?Val?Ala?Leu

690 695 700

Met

705

<210>20

<211>702

<212>PRT

<213>Homo?sapiens

<400>20

Met?Glu?Ser?Pro?Ser?Ala?Pro?Pro?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Arg?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Lys?Leu?Thr?Ile?Glu?Ser?Thr?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Val?His?Asn?Leu?Pro?Gln?His?Leu?Phe?Gly

50 55 60

Tyr?Ser?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Gly?Asn?Arg?Gln?Ile?Ile

65 70 75 80

Gly?Tyr?Val?Ile?Gly?Thr?Gln?Gln?Ala?Thr?Pro?Gly?Pro?Ala?Tyr?Ser

85 90 95

Gly?Arg?Glu?Ile?Ile?Tyr?Pro?Asn?Ala?Ser?Leu?Leu?Ile?Gln?Asn?Ile

100 105 110

Ile?Gln?Asn?Asp?Thr?Gly?Phe?Tyr?Thr?Leu?His?Val?Ile?Lys?Ser?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Ala?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Gln

180 185 190

Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn?Val?Thr?Arg?Asn

195 200 205

Asp?Thr?Ala?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Ala?Arg

210 215 220

Arg?Ser?Asp?Ser?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Ser?Tyr?Arg?Ser?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Thr?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Asp?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Thr?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Pro?Pro?Lys?Pro?Phe?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu

325 330 335

Asp?Glu?Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Ile?Gln?Asn?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg

355 360 365

Leu?Gln?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Leu?Leu?Ser?Val?Thr

370 375 380

Arg?Asn?Asp?Val?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Glu?Leu?Ser

385 390 395 400

Val?Asp?His?Ser?Asp?Pro?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Asp?Pro?Thr?Ile?Ser?Pro?Ser?Tyr?Thr?Tyr?Tyr?Arg?Pro?Gly?Val?Asn

420 425 430

Leu?Ser?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser

435 440 445

Trp?Leu?Ile?Asp?Gly?Asn?Ile?Gln?Gln?His?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Ser?Asn?Ile?Thr?Glu?Lys?Asn?Ser?Gly?Leu?Tyr?Thr?Cys?Gln?Ala?Asn

465 470 475 480

Asn?Ser?Ala?Ser?Gly?His?Ser?Arg?Thr?Thr?Val?Lys?Thr?Ile?Thr?Val

485 490 495

Ser?Ala?Glu?Leu?Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro

500 505 510

Val?Glu?Asp?Lys?Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Ala?Gln

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Gly?Gln?Ser?Leu?Pro?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn

545 550 555 560

Val?Thr?Arg?Asn?Asp?Ala?Arg?Ala?Tyr?Val?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Val?Ser?Ala?Asn?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asp?Val?Leu?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Ser?Ser?Tyr?Leu?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Ala?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Arg?Ile?Asn?Gly?Ile?Pro?Gln?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ala?Lys?Ile?Thr?Pro?Asn?Asn?Asn?Gly?Thr?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Ser?Ile

660 665 670

Thr?Val?Ser?Ala?Ser?Gly?Thr?Ser?Pro?Gly?Leu?Ser?Ala?Gly?Ala?Thr

675 680 685

Val?Gly?Ile?Met?Ile?Gly?Val?Leu?Val?Gly?Val?Ala?Leu?Ile

690 695 700

<210>21

<211>2859

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEAoptDOMopt fusions

<400>21

atggagagcc?ccagcgcccc?cccccaccgc?tggtgcatcc?cctggcagcg?cctgctgctg?60

accgccagcc?tgctgacctt?ctggaacccc?cccaccaccg?ccaagctgac?catcgagagc?120

acccccttca?acgtggccga?gggcaaggag?gtgctgctgc?tggtgcacaa?cctgccccag?180

cacctgttcg?gctacagctg?gtacaagggc?gagcgcgtgg?acggcaaccg?ccagatcatc?240

ggctacgtga?tcggcaccca?gcaggccacc?cccggccccg?cctacagcgg?ccgcgagatc?300

atctacccca?acgccagcct?gctgatccag?aacatcatcc?agaacgacac?cggcttctac?360

accctgcacg?tgatcaagag?cgacctggtg?aacgaggagg?ccaccggcca?gttccgcgtg?420

taccccgagc?tgcccaagcc?cagcatcagc?agcaacaaca?gcaagcccgt?ggaggacaag?480

gacgccgtgg?ccttcacctg?cgagcccgag?acccaggacg?ccacctacct?gtggtgggtg?540

aacaaccaga?gcctgcccgt?gagcccccgc?ctgcagctga?gcaacggcaa?ccgcaccctg?600

accctgttca?acgtgacccg?caacgacacc?gccagctaca?agtgcgagac?ccagaacccc?660

gtgagcgccc?gccgcagcga?cagcgtgatc?ctgaacgtgc?tgtacggccc?cgacgccccc?720

accatcagcc?ccctgaacac?cagctaccgc?agcggcgaga?acctgaacct?gagctgccac?780

gccgccagca?acccccccgc?ccagtacagc?tggttcgtga?acggcacctt?ccagcagagc?840

acccaggagc?tgttcatccc?caacatcacc?gtgaacaaca?gcggcagcta?cacctgccag?900

gcccacaaca?gcgacaccgg?cctgaaccgc?accaccgtga?ccaccatcac?cgtgtacgcc?960

gagcccccca?agcccttcat?caccagcaac?aacagcaacc?ccgtggagga?cgaggacgcc?1020

gtggccctga?cctgcgagcc?cgagatccag?aacaccacct?acctgtggtg?ggtgaacaac?1080

cagagcctgc?ccgtgagccc?ccgcctgcag?ctgagcaacg?acaaccgcac?cctgaccctg?1140

ctgagcgtga?cccgcaacga?cgtgggcccc?tacgagtgcg?gcatccagaa?cgagctgagc?1200

gtggaccaca?gcgaccccgt?gatcctgaac?gtgctgtacg?gccccgacga?ccccaccatc?1260

agccccagct?acacctacta?ccgccccggc?gtgaacctga?gcctgagctg?ccacgccgcc?1320

agcaaccccc?ccgcccagta?cagctggctg?atcgacggca?acatccagca?gcacacccag?1380

gagctgttca?tcagcaacat?caccgagaag?aacagcggcc?tgtacacctg?ccaggccaac?1440

aacagcgcca?gcggccacag?ccgcaccacc?gtgaagacca?tcaccgtgag?cgccgagctg?1500

cccaagccca?gcatcagcag?caacaacagc?aagcccgtgg?aggacaagga?cgccgtggcc?1560

ttcacctgcg?agcccgaggc?ccagaacacc?acctacctgt?ggtgggtgaa?cggccagagc?1620

ctgcccgtga?gcccccgcct?gcagctgagc?aacggcaacc?gcaccctgac?cctgttcaac?1680

gtgacccgca?acgacgcccg?cgcctacgtg?tgcggcatcc?agaacagcgt?gagcgccaac?1740

cgcagcgacc?ccgtgaccct?ggacgtgctg?tacggccccg?acacccccat?catcagcccc?1800

cccgacagca?gctacctgag?cggcgccaac?ctgaacctga?gctgccacag?cgccagcaac?1860

cccagccccc?agtacagctg?gcgcatcaac?ggcatccccc?agcagcacac?ccaggtgctg?1920

ttcatcgcca?agatcacccc?caacaacaac?ggcacctacg?cctgcttcgt?gagcaacctg?1980

gccaccggcc?gcaacaacag?catcgtgaag?agcatcaccg?tgagcgccag?cggcacctct?2040

agaagcaccc?ccatcccatt?cagctacagc?aagaacctgg?actgctgggt?ggacaacgag?2100

gaggacatcg?acgtgatcct?gaagaagagc?accatcctga?acctggacat?caacaacgac?2160

atcatcagcg?acatcagcgg?cttcaacagc?agcgtgatca?cctaccccga?cgcccagctg?2220

gtgcccggca?tcaacggcaa?ggccatccac?ctggtgaaca?acgagagcag?cgaggtgatc?2280

gtgcacaagg?ccatggacat?cgagtacaac?gacatgttca?acaacttcac?cgtgagcttc?2340

tggctgagag?tgcctaaggt?gagcgccagc?cacctggagc?agtacggcac?caacgagtac?2400

agcatcatca?gcagcatgaa?gaagcacagc?ctgagcatcg?gcagcggctg?gagcgtgagc?2460

ctgaagggca?acaacctcat?ctggaccctg?aaggatagcg?ccggagaggt?gagacagatc?2520

accttcagag?acctgcccga?caagttcaat?gcctacctgg?ccaacaagtg?ggtgttcatc?2580

accatcacca?acgacagact?gagcagcgcc?aacctgtaca?tcaacggcgt?gctcatgggc?2640

agcgccgaga?tcaccggcct?gggcgccatc?agagaggaca?acaacatcac?cctgaagctg?2700

gacagatgca?acaacaacaa?ccagtacgtg?agcatcgaca?agttccggat?cttctgcaag?2760

gccctgaacc?ccaaggagat?cgagaagctg?tacaccagct?acctgagcat?caccttcctg?2820

agagacttct?ggggcaaccc?cctgagatac?gacacctag 2859

<210>22

<211>2037

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Shan Chu human CEA

<400>22

atggagtctc?cctcggcccc?tccccacaga?tggtgcatcc?cctggcagag?gctcctgctc?60

acagcctcac?ttctaacctt?ctggaacccg?cccaccactg?ccaagctcac?tattgaatcc?120

acgccgttca?atgtcgcaga?ggggaaggag?gtgcttctac?ttgtccacaa?tctgccccag?180

catctttttg?gctacagctg?gtacaaaggt?gaaagagtgg?atggcaaccg?tcaaattata?240

ggatatgtaa?taggaactca?acaagctacc?ccagggcccg?catacagtgg?tcgagagata?300

atatacccca?atgcatccct?gctgatccag?aacatcatcc?agaatgacac?aggattctac?360

accctacacg?tcataaagtc?agatcttgtg?aatgaagaag?caactggcca?gttccgggta?420

tacccggagc?tgcccaagcc?ctccatctcc?agcaacaact?ccaaacccgt?ggaggacaag?480

gatgctgtgg?ccttcacctg?tgaacctgag?actcaggacg?caacctacct?gtggtgggta?540

aacaatcaga?gcctcccggt?cagtcccagg?ctgcagctgt?ccaatggcaa?caggaccctc?600

actctattca?atgtcacaag?aaatgacaca?gcaagctaca?aatgtgaaac?ccagaaccca?660

gtgagtgcca?ggcgcagtga?ttcagtcatc?ctgaatgtcc?tctatggccc?ggatgccccc?720

accatttccc?ctctaaacac?atcttacaga?tcaggggaaa?atctgaacct?ctcctgccac?780

gcagcctcta?acccacctgc?acagtactct?tggtttgtca?atgggacttt?ccagcaatcc?840

acccaagagc?tctttatccc?caacatcact?gtgaataata?gtggatccta?tacgtgccaa?900

gcccataact?cagacactgg?cctcaatagg?accacagtca?cgacgatcac?agtctatgca?960

gagccaccca?aacccttcat?caccagcaac?aactccaacc?ccgtggagga?tgaggatgct?1020

gtagccttaa?cctgtgaacc?tgagattcag?aacacaacct?acctgtggtg?ggtaaataat?1080

cagagcctcc?cggtcagtcc?caggctgcag?ctgtccaatg?acaacaggac?cctcactcta?1140

ctcagtgtca?caaggaatga?tgtaggaccc?tatgagtgtg?gaatccagaa?cgaattaagt?1200

gttgaccaca?gcgacccagt?catcctgaat?gtcctctatg?gcccagacga?ccccaccatt?1260

tccccctcat?acacctatta?ccgtccaggg?gtgaacctca?gcctctcctg?ccatgcagcc?1320

tctaacccac?ctgcacagta?ttcttggctg?attgatggga?acatccagca?acacacacaa?1380

gagctcttta?tctccaacat?cactgagaag?aacagcggac?tctatacctg?ccaggccaat?1440

aactcagcca?gtggccacag?caggactaca?gtcaagacaa?tcacagtctc?tgcggagctg?1500

cccaagccct?ccatctccag?caacaactcc?aaacccgtgg?aggacaagga?tgctgtggcc?1560

ttcacctgtg?aacctgaggc?tcagaacaca?acctacctgt?ggtgggtaaa?tggtcagagc?1620

ctcccagtca?gtcccaggct?gcagctgtcc?aatggcaaca?ggaccctcac?tctattcaat?1680

gtcacaagaa?atgacgcaag?agcctatgta?tgtggaatcc?agaactcagt?gagtgcaaac?1740

cgcagtgacc?cagtcaccct?ggatgtcctc?tatgggccgg?acacccccat?catttccccc?1800

ccagactcgt?cttacctttc?gggagcgaac?ctcaacctct?cctgccactc?ggcctctaac?1860

ccatccccgc?agtattcttg?gcgtatcaat?gggataccgc?agcaacacac?acaagttctc?1920

tttatcgcca?aaatcacgcc?aaataataac?gggacctatg?cctgttttgt?ctctaacttg?1980

gctactggcc?gcaataattc?catagtcaag?agcatcacag?tctctgcatc?tggaact 2037

<210>23

<211>679

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Shan Chu human CEA

<400>23

Met?Glu?Ser?Pro?Ser?Ala?Pro?Pro?His?Arg?Trp?Gys?Ile?Pro?Trp?Gln

1 5 10 15

Arg?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Lys?Leu?Thr?Ile?Glu?Ser?Thr?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Val?His?Asn?Leu?Pro?Gln?His?Leu?Phe?Gly

