CN100358917C - Tumor tag and the use thereof - Google Patents

Tumor tag and the use thereof Download PDF

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CN100358917C
CN100358917C CNB028284321A CN02828432A CN100358917C CN 100358917 C CN100358917 C CN 100358917C CN B028284321 A CNB028284321 A CN B028284321A CN 02828432 A CN02828432 A CN 02828432A CN 100358917 C CN100358917 C CN 100358917C
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吴骏
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Shanghai Genomics Inc
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Abstract

The present invention provides a new tumor marker-ULBP-4 protein, polynucleotide for coding the ULBP-4 protein and a method for producing the ULBP-4 protein by the recombination technology. The present invention also discloses the use of the ULBP-4 protein and coding sequences thereof, for example, for the diagnosis and therapy of tumors. The present invention also provides a medicine composition containing the ULBP-4 protein or antibodies thereof.

Description

Tumor markers and uses thereof
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to a kind of new tumor markers-ULBP-4 albumen, coding proteic polynucleotide of ULBP-4 and produce the proteic method of this ULBP-4 through recombinant technology.The invention also discloses the purposes of ULBP-4 albumen and encoding sequence thereof, as be used for diagnosis and treatment tumour, and the pharmaceutical composition that contains ULBP-4 albumen or its antibody.
Background technology
Twentieth century since the fifties medical science development of leap has been arranged, the progress of cancer therapy also is fruitful, and found many directly with relevant oncogene and the cancer suppressor gene of cancer generation.
Before 40 years, Lewis Thomas ﹠amp; Macfarlane Burnet has just proposed the effect that monitoring arranged and suppress of immunity system to cancer.Result of study about NK cell and T cell over nearly 1 year provides new supporting evidence for this hypothesis, and has disclosed the possible molecular mechanism that participates in this process.
NK cell (natural killer cell) is the first line of defence of immunity.Their target cell can be discerned, be killed to NK emiocytosis cytotoxin apace.Because the effect of NK cell need not antigen or mitotic division primary stimuli, does not also rely on antibody or complement, so itself should be able to be differentiated unusual and healthy tissues.It is generally acknowledged that at present the NK cell activity is to regulate by the balance of inhibition on the surface and activation receptor.The receptors bind of MHC-I and NK cell surface, comprise mouse Ly49 (identification H-2K, H-2D) and people KIR (Immunoglobulin-likereceptor, identification HLA-A ,-B, or-C) interior, thereby suppress its activity.In virus infected cell and tumour cell, the expression of MHC-I is destroyed usually, thereby can not kill with the inhibition receptors bind activation NK cell of NK cell surface and by it.
Except the NK cell, the T cell can stop the generation of the dermatoma that carcinogenic factor brings out.In human and mouse, (T Cell Receptor, TCR) male T cell is positioned at skin, digestive tube epithelium and mucomembranous surface to γ δ-TXi Baoshouti, participates in the locality immunity.Present experimental result shows that these T cells also have important effect in eliminating extraneous carcinogen cell transformed.Research shows that also γ δ-TCR+T cell can stop the generation of gut epithelium tumour.Two MHC-I associated molecules, promptly MICA/MICB is found the signal that can be used as cellular abnormality, causes immunne response.
People such as Bauer (Bauer S, et al., Science 1999 Jul 30; 285 (5428): 727-9) by representative variance analysis technology (representational difference analysis, RDA) identify the acceptor NKG2D of MICA/MICB, a kind ofly before this its expression and function is unknown orphan C type agglutinin NK cell receptor (orphan C-type lectin-like NKcell receptor).
Had been found that at present several MHC-I with and the specific NK cell receptor of associated molecule different with other NK cell receptor, NKG2D is a kind of activation receptor that is positioned at all NK cells, γ δ-TCR+T cell, CD8+ α β-TCR+T cell surface, with stride film switching protein D AP10 and act on (Wu J mutually, et al., Science 1999 Jul 30; 285 (5428): 730-2).The proteic kytoplasm of DAP10 partly contains a YxxM motif, can activate PI3 kinases pathway.
Do not have human MICA/B homologous gene in the mouse.Yet, find that in mouse another is called as glycoprotein family (Cerwenka A, et al., the Immunity2000 Jun of RAE-1; 12 (6): 721-7), act on equally, show the homology more weak with MHC-I as the part (ligand) of NKG2D.So far, had been found that 5 kinds of mouse RAE-1 paraproteins (RAE-1-α ,-β ,-γ ,-δ ,-ε) and an associated protein, H60.Expression analysis demonstration RAE-1 gene is not expressed or expresses very low in normal adult tissue, and in the expression of some tumor tissues constitutive characters, its expression amount improves under the effect of vitamin A acid (retinoic acid).In normal skin, do not have the expression of RAE-1, after carcinogen TPA induces, in the tumour of mouse, express.(Diefenbach A, et al., Nature 2001 Sep 13 such as recent Diefenbach; 413 (6852): 165-71) and people (Cerwenka A, et al., Proc Natl Acad Sci USA 2001Sep 25 such as Cerwenka; 98 (20): studies show that 11521-6), in the tumour of expressing MHC Class I, if express RAE-1 simultaneously, this tumour also can be external by the NK cells exclude.Identical with the NK cell, mouse γ δ-TCR+T cell can kill the squamous cell cancer cells (Squamous cell carcinomaline) of culturing in vivo, and under specific circumstances, RAE-1 also can cause the memory of γ δ-TCR+T cell to transplantation tumor.
In the mankind, also found the homologous gene of Rae-1, i.e. people ULBP-1 ,-2 ,-3.As the human cytomegalovirus (Cytomegalovirus, CMV) the conjugated protein of glycoprotein UL16 at first is found, the ULBP gene is relevant with MHC-I family, but and equally can with not very near getting in touch of UL16 bonded MICB.ULBP also is the part of NKG2D, can stimulate NK cell expressing cytokine (cytokine) and chemokines (chemokine).The expression of ULBP in the target cell of anti-NK cell can make it be subjected to the attack of NK cell.Thereby ULBP or MIC antigen that the cell of cytomegalovirus infection may be have just covered cell surface by UL16 albumen are escaped immune attack.
In sum, these that carry out in human and mouse studies show that, no matter at be virus or tumour, in the cell-mediated immune protection of NK cell, γ δ-TCR+T cell, CD8+ α β-TCR+T, NKG2D has played the effect of a key.Yet up to the present, the activation mechanism of NKG2D is also unclear.
Since the nineties, the research of tumor immunology has obtained some breakthroughs, and immunotherapy of tumors enters a new climax again.A lot of immunotherapy methods have entered clinical trial, mainly be by several different methods in vivo or external training, activate the patient immunocyte with the identification malignant cell, and make its a large amount of amplifications, finally remove or suppress malignant cell.
Aspect tumor immunology research, found human tumor immunological rejection antigen in 1991 first.Later a lot of researchs prove that most of tumour cells have the composition different with normal cell, can cause immunity system to identification and attack, be referred to as immunologic tumor rejection antigen.But utilize immunologic tumor rejection antigen can induce the immunocyte that produces killing tumor cells in the patient inside and outside.Therefore immunologic tumor rejection antigen is composition the most key in the immunotherapy of tumors.
Up to now, in melanoma and comprise in the various tumor tissues of prostate cancer, thymic carcinoma, ovarian cancer, stomach cancer and found many tumour antigens.The human tumor immunological rejection antigen of having found at present can be divided into four types.First kind antigen comes from the somatic mutation of normal gene product, and the second class antigen comes from the transgenation relevant with oncogenic process.This two classes antigen is patient-specific antigen, is not suitable for the ubiquity treatment.The 3rd class antigen is the sort of expression to be arranged also in healthy tissues, but the product of the gene that expression amount raises in tumour.If do not relate to sudden change, this class antigen has ubiquity in various cancer patients, but they are normally tissue-specific, and is not peculiar by tumour, has little significance in clinical application.The 4th class antigen has strict tumour-specific, and is relevant with general oncogenic process.Therefore, this class antigen is expressed in human tumor widely, is suitable for the target spot as tumor markers and antineoplastic immune attack most.Yet, seldom belong to this class in the antigen of having found at present.