50 55 60

Tyr?Ser?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Gly?Asn?Arg?Gln?Ile?Ile

65 70 75 80

Gly?Tyr?Val?Ile?Gly?Thr?Gln?Gln?Ala?Thr?Pro?Gly?Pro?Ala?Tyr?Ser

85 90 95

Gly?Arg?Glu?Ile?Ile?Tyr?Pro?Asn?Ala?Ser?Leu?Leu?Ile?Gln?Asn?Ile

100 105 110

Ile?Gln?Asn?Asp?Thr?Gly?Phe?Tyr?Thr?Leu?His?Val?Ile?Lys?Ser?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Ala?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Gln

180 185 190

Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn?Val?Thr?Arg?Asn

195 200 205

Asp?Thr?Ala?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Ala?Arg

210 215 220

Arg?Ser?Asp?Ser?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Ser?Tyr?Arg?Ser?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Thr?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Asp?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Thr?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Pro?Pro?Lys?Pro?Phe?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu

325 330 335

Asp?Glu?Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Ile?Gln?Asn?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg

355 360 365

Leu?Gln?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Leu?Leu?Ser?Val?Thr

370 375 380

Arg?Asn?Asp?Val?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Glu?Leu?Ser

385 390 395 400

Val?Asp?His?Ser?Asp?Pro?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Asp?Pro?Thr?Ile?Ser?Pro?Ser?Tyr?Thr?Tyr?Tyr?Arg?Pro?Gly?Val?Asn

420 425 430

Leu?Ser?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser

435 440 445

Trp?Leu?Ile?Asp?Gly?Asn?Ile?Gln?Gln?His?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Ser?Asn?Ile?Thr?Glu?Lys?Asn?Ser?Gly?Leu?Tyr?Thr?Cys?Gln?Ala?Asn

465 470 475 480

Asn?Ser?Ala?Ser?Gly?His?Ser?Arg?Thr?Thr?Val?Lys?Thr?Ile?Thr?Val

485 490 495

Ser?Ala?Glu?Leu?Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro

500 505 510

Val?Glu?Asp?Lys?Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Ala?Gln

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Gly?Gln?Ser?Leu?Pro?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn

545 550 555 560

Val?Thr?Arg?Asn?Asp?Ala?Arg?Ala?Tyr?Val?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Val?Ser?Ala?Asn?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asp?Val?Leu?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Ser?Ser?Tyr?Leu?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Ala?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Arg?Ile?Asn?Gly?Ile?Pro?Gln?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ala?Lys?Ile?Thr?Pro?Asn?Asn?Asn?Gly?Thr?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Ser?Ile