Therefore, this area presses for the 4th new class immunologic tumor rejection antigen of exploitation as tumor markers, is used for the diagnosis and the treatment of tumour.
Summary of the invention
The purpose of this invention is to provide a kind of new human tumor marker thing ULBP-4 albumen with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
The present invention has found and has isolated the new antigen gene that can be used as tumor markers: ULBP-4 through extensive and deep research.This gene in healthy tissues, do not express or expression amount very low, wide expression in tumor tissues.Its expression product is a membranin, also may be secreted into the extracellular.The ULBP-4 membranin that is expressed in cell surface can be efficiently and the NKG2D receptors bind.Finished the present invention on this basis.
In a first aspect of the present invention, novel isolated ULBP-4 polypeptide is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ IDNO:2 or 4 aminoacid sequences.Preferably, this polypeptide has the aminoacid sequence of 1-263 position in SEQ ID NO:2 or 4 or 24-263 position.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people ULBP-4 polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has 1-263 position or 24-263 amino acids polypeptide of sequence in SEQ ID NO:2 or 4.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 403-1194 position among the SEQ IDNO:1; (b) has the sequence of 1-1434 position among the SEQ ID NO:1; (c)
Sequence with 472-1194 position among the SEQID NO:1; (d) has the sequence of 1-792 position among the SEQ ID NO:3; (e) has the sequence of 70-792 position among the SEQ ID NO:3.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, preparation ULBP-4 is provided proteic method, this method comprises: (a) under the condition that is fit to expressing protein, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate ULBP-4 albumen.
In a fifth aspect of the present invention, provide and above-mentioned people ULBP-4 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used as primer or probe also is provided, and it contains a successive 15-1434 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people ULBP-4 polypeptide active is provided, and the compound that suppresses people ULBP-4 polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people ULBP-4 polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of ULBP-4 in the test sample, it comprises: sample is contacted with the proteic specific antibody of ULBP-4, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample ULBP-4 albumen.A kind of method that detects tumour also is provided, has comprised step: whether had ULBP-4 albumen in detection patient's the sample (as blood, urine, body fluid, saliva etc.).
In a eighth aspect of the present invention, the present invention also provides a kind of test kit that detects tumour, the primer that it contains specific amplification ULBP-4 to and/or the ULBP-4 specific antibody.In addition, also can contain specific probe and/or PCR damping fluid etc.
In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes people ULBP-4 polypeptide active, perhaps suppresses the antagonist of people ULBP-4 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people ULBP-4 of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.
In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the people ULBP-4 antagonist of the present invention and the pharmaceutically acceptable carrier of safe and effective amount.Preferred ULBP-4 antagonist is antibody and the ULBP-4 antisense sequences of anti-ULBP-4.These pharmaceutical compositions can be treated tumour.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 is the electrophorogram of ULBP-4.
Fig. 2 has shown the cDNA encoding sequence (SEQ ID NO:3) of a kind of people ULBP-4, and the difference of comparing with 403-1194 bit sequence among the SEQ ID NO:1 marks with underscore.
Fig. 3 has shown the aminoacid sequence (SEQ ID NO:4) of a kind of people ULBP-4, and the difference of comparing with SEQ ID NO:2 sequence marks with underscore.
Fig. 4 has shown the express spectra of ULBP-4 in various tumor cell strains.Each swimming lane is: 1, U373 (astrocytoma); 2, JURKAT (acute T chronic myeloid leukemia); 3, LOVO (colorectal carcinoma); 4, HELA (adenocarcinoma of cervix); 5, SW480 (colorectal carcinoma); 6, DU145 (prostate cancer); M: molecular weight standard.
Fig. 5 has shown the express spectra of ULBP-4 in multiple tissue.Wherein each swimming lane is: 1. liver; 2. skeletal muscle; 3. placenta; 4. spleen; 5. white corpuscle; 6. pancreas; 7. lymphoglandula; 8. heart; 9. lung; 10. brain; 11. thymus gland; 12. marrow; M: molecular weight standard.
Fig. 6 has shown that the ULBP-4 specificity is incorporated into NKG2D.