660 665 670

Thr?Val?Ser?Ala?Ser?Gly?Thr

675

<210>24

<211>3426

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEA-FRC fusions

<400>24

atggagtctc?cctcggcccc?tccccacaga?tggtgcatcc?cctggcagag?gctcctgctc?60

acagcctcac?ttctaacctt?ctggaacccg?cccaccactg?ccaagctcac?tattgaatcc?120

acgccgttca?atgtcgcaga?ggggaaggag?gtgcttctac?ttgtccacaa?tctgccccag?180

catctttttg?gctacagctg?gtacaaaggt?gaaagagtgg?atggcaaccg?tcaaattata?240

ggatatgtaa?taggaactca?acaagctacc?ccagggcccg?catacagtgg?tcgagagata?300

atatacccca?atgcatccct?gctgatccag?aacatcatcc?agaatgacac?aggattctac?360

accctacacg?tcataaagtc?agatcttgtg?aatgaagaag?caactggcca?gttccgggta?420

tacccggagc?tgcccaagcc?ctccatctcc?agcaacaact?ccaaacccgt?ggaggacaag?480

gatgctgtgg?ccttcacctg?tgaacctgag?actcaggacg?caacctacct?gtggtgggta?540

aacaatcaga?gcctcccggt?cagtcccagg?ctgcagctgt?ccaatggcaa?caggaccctc?600

actctattca?atgtcacaag?aaatgacaca?gcaagctaca?aatgtgaaac?ccagaaccca?660

gtgagtgcca?ggcgcagtga?ttcagtcatc?ctgaatgtcc?tctatggccc?ggatgccccc?720

accatttccc?ctctaaacac?atcttacaga?tcaggggaaa?atctgaacct?ctcctgccac?780

gcagcctcta?acccacctgc?acagtactct?tggtttgtca?atgggacttt?ccagcaatcc?840

acccaagagc?tctttatccc?caacatcact?gtgaataata?gtggatccta?tacgtgccaa?900

gcccataact?cagacactgg?cctcaatagg?accacagtca?cgacgatcac?agtctatgca?960

gagccaccca?aacccttcat?caccagcaac?aactccaacc?ccgtggagga?tgaggatgct?1020

gtagccttaa?cctgtgaacc?tgagattcag?aacacaacct?acctgtggtg?ggtaaataat?1080

cagagcctcc?cggtcagtcc?caggctgcag?ctgtccaatg?acaacaggac?cctcactcta?1140

ctcagtgtca?caaggaatga?tgtaggaccc?tatgagtgtg?gaatccagaa?cgaattaagt?1200

gttgaccaca?gcgacccagt?catcctgaat?gtcctctatg?gcccagacga?ccccaccatt?1260

tccccctcat?acacctatta?ccgtccaggg?gtgaacctca?gcctctcctg?ccatgcagcc?1320

tctaacccac?ctgcacagta?ttcttggctg?attgatggga?acatccagca?acacacacaa?1380

gagctcttta?tctccaacat?cactgagaag?aacagcggac?tctatacctg?ccaggccaat?1440

aactcagcca?gtggccacag?caggactaca?gtcaagacaa?tcacagtctc?tgcggagctg?1500

cccaagccct?ccatctccag?caacaactcc?aaacccgtgg?aggacaagga?tgctgtggcc?1560

ttcacctgtg?aacctgaggc?tcagaacaca?acctacctgt?ggtgggtaaa?tggtcagagc?1620

ctcccagtca?gtcccaggct?gcagctgtcc?aatggcaaca?ggaccctcac?tctattcaat?1680

gtcacaagaa?atgacgcaag?agcctatgta?tgtggaatcc?agaactcagt?gagtgcaaac?1740

cgcagtgacc?cagtcaccct?ggatgtcctc?tatgggccgg?acacccccat?catttccccc?1800

ccagactcgt?cttacctttc?gggagcgaac?ctcaacctct?cctgccactc?ggcctctaac?1860

ccatccccgc?agtattcttg?gcgtatcaat?gggataccgc?agcaacacac?acaagttctc?1920

tttatcgcca?aaatcacgcc?aaataataac?gggacctatg?cctgttttgt?ctctaacttg?1980

gctactggcc?gcaataattc?catagtcaag?agcatcacag?tctctgcatc?tggaactcta?2040

gattcaacac?caattccatt?ttcttattct?aaaaatctgg?attgttgggt?tgataatgaa?2100

gaagatatag?atgttatatt?aaaaaagagt?acaattttaa?atttagatat?taataatgat?2160

attatatcag?atatatctgg?gtttaattca?tctgtaataa?catatccaga?tgctcaattg?2220

gtgcccggaa?taaatggcaa?agcaatacat?ttagtaaaca?atgaatcttc?tgaagttata?2280

gtgcataaag?ctatggatat?tgaatataat?gatatgttta?ataattttac?cgttagcttt?2340

tggttgaggg?ttcctaaagt?atctgctagt?catttagaac?aatatggcac?aaatgagtat?2400

tcaataatta?gctctatgaa?aaaacatagt?ctatcaatag?gatctggttg?gagtgtatca?2460

cttaaaggta?ataacttaat?atggacttta?aaagattccg?cgggagaagt?tagacaaata?2520

acttttaggg?atttacctga?taaatttaat?gcttatttag?caaataaatg?ggtttttata?2580

actattacta?atgatagatt?atcttctgct?aatttgtata?taaatggagt?acttatggga?2640

agtgcagaaa?ttactggttt?aggagctatt?agagaggata?ataatataac?attaaaacta?2700

gatagatgta?ataataataa?tcaatacgtt?tctattgata?aatttaggat?attttgcaaa?2760

gcattaaatc?caaaagagat?tgaaaaatta?tacacaagtt?atttatctat?aaccttttta?2820

agagacttct?ggggaaaccc?tttacgatat?gatacagaat?attatttaat?accagtagct?2880

tctagttcta?aagatgttca?attgaaaaat?ataacagatt?atatgtattt?gacaaatgcg?2940

ccatcgtata?ctaacggaaa?attgaatata?tattatagaa?ggttatataa?tggactaaaa?3000

tttattataa?aaagatatac?acctaataat?gaaatagatt?cttttgttaa?atcaggtgat?3060

tttattaaat?tatatgtatc?atataacaat?aatgagcaca?ttgtaggtta?tccgaaagat?3120

ggaaatgcct?ttaataatct?tgatagaatt?ctaagagtag?gttataatgc?cccaggtatc?3180

cctctttata?aaaaaatgga?agcagtaaaa?ttgcgtgatt?taaaaaccta?ttctgtacaa?3240

cttaaattat?atgatgataa?aaatgcatct?ttaggactag?taggtaccca?taatggtcaa?3300

ataggcaacg?atccaaatag?ggatatatta?attgcaagca?actggtactt?taatcattta?3360

aaagataaaa?ttttaggatg?tgattggtac?tttgtaccta?cagatgaagg?atggacaaat?3420

gattaa 3426

<210>25

<211>2664

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEA-FcIgG fusions

<400>25

atggagagcc?ccagcgcccc?cccccaccgc?tggtgcatcc?cctggcagcg?cctgctgctg?60

accgccagcc?tgctgacctt?ctggaacccc?cccaccaccg?ccaagctgac?catcgagagc?120

acccccttca?acgtggccga?gggcaaggag?gtgctgctgc?tggtgcacaa?cctgccccag?180

cacctgttcg?gctacagctg?gtacaagggc?gagcgcgtgg?acggcaaccg?ccagatcatc?240

ggctacgtga?tcggcaccca?gcaggccacc?cccggccccg?cctacagcgg?ccgcgagatc?300

atctacccca?acgccagcct?gctgatccag?aacatcatcc?agaacgacac?cggcttctac?360

accctgcacg?tgatcaagag?cgacctggtg?aacgaggagg?ccaccggcca?gttccgcgtg?420

taccccgagc?tgcccaagcc?cagcatcagc?agcaacaaca?gcaagcccgt?ggaggacaag?480

gacgccgtgg?ccttcacctg?cgagcccgag?acccaggacg?ccacctacct?gtggtgggtg?540

aacaaccaga?gcctgcccgt?gagcccccgc?ctgcagctga?gcaacggcaa?ccgcaccctg?600

accctgttca?acgtgacccg?caacgacacc?gccagctaca?agtgcgagac?ccagaacccc?660

gtgagcgccc?gccgcagcga?cagcgtgatc?ctgaacgtgc?tgtacggccc?cgacgccccc?720

accatcagcc?ccctgaacac?cagctaccgc?agcggcgaga?acctgaacct?gagctgccac?780

gccgccagca?acccccccgc?ccagtacagc?tggttcgtga?acggcacctt?ccagcagagc?840

acccaggagc?tgttcatccc?caacatcacc?gtgaacaaca?gcggcagcta?cacctgccag?900

gcccacaaca?gcgacaccgg?cctgaaccgc?accaccgtga?ccaccatcac?cgtgtacgcc?960

gagcccccca?agcccttcat?caccagcaac?aacagcaacc?ccgtggagga?cgaggacgcc?1020

gtggccctga?cctgcgagcc?cgagatccag?aacaccacct?acctgtggtg?ggtgaacaac?1080

cagagcctgc?ccgtgagccc?ccgcctgcag?ctgagcaacg?acaaccgcac?cctgaccctg?1140

ctgagcgtga?cccgcaacga?cgtgggcccc?tacgagtgcg?gcatccagaa?cgagctgagc?1200

gtggaccaca?gcgaccccgt?gatcctgaac?gtgctgtacg?gccccgacga?ccccaccatc?1260

agccccagct?acacctacta?ccgccccggc?gtgaacctga?gcctgagctg?ccacgccgcc?1320

agcaaccccc?ccgcccagta?cagctggctg?atcgacggca?acatccagca?gcacacccag?1380

gagctgttca?tcagcaacat?caccgagaag?aacagcggcc?tgtacacctg?ccaggccaac?1440

aacagcgcca?gcggccacag?ccgcaccacc?gtgaagacca?tcaccgtgag?cgccgagctg?1500

cccaagccca?gcatcagcag?caacaacagc?aagcccgtgg?aggacaagga?cgccgtggcc?1560

ttcacctgcg?agcccgaggc?ccagaacacc?acctacctgt?ggtgggtgaa?cggccagagc?1620

ctgcccgtga?gcccccgcct?gcagctgagc?aacggcaacc?gcaccctgac?cctgttcaac?1680

cgcagcgacc?ccgtgaccct?ggacgtgctg?tacggccccg?acacccccat?catcagcccc?1740

cccgacagca?gctacctgag?cggcgccaac?ctgaacctga?gctgccacag?cgccagcaac?1800

cccagccccc?agtacagctg?gcgcatcaac?ggcatccccc?agcagcacac?ccaggtgctg?1860

ttcatcgcca?agatcacccc?caacaacaac?ggcacctacg?cctgcttcgt?gagcaacctg?1920

gccaccggcc?gcaacaacag?catcgtgaag?agcatcaccg?tgagcgccag?cggcacctct?1980

agaaagaccc?acacctgccc?cccttgccct?gcccctgagc?tgctgggcgg?acccagcgtg?2040

ttcctgttcc?cccccaagcc?taaggacacc?ctcatgatca?gcagaacccc?cgaggtgacc?2100

tgcgtggtgg?tggacgtgag?ccacgaggat?cccgaggtga?agttcaactg?gtacgtggac?2160

ggcgtggagg?tgcacaatgc?caagaccaag?cccagagagg?agcagtacaa?cagcacctac?2220

agagtggtga?gcgtgctcac?cgtgctgcac?caggattggc?tgaacggcaa?ggagtacaag?2280

tgcaaggtga?gcaacaaggc?cctgcctgcc?cccatcgaga?aaaccatcag?caaggccaag?2340

ggccagccca?gagagcccca?ggtgtacacc?ctgcccccta?gcagagatga?gttgaccaag?2400

aaccaggtga?gcctcacatg?cctggtgaag?ggcttctacc?ccagcgacat?cgccgtggag?2460

tgggagagca?acggccagcc?cgagaacaac?tacaagacca?ccccccctgt?gctggacagc?2520

gatggcagct?tcttcctgta?cagcaagctc?accgtggaca?agagcagatg?gcagcagggc?2580

aacgtgttca?gctgcagcgt?gatgcacgag?gccctgcaca?atcactacac?ccagaagagc?2640

ctgagcctga?gccccggcaa?gtaa 2664

<210>26

<211>2167

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEA-LAMP fusions

<400>26

catggagtct?ccctcggccc?ctccccacag?atggtgcatc?ccctggcaga?ggctcctgct?60

cacagcctca?cttctaacct?tctggaaccc?gcccaccact?gccaagctca?ctattgaatc?120

cacgccgttc?aatgtcgcag?aggggaagga?ggtgcttcta?cttgtccaca?atctgcccca?180

gcatcttttt?ggctacagct?ggtacaaagg?tgaaagagtg?gatggcaacc?gtcaaattat?240

aggatatgta?ataggaactc?aacaagctac?cccagggccc?gcatacagtg?gtcgagagat?300

aatatacccc?aatgcatccc?tgctgatcca?gaacatcatc?cagaatgaca?caggattcta?360

caccctacac?gtcataaagt?cagatcttgt?gaatgaagaa?gcaactggcc?agttccgggt?420

atacccggag?ctgcccaagc?cctccatctc?cagcaacaac?tccaaacccg?tggaggacaa?480

ggatgctgtg?gccttcacct?gtgaacctga?gactcaggac?gcaacctacc?tgtggtgggt?540

aaacaatcag?agcctcccgg?tcagtcccag?gctgcagctg?tccaatggca?acaggaccct?600

cactctattc?aatgtcacaa?gaaatgacac?agcaagctac?aaatgtgaaa?cccagaaccc?660

agtgagtgcc?aggcgcagtg?attcagtcat?cctgaatgtc?ctctatggcc?cggatgcccc?720

caccatttcc?cctctaaaca?catcttacag?atcaggggaa?aatctgaacc?tctcctgcca?780

cgcagcctct?aacccacctg?cacagtactc?ttggtttgtc?aatgggactt?tccagcaatc?840

cacccaagag?ctctttatcc?ccaacatcac?tgtgaataat?agtggatcct?atacgtgcca?900

agcccataac?tcagacactg?gcctcaatag?gaccacagtc?acgacgatca?cagtctatgc?960

agagccaccc?aaacccttca?tcaccagcaa?caactccaac?cccgtggagg?atgaggatgc?1020

tgtagcctta?acctgtgaac?ctgagattca?gaacacaacc?tacctgtggt?gggtaaataa?1080

tcagagcctc?ccggtcagtc?ccaggctgca?gctgtccaat?gacaacagga?ccctcactct?1140

actcagtgtc?acaaggaatg?atgtaggacc?ctatgagtgt?ggaatccaga?acgaattaag?1200

tgttgaccac?agcgacccag?tcatcctgaa?tgtcctctat?ggcccagacg?accccaccat?1260

ttccccctca?tacacctatt?accgtccagg?ggtgaacctc?agcctctcct?gccatgcagc?1320

ctctaaccca?cctgcacagt?attcttggct?gattgatggg?aacatccagc?aacacacaca?1380

agagctcttt?atctccaaca?tcactgagaa?gaacagcgga?ctctatacct?gccaggccaa?1440

taactcagcc?agtggccaca?gcaggactac?agtcaagaca?atcacagtct?ctgcggagct?1500

gcccaagccc?tccatctcca?gcaacaactc?caaacccgtg?gaggacaagg?atgctgtggc?1560

cttcacctgt?gaacctgagg?ctcagaacac?aacctacctg?tggtgggtaa?atggtcagag?1620

cctcccagtc?agtcccaggc?tgcagctgtc?caatggcaac?aggaccctca?ctctattcaa?1680

tgtcacaaga?aatgacgcaa?gagcctatgt?atgtggaatc?cagaactcag?tgagtgcaaa?1740

ccgcagtgac?ccagtcaccc?tggatgtcct?ctatgggccg?gacaccccca?tcatttcccc?1800

cccagactcg?tcttaccttt?cgggagcgaa?cctcaacctc?tcctgccact?cggcctctaa?1860

cccatccccg?cagtattctt?ggcgtatcaa?tgggataccg?cagcaacaca?cacaagttct?1920

ctttatcgcc?aaaatcacgc?caaataataa?cgggacctat?gcctgttttg?tctctaactt?1980

ggctactggc?cgcaataatt?ccatagtcaa?gagcatcaca?gtctctgcat?ctggaactct?2040

agatgatggt?aacaacatgt?tgatccccat?tgctgtgggc?ggtgccctgg?cagggctgat?2100

cctcatcgtc?ctcattgcct?acctcattgg?caggaagagg?agtcacgccg?gctatcagac?2160

catctag 2167

<210>27

<211>3921

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEA-HSP70 fusions

<400>27

atggagtctc?cctcggcccc?tccccacaga?tggtgcatcc?cctggcagag?gctcctgctc?60

acagcctcac?ttctaacctt?ctggaacccg?cccaccactg?ccaagctcac?tattgaatcc?120

acgccgttca?atgtcgcaga?ggggaaggag?gtgcttctac?ttgtccacaa?tctgccccag?180

catctttttg?gctacagctg?gtacaaaggt?gaaagagtgg?atggcaaccg?tcaaattata?240

ggatatgtaa?taggaactca?acaagctacc?ccagggcccg?catacagtgg?tcgagagata?300

atatacccca?atgcatccct?gctgatccag?aacatcatcc?agaatgacac?aggattctac?360

accctacacg?tcataaagtc?agatcttgtg?aatgaagaag?caactggcca?gttccgggta?420

tacccggagc?tgcccaagcc?ctccatctcc?agcaacaact?ccaaacccgt?ggaggacaag?480

gatgctgtgg?ccttcacctg?tgaacctgag?actcaggacg?caacctacct?gtggtgggta?540

aacaatcaga?gcctcccggt?cagtcccagg?ctgcagctgt?ccaatggcaa?caggaccctc?600

actctattca?atgtcacaag?aaatgacaca?gcaagctaca?aatgtgaaac?ccagaaccca?660

gtgagtgcca?ggcgcagtga?ttcagtcatc?ctgaatgtcc?tctatggccc?ggatgccccc?720

accatttccc?ctctaaacac?atcttacaga?tcaggggaaa?atctgaacct?ctcctgccac?780

gcagcctcta?acccacctgc?acagtactct?tggtttgtca?atgggacttt?ccagcaatcc?840

acccaagagc?tctttatccc?caacatcact?gtgaataata?gtggatccta?tacgtgccaa?900

gcccataact?cagacactgg?cctcaatagg?accacagtca?cgacgatcac?agtctatgca?960

gagccaccca?aacccttcat?caccagcaac?aactccaacc?ccgtggagga?tgaggatgct?1020

gtagccttaa?cctgtgaacc?tgagattcag?aacacaacct?acctgtggtg?ggtaaataat?1080

cagagcctcc?cggtcagtcc?caggctgcag?ctgtccaatg?acaacaggac?cctcactcta?1140

ctcagtgtca?caaggaatga?tgtaggaccc?tatgagtgtg?gaatccagaa?cgaattaagt?1200

gttgaccaca?gcgacccagt?catcctgaat?gtcctctatg?gcccagacga?ccccaccatt?1260

tccccctcat?acacctatta?ccgtccaggg?gtgaacctca?gcctctcctg?ccatgcagcc?1320

tctaacccac?ctgcacagta?ttcttggctg?attgatggga?acatccagca?acacacacaa?1380

gagctcttta?tctccaacat?cactgagaag?aacagcggac?tctatacctg?ccaggccaat?1440

aactcagcca?gtggccacag?caggactaca?gtcaagacaa?tcacagtctc?tgcggagctg?1500

cccaagccct?ccatctccag?caacaactcc?aaacccgtgg?aggacaagga?tgctgtggcc?1560

ttcacctgtg?aacctgaggc?tcagaacaca?acctacctgt?ggtgggtaaa?tggtcagagc?1620

ctcccagtca?gtcccaggct?gcagctgtcc?aatggcaaca?ggaccctcac?tctattcaat?1680

gtcacaagaa?atgacgcaag?agcctatgta?tgtggaatcc?agaactcagt?gagtgcaaac?1740

cgcagtgacc?cagtcaccct?ggatgtcctc?tatgggccgg?acacccccat?catttccccc?1800

ccagactcgt?cttacctttc?gggagcgaac?ctcaacctct?cctgccactc?ggcctctaac?1860

ccatccccgc?agtattcttg?gcgtatcaat?gggataccgc?agcaacacac?acaagttctc?1920

tttatcgcca?aaatcacgcc?aaataataac?gggacctatg?cctgttttgt?ctctaacttg?1980

gctactggcc?gcaataattc?catagtcaag?agcatcacag?tctctgcatc?tggaactcta?2040

gatatggctc?gtgcggtcgg?gatcgacctc?gggaccacca?actccgtcgt?ctcggttctg?2100

gaaggtggcg?acccggtcgt?cgtcgccaac?tccgagggct?ccaggaccac?cccgtcaatt?2160

gtcgcgttcg?cccgcaacgg?tgaggtgctg?gtcggccagc?ccgccaagaa?ccaggcggtg?2220

accaacgtcg?atcgcaccgt?gcgctcggtc?aagcgacaca?tgggcagcga?ctggtccata?2280

gagattgacg?gcaagaaata?caccgcgccg?gagatcagcg?cccgcattct?gatgaagctg?2340

aagcgcgacg?ccgaggccta?cctcggtgag?gacattaccg?acgcggttat?cacgacgccc?2400

gcctacttca?atgacgccca?gcgtcaggcc?accaaggacg?ccggccagat?cgccggcctc?2460

aacgtgctgc?ggatcgtcaa?cgagccgacc?gcggccgcgc?tggcctacgg?cctcgacaag?2520

ggcgagaagg?agcagcgaat?cctggtcttc?gacttgggtg?gtggcacttt?cgacgtttcc?2580

ctgctggaga?tcggcgaggg?tgtggttgag?gtccgtgcca?cttcgggtga?caaccacctc?2640

ggcggcgacg?actgggacca?gcgggtcgtc?gattggctgg?tggacaagtt?caagggcacc?2700

agcggcatcg?atctgaccaa?ggacaagatg?gcgatgcagc?ggctgcggga?agccgccgag?2760

aaggcaaagg?tcgagctgag?ttcgagtcag?tccacctcga?tcaacctgcc?ctacatcacc?2820

gtcgacgcag?acaagaaccc?gttgttctta?gacgagcagc?tgacccgcgc?ggagttccaa?2880

cggatcactc?aggacctgct?ggaccgcact?cgcaagccgt?tccagtcggt?gatcgctgac?2940

accggcattt?cggtgtcgga?gatcgatcac?gttgtgctcg?tgggtggttc?gacccggatg?3000

cccgcggtga?ccgatctggt?caaggaactc?accggcggca?aggaacccaa?caagggcgtc?3060

aaccccgatg?aggttgtcgc?ggtgggagcc?gctctgcagg?ccggcgtcct?caagggcgag?3120

gtgaaagacg?ttctgctgct?tgatgttacc?ccgctgagcc?tgggtatcga?gaccaagggc?3180

ggggtgatga?ccaggctcat?cgagcgcaac?accacgatcc?cctccaagcg?gtcggagact?3240

ttcaccaccg?ccgacgacaa?ccaaccgtcg?gtgcagatcc?aggtctatca?gggggagtgt?3300

gagatcgccg?cgcacaacaa?gttgcccggg?tccttcgagc?tgaccggcat?cccgccggcg?3360

ccgcggggga?ttccgcagat?cgaggtcact?ttcgacatcg?acgccaacgg?cattgtgcac?3420

gtcaccgcca?aggacaaggg?caccggcaag?gagaacacga?tccgaatcca?ggaaggctcg?3480

ggcctgtcca?aggaagacat?tgaccgcatg?atcaaggacg?ccgaagcgca?cgccgaggag?3540

gatcgcaagc?gtcgcgagga?ggccgatgtt?cgtaatcaag?ccgagacatt?ggtctaccag?3600

acggagaagt?tcgtcaaaga?acagcgtgag?gccgagggtg?gttcgaaggt?acctgaagac?3660

acgctgaaca?aggttgatgc?cgcggtggcg?gaagcgaagg?cggcacttgg?cggatcggat?3720

atttcggcca?tcaagtcggc?gatggagaag?ctgggccagg?agtcgcaggc?tctggggcaa?3780

gcgatctacg?aagcagctca?ggctgcgtca?ctggccactg?gcgctgccca?ccccggcggc?3840

gagccgggcg?gtgcccaccc?cggctcggct?gatgacgttg?tggacgcgga?ggtggtcgac?3900

gacggccggg?aggccaagtg?a 3921

<210>28

<211>3585

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEA-VSVG fusions

<400>28

atggagtctc?cctcggcccc?tccccacaga?tggtgcatcc?cctggcagag?gctcctgctc?60

acagcctcac?ttctaacctt?ctggaacccg?cccaccactg?ccaagctcac?tattgaatcc?120

acgccgttca?atgtcgcaga?ggggaaggag?gtgcttctac?ttgtccacaa?tctgccccag?180

catctttttg?gctacagctg?gtacaaaggt?gaaagagtgg?atggcaaccg?tcaaattata?240

ggatatgtaa?taggaactca?acaagctacc?ccagggcccg?catacagtgg?tcgagagata?300

atatacccca?atgcatccct?gctgatccag?aacatcatcc?agaatgacac?aggattctac?360

accctacacg?tcataaagtc?agatcttgtg?aatgaagaag?caactggcca?gttccgggta?420

tacccggagc?tgcccaagcc?ctccatctcc?agcaacaact?ccaaacccgt?ggaggacaag?480

gatgctgtgg?ccttcacctg?tgaacctgag?actcaggacg?caacctacct?gtggtgggta?540

aacaatcaga?gcctcccggt?cagtcccagg?ctgcagctgt?ccaatggcaa?caggaccctc?600

actctattca?atgtcacaag?aaatgacaca?gcaagctaca?aatgtgaaac?ccagaaccca?660

gtgagtgcca?ggcgcagtga?ttcagtcatc?ctgaatgtcc?tctatggccc?ggatgccccc?720

accatttccc?ctctaaacac?atcttacaga?tcaggggaaa?atctgaacct?ctcctgccac?780

gcagcctcta?acccacctgc?acagtactct?tggtttgtca?atgggacttt?ccagcaatcc?840

acccaagagc?tctttatccc?caacatcact?gtgaataata?gtggatccta?tacgtgccaa?900

gcccataact?cagacactgg?cctcaatagg?accacagtca?cgacgatcac?agtctatgca?960

gagccaccca?aacccttcat?caccagcaac?aactccaacc?ccgtggagga?tgaggatgct?1020

gtagccttaa?cctgtgaacc?tgagattcag?aacacaacct?acctgtggtg?ggtaaataat?1080

cagagcctcc?cggtcagtcc?caggctgcag?ctgtccaatg?acaacaggac?cctcactcta?1140

ctcagtgtca?caaggaatga?tgtaggaccc?tatgagtgtg?gaatccagaa?cgaattaagt?1200

gttgaccaca?gcgacccagt?catcctgaat?gtcctctatg?gcccagacga?ccccaccatt?1260

tccccctcat?acacctatta?ccgtccaggg?gtgaacctca?gcctctcctg?ccatgcagcc?1320

tctaacccac?ctgcacagta?ttcttggctg?attgatggga?acatccagca?acacacacaa?1380

gagctcttta?tctccaacat?cactgagaag?aacagcggac?tctatacctg?ccaggccaat?1440

aactcagcca?gtggccacag?caggactaca?gtcaagacaa?tcacagtctc?tgcggagctg?1500

cccaagccct?ccatctccag?caacaactcc?aaacccgtgg?aggacaagga?tgctgtggcc?1560

ttcacctgtg?aacctgaggc?tcagaacaca?acctacctgt?ggtgggtaaa?tggtcagagc?1620

ctcccagtca?gtcccaggct?gcagctgtcc?aatggcaaca?ggaccctcac?tctattcaat?1680

gtcacaagaa?atgacgcaag?agcctatgta?tgtggaatcc?agaactcagt?gagtgcaaac?1740

cgcagtgacc?cagtcaccct?ggatgtcctc?tatgggccgg?acacccccat?catttccccc?1800

ccagactcgt?cttacctttc?gggagcgaac?ctcaacctct?cctgccactc?ggcctctaac?1860

ccatccccgc?agtattcttg?gcgtatcaat?gggataccgc?agcaacacac?acaagttctc?1920

tttatcgcca?aaatcacgcc?aaataataac?gggacctatg?cctgttttgt?ctctaacttg?1980

gctactggcc?gcaataattc?catagtcaag?agcatcacag?tctctgcatc?tggaactcta?2040

gatgaattca?tgaagtgctt?tttgtactta?gcttttttat?tcatcggggt?gaattgcaag?2100

ttcaccatag?tttttccaca?caaccaaaaa?ggaaactgga?aaaatgttcc?ttccaattac?2160

cattattgcc?cgtcaagctc?agatttaaat?tggcataatg?acttaatagg?cacaggctta?2220

caagtcaaaa?tgcccaagag?tcacaaggct?attcaagcag?acggttggat?gtgtcatgct?2280

tccaaatggg?tcactacttg?tgatttccgc?tggtacggac?cgaagtatat?aacacattcc?2340

atccgatcct?tcactccatc?tgtagaacaa?tgcaaggaaa?gcattgaaca?aacgaaacaa?2400

ggaacttggc?tgaatccagg?cttccctcct?caaagttgtg?gatatgcaac?tgtgacggat?2460

gccgaagcag?tgattgtcca?ggtgactcct?caccatgtgc?ttgttgatga?atacacagga?2520

gaatgggttg?attcacagtt?catcaacgga?aaatgcagca?atgacatatg?ccccactgtc?2580

cataactcca?caacctggca?ttccgactat?aaggtcaaag?ggctatgtga?ttctaacctc?2640

atttccacgg?acatcacctt?cttctcagag?gacagagagc?tatcatccct?aggaaaggag?2700

ggcacagggt?tcagaagtaa?ctactttgct?tatgaaactg?gagacaaggc?ctgcaaaatg?2760

cagtactgca?agcattgggg?agtcagactc?ccatcaggtg?tctggttcga?gatggctgat?2820

aaggatctct?ttgctgcagc?cagattccct?gaatgcccag?aagggtcaag?tatctctgct?2880

ccatctcaga?cctcagtgga?tgtaagtctc?attcaggacg?ttgagaggat?cttggattat?2940

tccctctgcc?aagaaacctg?gagcaaaatc?agagcgggtc?ttcccatctc?tccagtggat?3000

ctcagctatc?ttgctcctaa?aaacccagga?accggtcctg?cctttaccat?aatcaatggt?3060

accctaaaat?actttgagac?cagatacatc?agagtcgata?ttgctgctcc?aatcctctca?3120

agaatggtcg?gaatgatcag?tggaactacc?acagaaaggg?aactgtggga?tgactgggct?3180

ccatatgaag?acgtggaaat?tggacccaat?ggagttctga?ggaccagttc?aggatataag?3240

tttcctttat?atatgattgg?acatggtatg?ttggactccg?gtcttcatct?tagctcaaag?3300

gctcaggtgt?ttgaacatcc?tcacattcaa?gacgctgctt?cgcagcttcc?tgatgatgag?3360

attttatttt?ttggtgatac?tgggctatcc?aaaaatccaa?tcgactttgt?cgaaggttgg?3420

ttcagtagtt?ggaagagctc?cattgcctct?tttttcttta?tcatagggtt?aatcattgga?3480

ctattcttgg?ttctccgagt?tggtatttat?ctttacatta?aattaaagca?caccaagaaa?3540

agacagattt?atacagacat?agagatgaac?cgacttggaa?ggtaa 3585

<210>29

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>29

tattctagat?tcaacaccaa?ttccattttc?ttattc 36

<210>30

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>30

gcggccgcta?gaatcatttg?tccatccttc?atc 33

<210>31

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>31

tattctagat?tcaacaccaa?ttccattttc?ttattc 36

<210>32

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>32

ttagcggccg?ctagttctgt?atcatatcgt?aaaggg 36

<210>33

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>33

tctagataaa?actcacacat?gccca 25

<210>34

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>34

gccgactcat?ttacccggag?acagggag 28

<210>35

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>35

tctagatttg?atccccattg?ctgtgggcgg?tgccctg 37

<210>36

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>36

ggcgtgactc?ctcttcctgc?caatgaggta?ggcaatgag 39

<210>37

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>37

atatctagat?ttcaccatag?tttttccaca?caacc 35

<210>38

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>38

gcggccgcct?tccttccaag?tcggttcatc?tctatg 36

<210>39

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>39

gctctagata?tggctcgtgc?ggtcgggatc?gacc 34

<210>40

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>40

gccgcggccg?ctcacttggc?ctcccggccg?tcgtcg 36

<210>41

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>41

gttatctaga?agcaccccca?tccc 24

<210>42

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>42

ttaagatctc?taagatctgg?tgtcgtatct?cagggg 36

<210>43

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>43

ttatctagaa?agacccacac?ctgcccccct?tgc 33

<210>44

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>44

tatagatctt?agggtacctt?acttgccggg?g 31

<210>45

<211>952

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CEA-Dom fusions

<400>45

Met?Glu?Ser?Pro?Ser?Ala?Pro?Pro?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Arg?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Lys?Leu?Thr?Ile?Glu?Ser?Thr?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Val?His?Asn?Leu?Pro?Gln?His?Leu?Phe?Gly