Detailed Description Of The Invention
In the present invention, term " ULBP-4 albumen ", " ULBP-4 polypeptide " or " tumor markers ULBP-4 " be used interchangeably, all refer to have human tumor marker thing ULBP-4 amino acid sequence (SEQ ID NO:2 or 4) Albumen or polypeptide. They also comprise the ripe ULBP-4 that does not contain signal peptide (1-23 position).
As used herein, " separation " refers to that material separates (if natural from its primal environment Material, primal environment namely are natural surroundingses). Such as the polynucleotides under the native state in the active somatic cell and polypeptide There is not separation and purification, but same polynucleotides or polypeptide other materials as from native state, existing together In separately, then for separation and purification.
As used herein, " ULBP-4 albumen or the polypeptide of separation " refers to that the ULBP-4 polypeptide is substantially free of the sky Right relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use standard Purified technology of protein purifying ULBP-4 albumen. Basically pure polypeptide is on non-reduced polyacrylamide gel Can produce single master tape.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide. This The polypeptide of invention can be the product of natural purifying, or the product of chemical synthesis, or use recombinant technique from Produce in protokaryon or the eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell). The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, or can right and wrong sugar Baseization. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analog of people ULBP-4 albumen. As used herein, term " fragment ", " derivative " and " analog " refer to basically keep natural human ULBP-4 egg of the present invention The biological function of Bai Xiangtong or active polypeptide. Polypeptide fragment of the present invention, derivative or analog can be (i) have one or more conservative or non-conservation amino acid residue (preferred conservative amino acid residue) is substituted Polypeptide, and the amino acid residue of such replacement can be also can not encoded by genetic code, or (ii) The polypeptide that in one or more amino acid residues, has substituted radical, or (iii) mature polypeptide and another change Compound (such as the compound that prolongs the polypeptide half-life, for example polyethylene glycol) merges formed polypeptide, or (iv) Additional amino acid sequence be fused to this peptide sequence and the polypeptide that forms (such as targeting sequencing or secretion sequence or usefulness Come sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion of the formation of antigen I gG fragment). Root According to the instruction of this paper, these fragments, derivative and analog belong to the known scope of those skilled in the art.
In the present invention, term " people ULBP-4 polypeptide " refers to have the SEQ ID NO. of people ULBP-4 protein active The full-length polypeptide of 2 or 4 sequences or mature polypeptide. This term also comprises having and people ULBP-4 albumen identical function , the variant form of SEQ ID NO.2 or 4 sequences. These variant forms comprise (but being not limited to): some Individual (be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose Disappearance, insert and/or replace, and add one or several and (be generally 20 in that C end and/or N are terminal In, preferably be in 10, more preferably be in 5) amino acid. For example, in the art, use When close the or similar amino acid of performance replaces, usually can not change the function of protein. Again such as, One of the terminal interpolation of C end and/or N or several amino acid also can not change the function of protein usually. This term The active fragment and the reactive derivative that also comprise people ULBP-4 albumen.
The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural sudden change Body, induced mutation body, under high or low stringency condition, can compile with the DNA of people ULBP-4 DNA hybridization The albumen of code and the polypeptide or the albumen that utilize the antiserum acquisition of anti-people ULBP-4 polypeptide. The present invention also carries Supply other polypeptide, as comprised the fusion of people ULBP-4 polypeptide or its fragment. Many except total length almost Outside the peptide, the present invention has also comprised the soluble fragments of people ULBP-4 polypeptide. Usually, this fragment has people ULBP-4 peptide sequences at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably extremely Few about 50 continuous amino acids are more preferably at least about 80 continuous amino acids, best at least about 100 companies Continuous amino acid.