50 55 60

Tyr?Ser?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Gly?Asn?Arg?Gln?Ile?Ile

65 70 75 80

Gly?Tyr?Val?Ile?Gly?Thr?Gln?Gln?Ala?Thr?Pro?Gly?Pro?Ala?Tyr?Ser

85 90 95

Gly?Arg?Glu?Ile?Ile?Tyr?Pro?Asn?Ala?Ser?Leu?Leu?Ile?Gln?Asn?Ile

100 105 110

Ile?Gln?Asn?Asp?Thr?Gly?Phe?Tyr?Thr?Leu?His?Val?Ile?Lys?Ser?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Ala?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Gln

180 185 190

Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn?Val?Thr?Arg?Asn

195 200 205

Asp?Thr?Ala?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Ala?Arg

210 215 220

Arg?Ser?Asp?Ser?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Ser?Tyr?Arg?Ser?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Thr?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Asp?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Thr?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Pro?Pro?Lys?Pro?Phe?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu

325 330 335

Asp?Glu?Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Ile?Gln?Asn?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg

355 360 365

Leu?Gln?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Leu?Leu?Ser?Val?Thr

370 375 380

Arg?Asn?Asp?Val?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Glu?Leu?Ser

385 390 395 400

Val?Asp?His?Ser?Asp?Pro?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Asp?Pro?Thr?Ile?Ser?Pro?Ser?Tyr?Thr?Tyr?Tyr?Arg?Pro?Gly?Val?Asn

420 425 430

Leu?Ser?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser

435 440 445

Trp?Leu?Ile?Asp?Gly?Asn?Ile?Gln?Gln?His?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Ser?Asn?Ile?Thr?Glu?Lys?Asn?Ser?Gly?Leu?Tyr?Thr?Cys?Gln?Ala?Asn

465 470 475 480

Asn?Ser?Ala?Ser?Gly?His?Ser?Arg?Thr?Thr?Val?Lys?Thr?Ile?Thr?Val

485 490 495

Ser?Ala?Glu?Leu?Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro

500 505 510

Val?Glu?Asp?Lys?Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Ala?Gln

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Gly?Gln?Ser?Leu?Pro?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn

545 550 555 560

Val?Thr?Arg?Asn?Asp?Ala?Arg?Ala?Tyr?Val?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Val?Ser?Ala?Asn?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asp?Val?Leu?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Ser?Ser?Tyr?Leu?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Ala?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Arg?Ile?Asn?Gly?Ile?Pro?Gln?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ala?Lys?Ile?Thr?Pro?Asn?Asn?Asn?Gly?Thr?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Ser?Ile

660 665 670

Thr?Val?Ser?Ala?Ser?Gly?Thr?Ser?Arg?Ser?Thr?Pro?Ile?Pro?Phe?Ser

675 680 685

Tyr?Ser?Lys?Asn?Leu?Asp?Cys?Trp?Val?Asp?Asn?Glu?Glu?Asp?Ile?Asp

690 695 700

Val?Ile?Leu?Lys?Lys?Ser?Thr?Ile?Leu?Asn?Leu?Asp?Ile?Asn?Asn?Asp

705 710 715 720

Ile?Ile?Ser?Asp?Ile?Ser?Gly?Phe?Asn?Ser?Ser?Val?Ile?Thr?Tyr?Pro

725 730 735

Asp?Ala?Gln?Leu?Val?Pro?Gly?Ile?Asn?Gly?Lys?Ala?Ile?His?Leu?Val

740 745 750

Asn?Asn?Glu?Ser?Ser?Glu?Val?Ile?Val?His?Lys?Ala?Met?Asp?Ile?Glu

755 760 765

Tyr?Asn?Asp?Met?Phe?Asn?Asn?Phe?Thr?Val?Ser?Phe?Trp?Leu?Arg?Val

770 775 780

Pro?Lys?Val?Ser?Ala?Ser?His?Leu?Glu?Gln?Tyr?Gly?Thr?Asn?Glu?Tyr

785 790 795 800

Ser?Ile?Ile?Ser?Ser?Met?Lys?Lys?His?Ser?Leu?Ser?Ile?Gly?Ser?Gly

805 810 815

Trp?Ser?Val?Ser?Leu?Lys?Gly?Asn?Asn?Leu?Ile?Trp?Thr?Leu?Lys?Asp

820 825 830

Ser?Ala?Gly?Glu?Val?Arg?Gln?Ile?Thr?Phe?Arg?Asp?Leu?Pro?Asp?Lys

835 840 845

Phe?Asn?Ala?Tyr?Leu?Ala?Asn?Lys?Trp?Val?Phe?Ile?Thr?Ile?Thr?Asn

850 855 860

Asp?Arg?Leu?Ser?Ser?Ala?Asn?Leu?Tyr?Ile?Asn?Gly?Val?Leu?Met?Gly

865 870 875 880

Ser?Ala?Glu?Ile?Thr?Gly?Leu?Gly?Ala?Ile?Arg?Glu?Asp?Asn?Asn?Ile

885 890 895

Thr?Leu?Lys?Leu?Asp?Arg?Cys?Asn?Asn?Asn?Asn?Gln?Tyr?Val?Ser?Ile

900 905 910

Asp?Lys?Phe?Arg?Ile?Phe?Cys?Lys?Ala?Leu?Asn?Pro?Lys?Glu?Ile?Glu

915 920 925

Lys?Leu?Tyr?Thr?Ser?Tyr?Leu?Ser?Ile?Thr?Phe?Leu?Arg?Asp?Phe?Trp

930 935 940

Gly?Asn?Pro?Leu?Arg?Tyr?Asp?Thr

945 950

<210>46

<211>907

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CEA-FcIgG fusions

<400>46

Met?Glu?Ser?Pro?Ser?Ala?Pro?Pro?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Arg?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Lys?Leu?Thr?Ile?Glu?Ser?Thr?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Val?His?Asn?Leu?Pro?Gln?His?Leu?Phe?Gly

50 55 60

Tyr?Ser?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Gly?Asn?Arg?Gln?Ile?Ile

65 70 75 80

Gly?Tyr?Val?Ile?Gly?Thr?Gln?Gln?Ala?Thr?Pro?Gly?Pro?Ala?Tyr?Ser

85 90 95

Gly?Arg?Glu?Ile?Ile?Tyr?Pro?Asn?Ala?Ser?Leu?Leu?Ile?Gln?Asn?Ile

100 105 110

Ile?Gln?Asn?Asp?Thr?Gly?Phe?Tyr?Thr?Leu?His?Val?Ile?Lys?Ser?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Ala?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Gln

180 185 190

Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn?Val?Thr?Arg?Asn

195 200 205

Asp?Thr?Ala?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Ala?Arg

210 215 220

Arg?Ser?Asp?Ser?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Ser?Tyr?Arg?Ser?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Thr?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Asp?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Thr?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Pro?Pro?Lys?Pro?Phe?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu

325 330 335

Asp?Glu?Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Ile?Gln?Asn?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg

355 360 365

Leu?Gln?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Leu?Leu?Ser?Val?Thr

370 375 380

Arg?Asn?Asp?Val?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Glu?Leu?Ser

385 390 395 400

Val?Asp?His?Ser?Asp?Pro?Val?Ile?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Asp?Pro?Thr?Ile?Ser?Pro?Ser?Tyr?Thr?Tyr?Tyr?Arg?Pro?Gly?Val?Asn

420 425 430

Leu?Ser?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Pro?Ala?Gln?Tyr?Ser

435 440 445

Trp?Leu?Ile?Asp?Gly?Asn?Ile?Gln?Gln?His?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Ser?Asn?Ile?Thr?Glu?Lys?Asn?Ser?Gly?Leu?Tyr?Thr?Cys?Gln?Ala?Asn

465 470 475 480

Asn?Ser?Ala?Ser?Gly?His?Ser?Arg?Thr?Thr?Val?Lys?Thr?Ile?Thr?Val

485 490 495

Ser?Ala?Glu?Leu?Pro?Lys?Pro?Ser?Ile?Ser?Ser?Asn?Asn?Ser?Lys?Pro

500 505 510

Val?Glu?Asp?Lys?Asp?Ala?Val?Ala?Phe?Thr?Cys?Glu?Pro?Glu?Ala?Gln

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Gly?Gln?Ser?Leu?Pro?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Thr?Leu?Thr?Leu?Phe?Asn

545 550 555 560

Val?Thr?Arg?Asn?Asp?Ala?Arg?Ala?Tyr?Val?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Val?Ser?Ala?Asn?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asp?Val?Leu?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Ser?Ser?Tyr?Leu?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Ala?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Arg?Ile?Asn?Gly?Ile?Pro?Gln?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ala?Lys?Ile?Thr?Pro?Asn?Asn?Asn?Gly?Thr?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Ser?Ile