Invention also provides the analog of people ULBP-4 albumen or polypeptide. These analogs and natural human ULBP-4 are many The difference of peptide can be the difference on the amino acid sequence, and can be affect poor on the modified forms of sequence yet Different, perhaps have both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can lead to Cross various technology and obtain, as by radiation or be exposed to mutagens and produce random mutagenesis, also can lure by fixed point Political reform or the biological technology of other known moleculars. Analog also comprises having that to be different from natural L-amino acid whose residual The analog of base (such as D-amino acid), and have that non-natural exists or synthetic amino acid analogue. Should manage Separate, polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(usually the not changing primary structure) form of modifying comprises: in the body or the chemically derived form of external polypeptide as Acetylation or carboxylated. Modify and also to comprise glycosylation, such as those in the synthetic and processing of polypeptide or further add Carry out glycosylation modified and polypeptide that produce during work step is rapid. This modification can be carried out sugar by polypeptide is exposed to The enzyme of baseization (such as mammiferous glycosylase or deglycosylating enzyme) and finishing. Modified forms also comprises having phosphorus The sequence of acidifying amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine). Also comprise and being repaiied Thereby decorations have improved its anti-proteolysis performance or have optimized the polypeptide of solubility property.
In the present invention, " people ULBP-4 albumen conservative variation polypeptide " refers to the ammonia with SEQ ID NO:2 or 4 The base acid sequence is compared, and has 10 at the most, and preferably at the most 8, more preferably at the most 5, at the most 3 best Amino acid is replaced by similar performance or close amino acid and is formed polypeptide. These conservative variation polypeptide are best Carrying out amino acid substitution according to table 1 produces.
Table 1
Initial residue Representational replacement The preferred replacement
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
Polynucleotides of the present invention can be dna form or rna form. Dna form comprises cDNA, genome DNA or artificial synthetic DNA. DNA can be strand or double-stranded. DNA can be coding strand or non-volume The code chain. The coding region sequence of encoding mature polypeptide can with the coding region sequence phase shown in SEQ ID NO:1 or 3 With or the variant of degeneracy. As used herein, " variant of degeneracy " refers to the tool of encoding in the present invention The protein that SEQ ID NO:2 or 4 are arranged, but with the coding region sequence shown in SEQ ID NO:1 or 3 difference is arranged Nucleotide sequence.
The polynucleotides of the mature polypeptide of coding ULBP-4 comprise: the coded sequence of an encoding mature polypeptide; Become The coded sequence of ripe polypeptide and various additional code sequence; The coded sequence of mature polypeptide is (with optional additional volume The code sequence) and non-coding sequence. Term " polynucleotides of coded polypeptide " can be to comprise this polypeptide of coding Polynucleotides, also can be the polynucleotides that also comprise additional code and/or non-coding sequence.
The invention still further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid sequence with the present invention Polypeptide or fragment, analog and the derivative of polypeptide. The variant of these polynucleotides can be natural generation The variant that allelic variant or non-natural take place. These nucleotide diversity bodies comprise that replacement variant, disappearance become Allosome and insertion variant. As known in the art, allelic variant is the replacement form of polynucleotides, It may be replacement, disappearance or the insertion of one or more nucleotides, but can be not many from changing in fact its coding The function of peptide.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, more preferably at least 80%, the polynucleotides of at least 90% or 95% homogeny best. The present invention relates to especially And under stringent condition with the interfertile polynucleotides of polynucleotides of the present invention. In the present invention, " strict Condition " refer to: (1) than the hybridization under LIS and the higher temperature and wash-out, such as 0.2 * SSC, 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% Just hybridize when above. And, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO:2 or 4 The mature polypeptide that shows has identical biological function and activity.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding ULBP-4.
People ULBP-4 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, derivative) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The method of using round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.The primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or ULBP-4 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
By the recombinant DNA technology of routine, can utilize polynucleotide sequence of the present invention to can be used to express or produce the ULBP-4 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people ULBP-4 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, people ULBP-4 polynucleotide sequence can be inserted in the recombinant expression vector.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains people ULBP-4 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, the bacterial cell of streptomyces; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS or 293 cells etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results 2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl 2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The people ULBP-4 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the antibody, polypeptide or other material that are used to screen antagonism ULBP-4 protein function.
On the other hand, the present invention also comprises people ULBP-4 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people ULBP-4 gene product or fragment.Preferably, refer to that those can combine with people ULBP-4 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) 2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.
The proteic antibody of anti-people ULBP-4 can be used in the immunohistochemistry technology, detects the people ULBP-4 albumen in the biopsy specimen.