660 665 670

Thr?Val?Ser?Ala?Ser?Gly?Thr?Ser?Arg?Lys?Thr?His?Thr?Cys?Pro?Pro

675 680 685

Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro

690 695 700

Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr

705 710 715 720

Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn

725 730 735

Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg

740 745 750

Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val

755 760 765

Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser

770 775 780

Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys

785 790 795 800

Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp

805 810 815

Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe

820 825 830

Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu

835 840 845

Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe

850 855 860

Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly

865 870 875 880

Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr

885 890 895

Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

900 905

<210>47

<211>825

<212>DNA

＜213〉artificial sequence

<220>

<223>DOM

<400>47

gattcaacac?caattccatt?ttcttattct?aaaaatctgg?attgttgggt?tgataatgaa?60

gaagatatag?atgttatatt?aaaaaagagt?acaattttaa?atttagatat?taataatgat?120

attatatcag?atatatctgg?gtttaattca?tctgtaataa?catatccaga?tgctcaattg?180

gtgcccggaa?taaatggcaa?agcaatacat?ttagtaaaca?atgaatcttc?tgaagttata?240

gtgcataaag?ctatggatat?tgaatataat?gatatgttta?ataattttac?cgttagcttt?300

tggttgaggg?ttcctaaagt?atctgctagt?catttagaac?aatatggcac?aaatgagtat?360

tcaataatta?gctctatgaa?aaaacatagt?ctatcaatag?gatctggttg?gagtgtatca?420

cttaaaggta?ataacttaat?atggacttta?aaagattccg?cgggagaagt?tagacaaata?480

acttttaggg?atttacctga?taaatttaat?gcttatttag?caaataaatg?ggtttttata?540

actattacta?atgatagatt?atcttctgct?aatttgtata?taaatggagt?acttatggga?600

agtgcagaaa?ttactggttt?aggagctatt?agagaggata?ataatataac?attaaaacta?660

gatagatgta?ataataataa?tcaatacgtt?tctattgata?aatttaggat?attttgcaaa?720

gcattaaatc?caaaagagat?tgaaaaatta?tacacaagtt?atttatctat?aaccttttta?780

agagacttct?ggggaaaccc?tttacgatat?gatacagata?ggtag 825

<210>48

<211>274

<212>PRT

＜213〉artificial sequence

<220>

<223>DOM

<400>48

Asp?Ser?Thr?Pro?Ile?Pro?Phe?Ser?Tyr?Ser?Lys?Asn?Leu?Asp?Cys?Trp

1 5 10 15

Val?Asp?Asn?Glu?Glu?Asp?Ile?Asp?Val?Ile?Leu?Lys?Lys?Ser?Thr?Ile

20 25 30

Leu?Asn?Leu?Asp?Ile?Asn?Asn?Asp?Ile?Ile?Ser?Asp?Ile?Ser?Gly?Phe

35 40 45

Asn?Ser?Ser?Val?Ile?Thr?Tyr?Pro?Asp?Ala?Gln?Leu?Val?Pro?Gly?Ile

50 55 60

Asn?Gly?Lys?Ala?Ile?His?Leu?Val?Asn?Asn?Glu?Ser?Ser?Glu?Val?Ile

65 70 75 80

Val?His?Lys?Ala?Met?Asp?Ile?Glu?Tyr?Asn?Asp?Met?Phe?Asn?Asn?Phe

85 90 95

Thr?Val?Ser?Phe?Trp?Leu?Arg?Val?Pro?Lys?Val?Ser?Ala?Ser?His?Leu

100 105 110

Glu?Gln?Tyr?Gly?Thr?Asn?Glu?Tyr?Ser?Ile?Ile?Ser?Ser?Met?Lys?Lys

115 120 125

His?Ser?Leu?Ser?Ile?Gly?Ser?Gly?Trp?Ser?Val?Ser?Leu?Lys?Gly?Asn

130 135 140

Asn?Leu?Ile?Trp?Thr?Leu?Lys?Asp?Ser?Ala?Gly?Glu?Val?Arg?Gln?Ile

145 150 155 160

Thr?Phe?Arg?Asp?Leu?Pro?Asp?Lys?Phe?Asn?Ala?Tyr?Leu?Ala?Asn?Lys

165 170 175

Trp?Val?Phe?Ile?Thr?Ile?Thr?Asn?Asp?Arg?Leu?Ser?Ser?Ala?Asn?Leu

180 185 190

Tyr?Ile?Asn?Gly?Val?Leu?Met?Gly?Ser?Ala?Glu?Ile?Thr?Gly?Leu?Gly

195 200 205

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

210 215 220

Asn?Asn?Asn?Gln?Tyr?Val?Ser?Ile?Asp?Lys?Phe?Arg?Ile?Phe?Cys?Lys

225 230 235 240

Ala?Leu?Asn?Pro?Lys?Glu?Ile?Glu?Lys?Leu?Tyr?Thr?Ser?Tyr?Leu?Ser

245 250 255

Ile?Thr?Phe?Leu?Arg?Asp?Phe?Trp?Gly?Asn?Pro?Leu?Arg?Tyr?Asp?Thr

260 265 270

Asp?Arg

<210>49

<211>2857

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CEA-DOM fusions

<400>49

atggagtctc?cctcggcccc?tccccacaga?tggtgcatcc?cctggcagag?gctcctgctc?60

acagcctcac?ttctaacctt?ctggaacccg?cccaccactg?ccaagctcac?tattgaatcc?120

acgccgttca?atgtcgcaga?ggggaaggag?gtgcttctac?ttgtccacaa?tctgccccag?180

catctttttg?gctacagctg?gtacaaaggt?gaaagagtgg?atggcaaccg?tcaaattata?240

ggatatgtaa?taggaactca?acaagctacc?ccagggcccg?catacagtgg?tcgagagata?300

atatacccca?atgcatccct?gctgatccag?aacatcatcc?agaatgacac?aggattctac?360

accctacacg?tcataaagtc?agatcttgtg?aatgaagaag?caactggcca?gttccgggta?420

tacccggagc?tgcccaagcc?ctccatctcc?agcaacaact?ccaaacccgt?ggaggacaag?480

gatgctgtgg?ccttcacctg?tgaacctgag?actcaggacg?caacctacct?gtggtgggta?540

aacaatcaga?gcctcccggt?cagtcccagg?ctgcagctgt?ccaatggcaa?caggaccctc?600

actctattca?atgtcacaag?aaatgacaca?gcaagctaca?aatgtgaaac?ccagaaccca?660

gtgagtgcca?ggcgcagtga?ttcagtcatc?ctgaatgtcc?tctatggccc?ggatgccccc?720

accatttccc?ctctaaacac?atcttacaga?tcaggggaaa?atctgaacct?ctcctgccac?780

gcagcctcta?acccacctgc?acagtactct?tggtttgtca?atgggacttt?ccagcaatcc?840

acccaagagc?tctttatccc?caacatcact?gtgaataata?gtggatccta?tacgtgccaa?900

gcccataact?cagacactgg?cctcaatagg?accacagtca?cgacgatcac?agtctatgca?960

gagccaccca?aacccttcat?caccagcaac?aactccaacc?ccgtggagga?tgaggatgct?1020

gtagccttaa?cctgtgaacc?tgagattcag?aacacaacct?acctgtggtg?ggtaaataat?1080

cagagcctcc?cggtcagtcc?caggctgcag?ctgtccaatg?acaacaggac?cctcactcta?1140

ctcagtgtca?caaggaatga?tgtaggaccc?tatgagtgtg?gaatccagaa?cgaattaagt?1200

gttgaccaca?gcgacccagt?catcctgaat?gtcctctatg?gcccagacga?ccccaccatt?1260

tccccctcat?acacctatta?ccgtccaggg?gtgaacctca?gcctctcctg?ccatgcagcc?1320

tctaacccac?ctgcacagta?ttcttggctg?attgatggga?acatccagca?acacacacaa?1380

gagctcttta?tctccaacat?cactgagaag?aacagcggac?tctatacctg?ccaggccaat?1440

aactcagcca?gtggccacag?caggactaca?gtcaagacaa?tcacagtctc?tgcggagctg?1500

cccaagccct?ccatctccag?caacaactcc?aaacccgtgg?aggacaagga?tgctgtggcc?1560

ttcacctgtg?aacctgaggc?tcagaacaca?acctacctgt?ggtgggtaaa?tggtcagagc?1620

ctcccagtca?gtcccaggct?gcagctgtcc?aatggcaaca?ggaccctcac?tctattcaat?1680

gtcacaagaa?atgacgcaag?agcctatgta?tgtggaatcc?agaactcagt?gagtgcaaac?1740

cgcagtgacc?cagtcaccct?ggatgtcctc?tatgggccgg?acacccccat?catttccccc?1800

ccagactcgt?cttacctttc?gggagcgaac?ctcaacctct?cctgccactc?ggcctctaac?1860

ccatccccgc?agtattcttg?gcgtatcaat?gggataccgc?agcaacacac?acaagttctc?1920

tttatcgcca?aaatcacgcc?aaataataac?gggacctatg?cctgttttgt?ctctaacttg?1980

gctactggcc?gcaataattc?catagtcaag?agcatcacag?tctctgcatc?tggaactcta?2040

gattcaacac?caattccatt?ttcttattct?aaaaatctgg?attgttgggt?tgataatgaa?2100

gaagatatag?atgttatatt?aaaaaagagt?acaattttaa?atttagatat?taataatgat?2160

attatatcag?atatatctgg?gtttaattca?tctgtaataa?catatccaga?tgctcaattg?2220

gtgcccggaa?taaatggcaa?agcaatacat?ttagtaaaca?atgaatcttc?tgaagttata?2280

gtgcataaag?ctatggatat?tgaatataat?gatatgttta?ataattttac?cgttagcttt?2340

tggttgaggg?ttcctaaagt?atctgctagt?catttagaac?aatatggcac?aaatgagtat?2400

tcaataatta?gctctatgaa?aaaacatagt?ctatcaatag?gatctggttg?gagtgtatca?2460

cttaaaggta?ataacttaat?atggacttta?aaagattccg?cgggagaagt?tagacaaata?2520

acttttaggg?atttacctga?taaatttaat?gcttatttag?caaataaatg?ggtttttata?2580

actattacta?atgatagatt?atcttctgct?aatttgtata?taaatggagt?acttatggga?2640

agtgcagaaa?ttactggttt?aggagctatt?agagaggata?ataatataac?attaaaacta?2700

gatagatgta?ataataataa?tcaatacgtt?tctattgata?aatttaggat?attttgcaaa?2760

gcattaaatc?caaaagagat?tgaaaaatta?tacacaagtt?atttatctat?aaccttttta?2820

agagacttct?ggggaaaccc?tttacgatat?gatatag 2857

<210>50

<211>2859

<212>DNA

＜213〉artificial sequence

<220>

＜223〉rhCEA-DOMopt fusions

<400>50

atgggcagcc?ccagcgcccc?cctgcaccgc?tggtgcatcc?cctggcagac?cctgctgctg?60

accgccagcc?tgctgacctt?ctggaacccc?cccaccaccg?cccagctgac?catcgagagc?120

cgccccttca?acgtggccga?gggcaaggag?gtgctgctgc?tggcccacaa?cgtgagccag?180

aacctgttcg?gctacatctg?gtacaagggc?gagcgcgtgg?acgccagccg?ccgcatcggc?240

agctgcgtga?tccgcaccca?gcagatcacc?cccggccccg?cccacagcgg?ccgcgagacc?300

atcgacttca?acgccagcct?gctgatccac?aacgtgaccc?agagcgacac?cggcagctac?360

accatccagg?tgatcaagga?ggacctggtg?aacgaggagg?ccaccggcca?gttccgcgtg?420

taccccgagc?tgcccaagcc?ctacatcagc?agcaacaaca?gcaaccccgt?ggaggacaag?480

gacgccgtgg?ccctgacctg?cgagcccgag?acccaggaca?ccacctacct?gtggtgggtg?540

aacaaccaga?gcctgcccgt?gagcccccgc?ctggagctga?gcagcgacaa?ccgcaccctg?600

accgtgttca?acatcccccg?caacgacacc?accagctaca?agtgcgagac?ccagaacccc?660

gtgagcgtgc?gccgcagcga?ccccgtgacc?ctgaacgtgc?tgtacggccc?cgacgccccc?720

accatcagcc?ccctgaacac?cccctaccgc?gccggcgaga?acctgaacct?gacctgccac?780

gccgccagca?accccaccgc?ccagtacttc?tggttcgtga?acggcacctt?ccagcagagc?840

acccaggagc?tgttcatccc?caacatcacc?gtgaacaaca?gcggcagcta?catgtgccag?900

gcccacaaca?gcgccaccgg?cctgaaccgc?accaccgtga?ccgccatcac?cgtgtacgcc?960

gagctgccca?agccctacat?caccagcaac?aacagcaacc?ccatcgagga?caaggacgcc?1020

gtgaccctga?cctgcgagcc?cgagacccag?gacaccacct?acctgtggtg?ggtgaacaac?1080

cagagcctga?gcgtgagcag?ccgcctggag?ctgagcaacg?acaaccgcac?cctgaccgtg?1140

ttcaacatcc?cccgcaacga?caccaccttc?tacgagtgcg?agacccagaa?ccccgtgagc?1200

gtgcgccgca?gcgaccccgt?gaccctgaac?gtgctgtacg?gccccgacgc?ccccaccatc?1260

agccccctga?acacccccta?ccgcgccggc?gagaacctga?acctgagctg?ccacgccgcc?1320

agcaaccccg?ccgcccagta?cagctggttc?gtgaacggca?ccttccagca?gagcacccag?1380

gagctgttca?tccccaacat?caccgtgaac?aacagcggca?gctacatgtg?ccaggcccac?1440

aacagcgcca?ccggcctgaa?ccgcaccacc?gtgaccgcca?tcaccgtgta?cgtggagctg?1500

cccaagccct?acatcagcag?caacaacagc?aaccccatcg?aggacaagga?cgccgtgacc?1560

ctgacctgcg?agcccgtggc?cgagaacacc?acctacctgt?ggtgggtgaa?caaccagagc?1620

ctgagcgtga?gcccccgcct?gcagctgagc?aacggcaacc?gcatcctgac?cctgctgagc?1680

gtgacccgca?acgacaccgg?cccctacgag?tgcggcatcc?agaacagcga?gagcgccaag?1740

cgcagcgacc?ccgtgaccct?gaacgtgacc?tacggccccg?acacccccat?catcagcccc?1800

cccgacctga?gctaccgcag?cggcgccaac?ctgaacctga?gctgccacag?cgacagcaac?1860

cccagccccc?agtacagctg?gctgatcaac?ggcaccctgc?gccagcacac?ccaggtgctg?1920

ttcatcagca?agatcaccag?caacaacagc?ggcgcctacg?cctgcttcgt?gagcaacctg?1980

gccaccggcc?gcaacaacag?catcgtgaag?aacatcagcg?tgagcagcgg?cgacagctct?2040

agaagcaccc?ccatcccatt?cagctacagc?aagaacctgg?actgctgggt?ggacaacgag?2100

gaggacatcg?acgtgatcct?gaagaagagc?accatcctga?acctggacat?caacaacgac?2160

atcatcagcg?acatcagcgg?cttcaacagc?agcgtgatca?cctaccccga?cgcccagctg?2220

gtgcccggca?tcaacggcaa?ggccatccac?ctggtgaaca?acgagagcag?cgaggtgatc?2280

gtgcacaagg?ccatggacat?cgagtacaac?gacatgttca?acaacttcac?cgtgagcttc?2340

tggctgagag?tgcctaaggt?gagcgccagc?cacctggagc?agtacggcac?caacgagtac?2400

agcatcatca?gcagcatgaa?gaagcacagc?ctgagcatcg?gcagcggctg?gagcgtgagc?2460

ctgaagggca?acaacctcat?ctggaccctg?aaggatagcg?ccggagaggt?gagacagatc?2520

accttcagag?acctgcccga?caagttcaat?gcctacctgg?ccaacaagtg?ggtgttcatc?2580

accatcacca?acgacagact?gagcagcgcc?aacctgtaca?tcaacggcgt?gctcatgggc?2640

agcgccgaga?tcaccggcct?gggcgccatc?agagaggaca?acaacatcac?cctgaagctg?2700

gacagatgca?acaacaacaa?ccagtacgtg?agcatcgaca?agttccggat?cttctgcaag?2760

gccctgaacc?ccaaggagat?cgagaagctg?tacaccagct?acctgagcat?caccttcctg?2820

agagacttct?ggggcaaccc?cctgagatac?gacacctag 2859

<210>51

<211>952

<212>PRT

＜213〉artificial sequence

<220>

＜223〉rhCEA-DOMopt fusions

<400>51

Met?Gly?Ser?Pro?Ser?Ala?Pro?Leu?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Thr?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Gln?Leu?Thr?Ile?Glu?Ser?Arg?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Ala?His?Asn?Val?Ser?Gln?Asn?Leu?Phe?Gly

50 55 60

Tyr?Ile?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Ala?Ser?Arg?Arg?Ile?Gly

65 70 75 80

Ser?Cys?Val?Ile?Arg?Thr?Gln?Gln?Ile?Thr?Pro?Gly?Pro?Ala?His?Ser

85 90 95

Gly?Arg?Glu?Thr?Ile?Asp?Phe?Asn?Ala?Ser?Leu?Leu?Ile?His?Asn?Val

100 105 110

Thr?Gln?Ser?Asp?Thr?Gly?Ser?Tyr?Thr?Ile?Gln?Val?Ile?Lys?Glu?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Glu

180 185 190

Leu?Ser?Ser?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro?Arg?Asn

195 200 205

Asp?Thr?Thr?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Val?Arg

210 215 220

Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Thr?Cys?His?Ala?Ala?Ser?Asn?Pro?Thr?Ala?Gln?Tyr?Phe?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Ile?Glu

325 330 335

Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser?Ser?Arg

355 360 365

Leu?Glu?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro

370 375 380

Arg?Asn?Asp?Thr?Thr?Phe?Tyr?Glu?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser

385 390 395 400

Val?Arg?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Ala?Pro?Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn

420 425 430

Leu?Asn?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Ala?Ala?Gln?Tyr?Ser

435 440 445

Trp?Phe?Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Pro?Asn?Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His

465 470 475 480

Asn?Ser?Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val

485 490 495

Tyr?Val?Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro

500 505 510

Ile?Glu?Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Val?Ala?Glu

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Ile?Leu?Thr?Leu?Leu?Ser

545 550 555 560

Val?Thr?Arg?Asn?Asp?Thr?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Glu?Ser?Ala?Lys?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Thr?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Leu?Ser?Tyr?Arg?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Asp?Ser?Asn?Pro?Ser?Pro?Gln

6l0 615 620

Tyr?Ser?Trp?Leu?Ile?Asn?Gly?Thr?Leu?Arg?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ser?Lys?Ile?Thr?Ser?Asn?Asn?Ser?Gly?Ala?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Asn?Ile

660 665 670

Ser?Val?Ser?Ser?Gly?Asp?Ser?Ser?Arg?Ser?Thr?Pro?Ile?Pro?Phe?Ser

675 680 685

Tyr?Ser?Lys?Asn?Leu?Asp?Cys?Trp?Val?Asp?Asn?Glu?Glu?Asp?Ile?Asp

690 695 700

Val?Ile?Leu?Lys?Lys?Ser?Thr?Ile?Leu?Asn?Leu?Asp?Ile?Asn?Asn?Asp

705 710 715 720

Ile?Ile?Ser?Asp?Ile?Ser?Gly?Phe?Asn?Ser?Ser?Val?Ile?Thr?Tyr?Pro

725 730 735

Asp?Ala?Gln?Leu?Val?Pro?Gly?Ile?Asn?Gly?Lys?Ala?Ile?His?Leu?Val

740 745 750

Asn?Asn?Glu?Ser?Ser?Glu?Val?Ile?Val?His?Lys?Ala?Met?Asp?Ile?Glu

755 760 765

Tyr?Asn?Asp?Met?Phe?Asn?Asn?Phe?Thr?Val?Ser?Phe?Trp?Leu?Arg?Val

770 775 780

Pro?Lys?Val?Ser?Ala?Ser?His?Leu?Glu?Gln?Tyr?Gly?Thr?Asn?Glu?Tyr

785 790 795 800

Ser?Ile?Ile?Ser?Ser?Met?Lys?Lys?His?Ser?Leu?Ser?Ile?Gly?Ser?Gly

805 810 815

Trp?Ser?Val?Ser?Leu?Lys?Gly?Asn?Asn?Leu?Ile?Trp?Thr?Leu?Lys?Asp

820 825 830

Ser?Ala?Gly?Glu?Val?Arg?Gln?Ile?Thr?Phe?Arg?Asp?Leu?Pro?Asp?Lys

835 840 845

Phe?Asn?Ala?Tyr?Leu?Ala?Asn?Lys?Trp?Val?Phe?Ile?Thr?Ile?Thr?Asn

850 855 860

Asp?Arg?Leu?Ser?Ser?Ala?Asn?Leu?Tyr?Ile?Asn?Gly?Val?Leu?Met?Gly

865 870 875 880

Ser?Ala?Glu?Ile?Thr?Gly?Leu?Gly?Ala?Ile?Arg?Glu?Asp?Asn?Asn?Ile

885 890 895

Thr?Leu?Lys?Leu?Asp?Arg?Cys?Asn?Asn?Asn?Asn?Gln?Tyr?Val?Ser?Ile

900 905 910

Asp?Lys?Phe?Arg?Ile?Phe?Cys?Lys?Ala?Leu?Asn?Pro?Lys?Glu?Ile?Glu

915 920 925

Lys?Leu?Tyr?Thr?Ser?Tyr?Leu?Ser?Ile?Thr?Phe?Leu?Arg?Asp?Phe?Trp

930 935 940

Gly?Asn?Pro?Leu?Arg?Tyr?Asp?Thr

945 950

<210>52

<211>2359

<212>DNA

＜213〉artificial sequence

<220>

＜223〉rhCEA-CTBopt fusions

<400>52

atgggcagcc?ccagcgcccc?cctgcaccgc?tggtgcatcc?cctggcagac?cctgctgctg?60

accgccagcc?tgctgacctt?ctggaacccc?cccaccaccg?cccagctgac?catcgagagc?120

cgccccttca?acgtggccga?gggcaaggag?gtgctgctgc?tggcccacaa?cgtgagccag?180

aacctgttcg?gctacatctg?gtacaagggc?gagcgcgtgg?acgccagccg?ccgcatcggc?240

agctgcgtga?tccgcaccca?gcagatcacc?cccggccccg?cccacagcgg?ccgcgagacc?300

atcgacttca?acgccagcct?gctgatccac?aacgtgaccc?agagcgacac?cggcagctac?360

accatccagg?tgatcaagga?ggacctggtg?aacgaggagg?ccaccggcca?gttccgcgtg?420

taccccgagc?tgcccaagcc?ctacatcagc?agcaacaaca?gcaaccccgt?ggaggacaag?480

gacgccgtgg?ccctgacctg?cgagcccgag?acccaggaca?ccacctacct?gtggtgggtg?540

aacaaccaga?gcctgcccgt?gagcccccgc?ctggagctga?gcagcgacaa?ccgcaccctg?600

accgtgttca?acatcccccg?caacgacacc?accagctaca?agtgcgagac?ccagaacccc?660

gtgagcgtgc?gccgcagcga?ccccgtgacc?ctgaacgtgc?tgtacggccc?cgacgccccc?720

accatcagcc?ccctgaacac?cccctaccgc?gccggcgaga?acctgaacct?gacctgccac?780

gccgccagca?accccaccgc?ccagtacttc?tggttcgtga?acggcacctt?ccagcagagc?840

acccaggagc?tgttcatccc?caacatcacc?gtgaacaaca?gcggcagcta?catgtgccag?900

gcccacaaca?gcgccaccgg?cctgaaccgc?accaccgtga?ccgccatcac?cgtgtacgcc?960

gagctgccca?agccctacat?caccagcaac?aacagcaacc?ccatcgagga?caaggacgcc?1020

gtgaccctga?cctgcgagcc?cgagacccag?gacaccacct?acctgtggtg?ggtgaacaac?1080

cagagcctga?gcgtgagcag?ccgcctggag?ctgagcaacg?acaaccgcac?cctgaccgtg?1140

ttcaacatcc?cccgcaacga?caccaccttc?tacgagtgcg?agacccagaa?ccccgtgagc?1200

gtgcgccgca?gcgaccccgt?gaccctgaac?gtgctgtacg?gccccgacgc?ccccaccatc?1260

agccccctga?acacccccta?ccgcgccggc?gagaacctga?acctgagctg?ccacgccgcc?1320

agcaaccccg?ccgcccagta?cagctggttc?gtgaacggca?ccttccagca?gagcacccag?1380

gagctgttca?tccccaacat?caccgtgaac?aacagcggca?gctacatgtg?ccaggcccac?1440

aacagcgcca?ccggcctgaa?ccgcaccacc?gtgaccgcca?tcaccgtgta?cgtggagctg?1500

cccaagccct?acatcagcag?caacaacagc?aaccccatcg?aggacaagga?cgccgtgacc?1560

ctgacctgcg?agcccgtggc?cgagaacacc?acctacctgt?ggtgggtgaa?caaccagagc?1620

ctgagcgtga?gcccccgcct?gcagctgagc?aacggcaacc?gcatcctgac?cctgctgagc?1680

gtgacccgca?acgacaccgg?cccctacgag?tgcggcatcc?agaacagcga?gagcgccaag?1740

cgcagcgacc?ccgtgaccct?gaacgtgacc?tacggccccg?acacccccat?catcagcccc?1800

cccgacctga?gctaccgcag?cggcgccaac?ctgaacctga?gctgccacag?cgacagcaac?1860

cccagccccc?agtacagctg?gctgatcaac?ggcaccctgc?gccagcacac?ccaggtgctg?1920

ttcatcagca?agatcaccag?caacaacagc?ggcgcctacg?cctgcttcgt?gagcaacctg?1980

gccaccggcc?gcaacaacag?catcgtgaag?aacatcagcg?tgagcagcgg?cgacagctct?2040

agaacccctc?agaacatcac?cgatctgtgc?gccgagtacc?acaacaccca?gatctacacc?2100

ctgaacgaca?agatcttcag?ctacaccgag?agcctggccg?gcaagagaga?gatggccatc?2160

atcaccttca?agaacggcgc?catcttccag?gtggaggtgc?ccggcagcca?gcacatcgac?2220

agccagaaga?aggccatcga?gcggatgaag?gacaccctgc?ggatcgccta?cctcaccgag?2280

gccaaggtgg?agaagctgtg?cgtgtggaac?aacaagaccc?ctcacgccat?cgccgccatc?2340

agcatggcca?attgataag 2359

<210>53

<211>784

<212>PRT

＜213〉artificial sequence

<220>

＜223〉rhCEA-CTBopt fusions

<400>53

Met?Gly?Ser?Pro?Ser?Ala?Pro?Leu?His?Arg?Trp?Cys?Ile?Pro?Trp?Gln

1 5 10 15

Thr?Leu?Leu?Leu?Thr?Ala?Ser?Leu?Leu?Thr?Phe?Trp?Asn?Pro?Pro?Thr

20 25 30

Thr?Ala?Gln?Leu?Thr?Ile?Glu?Ser?Arg?Pro?Phe?Asn?Val?Ala?Glu?Gly

35 40 45

Lys?Glu?Val?Leu?Leu?Leu?Ala?His?Asn?Val?Ser?Gln?Asn?Leu?Phe?Gly

50 55 60

Tyr?Ile?Trp?Tyr?Lys?Gly?Glu?Arg?Val?Asp?Ala?Ser?Arg?Arg?Ile?Gly

65 70 75 80

Ser?Cys?Val?Ile?Arg?Thr?Gln?Gln?Ile?Thr?Pro?Gly?Pro?Ala?His?Ser

85 90 95

Gly?Arg?Glu?Thr?Ile?Asp?Phe?Asn?Ala?Ser?Leu?Leu?Ile?His?Asn?Val

100 105 110

Thr?Gln?Ser?Asp?Thr?Gly?Ser?Tyr?Thr?Ile?Gln?Val?Ile?Lys?Glu?Asp

115 120 125

Leu?Val?Asn?Glu?Glu?Ala?Thr?Gly?Gln?Phe?Arg?Val?Tyr?Pro?Glu?Leu

130 135 140

Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro?Val?Glu?Asp?Lys

145 150 155 160

Asp?Ala?Val?Ala?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr?Thr?Tyr

165 170 175

Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Pro?Val?Ser?Pro?Arg?Leu?Glu

180 185 190

Leu?Ser?Ser?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro?Arg?Asn

195 200 205

Asp?Thr?Thr?Ser?Tyr?Lys?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser?Val?Arg