Antibody of the present invention can be used for treatment or prevention people's ULBP-4 protein related diseases such as tumour.A kind of method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell of people ULBP-4 protein positive, as tumour cell.
Utilize albumen of the present invention,, can filter out with ULBP-4 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
ULBP-4 antagonist (as antibody and antisense sequences) can be directly used in disease treatment, for example, is used for the treatment of tumour aspect.In addition, but also coupling other treatment agent, as TNF-α, TNF-β etc.
The present invention also provides a kind of pharmaceutical composition, and it contains ULBP-4 antagonist of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents be the treatment significant quantity, for example every day about 1 microgram-10 mg/kg body weight.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people ULBP-4 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people ULBP-4 protein level that is detected in the test can be used for diagnosing tumour.
Whether having the proteic method of ULBP-4 in a kind of test sample is to utilize the proteic specific antibody of ULBP-4 to detect, and it comprises: sample is contacted with the ULBP-4 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample ULBP-4 albumen.
The proteic polynucleotide of ULBP-4 can be used for the diagnosis and the treatment of ULBP-4 protein related diseases.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray or DNA chip, is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of ULBP-4 albumen and also can detect the proteic transcription product of ULBP-4.
The present invention also provides a kind of test kit that detects tumour, the primer that it contains specific amplification ULBP-4 to and/or the ULBP-4 specific antibody.In addition, also can contain specific probe and/or PCR damping fluid etc.
Sequencing result to ULBP-4 shows, it and ULBP-1 ,-2 and the homology of-3 three genes relatively poor, but all can with the NKG2D combination, be positioned at the same position on the karyomit(e).Expression analysis is the result show, the ULBP-4 gene is only expressed in tumor tissues, in normal cell, do not express or expression amount very low.The comparison result shows of RAE-1 family and H60 gene in ULBP-4 gene and the mouse, ULBP-4 may be exactly the H60 homologous gene among the mankind.Existing report (O ' Callaghan CA, et al., Immunity 2001 Aug; 15 (2): 201-11) show, mouse H60 and RAE-1 family competitively with the NKG2D receptors bind, and H60 is higher about 10 times than RAE-1 family with the avidity of NKG2D.Since human ULBP-1 ,-2, the-the 3rd, the homologous gene of RAE-1 gene, so the binding ability of ULBP-4 and NKG2D acceptor may also can be higher than ULBP-1 ,-2 ,-3.
In addition, interested is that the ULBP gene is the same with the RAE-1 gene family, is the membranin of GPI-grappling.Under the effect of Phospholipase C, can be secreted in the body fluid.Therefore, ULBP-4 may be an effective tumor markers, plays a role in the diagnosis of numerous cancers and treatment.
In view of ULBP-4 may be the distinctive immunological rejection antigen of tumour cell, be secreted in the body fluid.Therefore, the direct mensuration of the ULBP-4 in blood sample or the urine also can be used as the foundation of early diagnosis of tumor except the auxiliary diagnosis that can be used as tumour with the observation index more.
Except as diagnostic means, ULBP-4 also has very big using value on the surgical operation of tumour, helps to determine the degree of depth, concrete edge and the part of hiding of the infiltration of tumour.Radioimmunity instructs surgery, and (radioimmunoguided surgery RIGS) is antibody with the tumor associated antigen of mark labelled with radioisotope, is expelled to and carries out the video picture analysis in the body, and it is the positioning tumor scope of soaking into accurately.
Aspect the immunotherapy of tumour, as immunological rejection antigen, the fusion rotein of ULBP-4 or ULBP4 can with the NKG2D receptors bind that is positioned at NK cell, γ δ-TCR+T cell, CD8+ α β-TCR+T cell surface, activate these cell activity, discharge cytotoxin and kill tumor cell, this class antitumor cell quantity in vivo simultaneously increases.Because ULBP-4 only is positioned at tumor cell surface, it also can be used as the guidance system of biological missile, when the treatment tumour medicine is concentrated on tumor region and improves curative ratio, reduces the injury of medicine to whole body simultaneously.
Aspect tumor vaccine, ULBP-4 can be used for preparing molecule knurl seedling.Tumor vaccine is the main contents of active immunity, is divided into four types of glucagonoma seedling, ubcellular knurl seedling, molecule knurl seedling and gene knurl seedlings.The former two is early stage tumor vaccine, and clinical effectiveness is uneven, and the late result majority is undesirable.Molecule knurl seedling is according to antibody and antigenic interactively, prepares the anti-antibody of antitumor former idiotype, as idiotype knurl seedling, also sees certain curative effect clinically.Tumor antigen peptide can industrialization production, does not have the danger of tumor planting, does not have the inhibition composition of tumour cell.As tumor antigen peptide, the molecular vaccine that obtains is not limited by MHC, and can be applied to kinds of tumors with ULBP-4.Except molecule knurl seedling, the ULBP-4 gene is cascaded, and to insert virus vector also be a kind of seedling of knurl preferably mode as gene knurl seedling.
In addition, the film outside part of ULBP-4 can be used as the immunostimulation factor, combines with NKG2D, thereby activates immunocytes such as NK cell, T cell, reaches the effect of strengthening immunity.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, condition described in the molecular cloning laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
The acquisition and the location of embodiment 1:ULBP-4 gene
With three gene ULBP1 of ULBP family, ULBP2, the protein sequence of ULBP3 is tblastn to the HUMAN-EST storehouse, to seek relevant EST.The result finds three EST:BI258059, BE545401, AL602601 in the section upstream of the localized karyomit(e) contig of ULBP123 (NT007285.7).With the splicing of assembly software, same then contig (NT007285.6) analyzes together, to obtain the ULBP-4 coding region.The splicing sequence is carried out the proteins encoded prediction, and finding has a big frame since 403 bases, but does not have stop code, estimates that 3 ' end is incomplete.Utilize this chromosome segment exon prediction case of genescan tool analysis of ensembl, find that this chromosome segment has an exon compact district, and one section successive exon is arranged, show that 3 ' end of this gene can extend on karyomit(e), the result obtains the complete coding region of this gene.Concrete sequence is shown in SEQ ID NO:1.Its ORF is positioned at the 403-1194 position, one the 263 amino acid whose protein (SEQ ID NO:2) of encoding, and wherein amino acid/11-23 is a signal peptide.EST information according to ULBP-4 is located in karyomit(e) 6, q25.1.
Embodiment 2; The polymorphism of ULBP-4
Synthetic following primer is a template with the human lymphocyte strain Jurkat cDNA library that makes up with ordinary method, clones gene ULBP-4 by conventional PCR method.
5′-cgggatccatgcgaagaatatccctgacttctag-3′(SEQ?ID?NO：5)
5′-gccaagcttagacgtcctcaagggccagagac-3′(SEQ?ID?NO：6)
The result finds out from Fig. 1, and the amplified fragments size is about 800bp, and is consistent with the 792bp of prediction.
Check order after the pcr amplification segment is cloned into carrier, obtain sequence shown in the SEQ ID NO:3, compare, 7 site Nucleotide differences (Fig. 2) are arranged with SEQ IDNO:1.The ULBP-4 albumen of coding has 6 amino acid differences (Fig. 3).With MICA/B, ULPB family has polymorphism and conforms to, and this confirms that ULBP-4 also has polymorphism.
Embodiment 3: to the sequential analysis of ULBP-4
ULBP-4 and RAE-1 family and H60 are carried out the homology comparison, find that ULBP-4 and RAE-1 do not have homology, but 23.8% homology is arranged with H60.This hint people's ULBP-4 may be the homologous gene of H60.
The expression of embodiment 4:ULBP-4
Added one section monoclonal antibody recognition sequence (FLAG) behind the signal peptide sequence of ULBP-4, improved cDNA is inserted into retrovirus expression vector, be transfected into mouse BaF/3 cell then, utilize immunoprecipitation and protein electrophoresis method to confirm the expression of ULBP-4 cDNA in mammalian cell, experiment molecular weight of gained and matching of prediction, about 32kDa.