210 215 220

Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp?Ala?Pro

225 230 235 240

Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn?Leu?Asn

245 250 255

Leu?Thr?Cys?His?Ala?Ala?Ser?Asn?Pro?Thr?Ala?Gln?Tyr?Phe?Trp?Phe

260 265 270

Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile?Pro?Asn

275 280 285

Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His?Asn?Ser

290 295 300

Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val?Tyr?Ala

305 310 315 320

Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Thr?Ser?Asn?Asn?Ser?Asn?Pro?Ile?Glu

325 330 335

Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Glu?Thr?Gln?Asp?Thr

340 345 350

Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser?Ser?Arg

355 360 365

Leu?Glu?Leu?Ser?Asn?Asp?Asn?Arg?Thr?Leu?Thr?Val?Phe?Asn?Ile?Pro

370 375 380

Arg?Asn?Asp?Thr?Thr?Phe?Tyr?Glu?Cys?Glu?Thr?Gln?Asn?Pro?Val?Ser

385 390 395 400

Val?Arg?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Leu?Tyr?Gly?Pro?Asp

405 410 415

Ala?Pro?Thr?Ile?Ser?Pro?Leu?Asn?Thr?Pro?Tyr?Arg?Ala?Gly?Glu?Asn

420 425 430

Leu?Asn?Leu?Ser?Cys?His?Ala?Ala?Ser?Asn?Pro?Ala?Ala?Gln?Tyr?Ser

435 440 445

Trp?Phe?Val?Asn?Gly?Thr?Phe?Gln?Gln?Ser?Thr?Gln?Glu?Leu?Phe?Ile

450 455 460

Pro?Asn?Ile?Thr?Val?Asn?Asn?Ser?Gly?Ser?Tyr?Met?Cys?Gln?Ala?His

465 470 475 480

Asn?Ser?Ala?Thr?Gly?Leu?Asn?Arg?Thr?Thr?Val?Thr?Ala?Ile?Thr?Val

485 490 495

Tyr?Val?Glu?Leu?Pro?Lys?Pro?Tyr?Ile?Ser?Ser?Asn?Asn?Ser?Asn?Pro

500 505 510

Ile?Glu?Asp?Lys?Asp?Ala?Val?Thr?Leu?Thr?Cys?Glu?Pro?Val?Ala?Glu

515 520 525

Asn?Thr?Thr?Tyr?Leu?Trp?Trp?Val?Asn?Asn?Gln?Ser?Leu?Ser?Val?Ser

530 535 540

Pro?Arg?Leu?Gln?Leu?Ser?Asn?Gly?Asn?Arg?Ile?Leu?Thr?Leu?Leu?Ser

545 550 555 560

Val?Thr?Arg?Asn?Asp?Thr?Gly?Pro?Tyr?Glu?Cys?Gly?Ile?Gln?Asn?Ser

565 570 575

Glu?Ser?Ala?Lys?Arg?Ser?Asp?Pro?Val?Thr?Leu?Asn?Val?Thr?Tyr?Gly

580 585 590

Pro?Asp?Thr?Pro?Ile?Ile?Ser?Pro?Pro?Asp?Leu?Ser?Tyr?Arg?Ser?Gly

595 600 605

Ala?Asn?Leu?Asn?Leu?Ser?Cys?His?Ser?Asp?Ser?Asn?Pro?Ser?Pro?Gln

610 615 620

Tyr?Ser?Trp?Leu?Ile?Asn?Gly?Thr?Leu?Arg?Gln?His?Thr?Gln?Val?Leu

625 630 635 640

Phe?Ile?Ser?Lys?Ile?Thr?Ser?Asn?Asn?Ser?Gly?Ala?Tyr?Ala?Cys?Phe

645 650 655

Val?Ser?Asn?Leu?Ala?Thr?Gly?Arg?Asn?Asn?Ser?Ile?Val?Lys?Asn?Ile

660 665 670

Ser?Val?Ser?Ser?Gly?Asp?Ser?Ser?Arg?Thr?Pro?Gln?Asn?Ile?Thr?Asp

675 680 685

Leu?Cys?Ala?Glu?Tyr?His?Asn?Thr?Gln?Ile?Tyr?Thr?Leu?Asn?Asp?Lys

690 695 700

Ile?Phe?Ser?Tyr?Thr?Glu?Ser?Leu?Ala?Gly?Lys?Arg?Glu?Met?Ala?Ile

705 710 715 720

Ile?Thr?Phe?Lys?Asn?Gly?Ala?Ile?Phe?Gln?Val?Glu?Val?Pro?Gly?Ser

725 730 735

Gln?His?Ile?Asp?Ser?Gln?Lys?Lys?Ala?Ile?Glu?Arg?Met?Lys?Asp?Thr

740 745 750

Leu?Arg?Ile?Ala?Tyr?Leu?Thr?Glu?Ala?Lys?Val?Glu?Lys?Leu?Cys?Val

755 760 765

Trp?Asn?Asn?Lys?Thr?Pro?His?Ala?Ile?Ala?Ala?Ile?Ser?Met?Ala?Asn

770 775 780

<210>54

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Toxoid,tetanus peptide

<400>54

Asn?Asn?Phe?Thr?Val?Ser?Phe?Trp?Leu?Arg?Val?Pro?Lys?Val?Ser?Ala

1 5 10 15

Ser?His?Leu?Glu

20

Claims (35)

1. the nucleic acid molecule that comprises the nucleotide sequence of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of DOM, FcIgG, CT, LTA and LTB; Can in Mammals, produce immune response with wherein said fusion rotein.

2. the nucleic acid molecule of claim 1, wherein said CEA albumen is human CEA albumen or its variant.

3. the nucleic acid molecule of claim 1, wherein said CEA albumen is rhesus monkey CEA albumen or its variant.

4. the nucleic acid molecule of claim 1, wherein said CEA albumen are the terminal brachymemmas of C-.

5. the nucleic acid molecule of claim 4, the terminal brachymemma of wherein said C-comprises the amino acid 679-702 of SEQ IDNO:20.

6. the nucleic acid molecule of claim 1, wherein said immunostimulant element comprise the entity part of subunit A of the heat labile enterotoxin (LT) of E.coli.

7. the nucleic acid molecule of claim 1, wherein said immunostimulant element comprise the entity part of subunit B of the heat labile enterotoxin (LT) of E.coli.

8. the nucleic acid molecule of claim 7, wherein said LT subunit B by brachymemma its signal sequence.

9. the nucleic acid molecule of claim 1, wherein said immunostimulant element comprises the entity part of DOM or FcIgG.

10. the nucleic acid molecule of claim 1, wherein said nucleotide sequence comprises the nucleotide sequence that is selected from by SEQID NO:7,9,11,12,14,21,25,49,50 and 52 groups that constitute.

11. the nucleic acid molecule of claim 11, wherein said nucleotide sequence are included in listed nucleotide sequence among SEQ IDNO:9, SEQ ID NO:11 or the SEQ ID NO:12.

12. comprise the nucleic acid molecule of the nucleotide sequence of coding CEA fusion rotein, wherein said nucleotides sequence is listed among the SEQ ID NO:12 and lists.

13. the nucleic acid molecule of claim 8, the proteic C-end of wherein said CEA is fused to the N-end of LT subunit B.

14. comprise the carrier of the nucleic acid molecule of claim 1.

15. the carrier of claim 14, wherein said carrier are adenovirus carrier or plasmid vector.

16. the carrier of claim 15, wherein said carrier are Ad 5 carriers.

17. the carrier of claim 15, wherein said carrier are Ad 6 carriers or Ad 24 carriers.

18. the carrier of claim 15, wherein said carrier are chimpanzee Ad carriers.

19. the carrier of claim 15, wherein said carrier is pV1JnsB.

20. comprise the host cell of the carrier of claim 15.

21. in recombinant host cell, express the method for CEA fusion rotein, comprising:

(a) will comprise in the host cell that the carrier of the nucleic acid molecule of claim 1 import to be fit to; With

(b) allowing the described host cell of cultivation under the condition of expressing described human CEA fusion rotein.

22. CEA fusion rotein by the purifying of the nucleic acid molecule encoding of claim 1.

23. the CEA fusion rotein of the purifying of claim 22, wherein said fusion rotein comprise the aminoacid sequence that is selected from by SEQ ID NO:8,10,13,15,45,46,51 and 53 groups that constitute.

24. prevention or treatment method for cancer comprise the vaccine carrier that comprises the nucleic acid molecule of claim 1 to administration.

25. according to the method for claim 24, wherein said Mammals is human.

26. according to the method for claim 25, wherein said carrier is adenovirus carrier or plasmid vector.

27. method according to claim 26, wherein said carrier is the adenovirus carrier that comprises the adenoviral gene group, described adenoviral gene group has deletion and the inset in the adenovirus E 1 zone in the adenovirus E 1 zone, and wherein said inset comprises expression cassette, and described expression cassette comprises:

(a) comprise the polynucleotide of the nucleotide sequence of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of DOM, FcIgG, CT, LTA and LTB; Can in Mammals, produce immune response with wherein said fusion rotein; And,

(b) promotor that can be operatively connected with described polynucleotide.

28. according to the method for claim 26, wherein said carrier is the plasmid vaccine carrier, it comprises plasmid part and effable box, and described box comprises

(a) comprise the polynucleotide of the nucleotide sequence of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of DOM, FcIgG, CT, LTA and LTB; Can in Mammals, produce immune response with wherein said fusion rotein; And,

(b) promotor that can be operatively connected with described polynucleotide.

29. comprise the adenovirus vaccine carrier of adenoviral gene group, described adenoviral gene group has deletion and the inset in the E1 zone in the E1 zone, wherein said inset comprises expression cassette, and described expression cassette comprises:

(a) comprise the polynucleotide of the nucleotide sequence of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of DOM, FcIgG, CT, LTA and LTB; Can in Mammals, produce immune response with wherein said fusion rotein; And,

(b) promotor that can be operatively connected with described polynucleotide.

30. according to the adenovirus carrier of claim 29, it is Ad 5 carriers.

31. according to the adenovirus carrier of claim 29, it is Ad 6 carriers or Ad 24 carriers.

32. comprise the vaccine plasmid of plasmid part and expression cassette part, described expression cassette partly comprises:

(a) comprise the polynucleotide of the nucleotide sequence of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of DOM, FcIgG, CT, LTA and LTB; Can in Mammals, produce immune response with wherein said fusion rotein; And,

(b) promotor that can be operatively connected with described polynucleotide.

33. treatment suffers from the CEA associated cancer or to the mammiferous method of CEA associated cancer susceptible, comprising:

(a) import first carrier in Mammals, described first carrier comprises:

(i) comprise the polynucleotide of the nucleotide sequence of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of DOM, FcIgG, CT, LTA and LTB; Can in Mammals, produce immune response with wherein said fusion rotein; And,

The (ii) promotor that can be operatively connected with described polynucleotide;

(b) allow through preset time; With

(c) import second carrier in this Mammals, described second carrier comprises:

(i) comprise the polynucleotide of the nucleotide sequence of coding CEA fusion rotein, wherein said CEA fusion rotein comprises CEA albumen or its variant, it is fused to the entity part of immunostimulant element, and described immunostimulant element is selected from the group that is made of DOM, FcIgG, CT, LTA and LTB; Can in Mammals, produce immune response with wherein said fusion rotein; And

The (ii) promotor that can be operatively connected with described polynucleotide.

34. according to the method for claim 33, wherein said first carrier is a plasmid, described second carrier is an adenovirus carrier.

35. according to the method for claim 33, wherein said first carrier is an adenovirus carrier, described second carrier is a plasmid.

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