The express spectra of embodiment 5:ULBP-4
With the inner primer in ULBP-4 coding region,, analyze the expression of ULBP-4 in kinds of tumor cells and healthy tissues by the RT-PCR method:
Forward: 5 '-atgcgaagaatatccctgacttctagc-3 ' (SEQ ID NO:7)
Oppositely: 5 '-attttgccaccagacacagatgagaa-3 ' (SEQ ID NO:8)
From Fig. 4 as seen, as over against photograph, in each JEG-3, all can obtain tangible positive fragment with house-keeping gene GAPDH (right side).And when carrying out PCR with ULBP-4 gene coding region inner primer, can amplify positive fragment at U373 (astrocytoma), JURKAT, HELA (SCC cell), SW480 (colorectal carcinoma), DU145 (prostate cancer) in these 5 cell strain libraries, illustrating has the ULBP-4 expression of gene in these cancer cells.
From Fig. 5 as seen, ULBP-4 does not express in healthy tissues.Because ULBP-4 is specific expressed in tumor tissues, therefore can be used as tumor marker.
Embodiment 6:ULBP-4 combines with NKG2D
Respectively with containing ULBP-1 ,-2 ,-3 and the retrovirus expression vector transfection mouse Ba/F3 cell strain of ULBP-4cDNA.Transfectional cell with the NKG2D-Ig fusion rotein (thick line is represented among the figure) of solubility or human Ig (in contrast, fine rule is represented among the figure) mark after by flow cytometer, immunofluorescence technique mark.
As can be seen from Fig. 6, the Ba/F3 cell strain of untransfected and NKG2D-Ig fusion rotein and the equal debond of human Ig, and transfection ULBP-1 ,-2 ,-3 cell strain can combine with the NKG2D-Ig fusion rotein, show that its product all is parts of NKG2D acceptor, coincide with existing report.Meanwhile, the cell strain of ULBP-4 gene transfection also can combine specifically with NKG2D, and this has not only confirmed the expression of ULBP-4 cDNA in mammalian cell, illustrates that simultaneously this gene product also is the part of NKG2D.
Recombinant expressed and the purifying of embodiment 7 ULBP-4 albumen
In this embodiment, be template with the pcr amplification product among the embodiment 2,5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people ULBP-4 DNA as inserting fragment.
5 ' end Oligonucleolide primers sequence is: 5 '-ccgggatccTTGGAGATCATGGTTG GTGGTCACT-3 ' (SEQ ID NO:9).This primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence that does not contain signal peptide sequence after this restriction enzyme site;
3 ' end primer sequence is: 5 '-ggccaagcttCCATCTATCTGGTAGACTAGAAGAA-3 ' (SEQ ID NO:10).This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people ULBP-4 of HindIII restriction enzyme.
People ULBP-4 albumen cDNA PCR product purification is after BamHI, the HindIII double digestion is recombinated according to a conventional method with plasmid pProEXHTa (GIBCOBRL) and is formed carrier pProEXHTa-ULBP-4 and be converted into the competence bacillus coli DH 5 alpha, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).Order-checking confirms to insert complete ULBP-4 encoding sequence.
Choosing the positive DH5 α clone who expresses ULBP-4 is inoculated in the 10ml LB substratum, 37 ℃ of 300rpm shaking culture are spent the night, be diluted at 1: 100 and be inoculated in LB substratum shaking culture 2.5hr, add behind the 100mM IPTG to 0.1mM 37 ℃ and induce 2-3hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml sample-loading buffer (0.5M NaCl on ice, 20mM imidazoles 2.7mM KCl, 10.1mM Na 2HPO 4, 1.8mM KH 2PO 4, pH8.0) resuspended, ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml Ni 2+Metal-chelating Sepharose 4B chromatography column behind the sample-loading buffer thorough washing, adds 500ul imidazoles elution buffer (0.5M NaCl, 500mM imidazoles 2.7mMKCl, 10.1mM Na 2HPO 4, 1.8mM KH 2PO 4, pH8.0) room temperature leaves standstill after 30 minutes and collects elutriant, repeats wash-out 2-3 time, obtains people ULBP-4 albumen.
The generation of embodiment 8 anti-ULBP-4 protein antibodies
The recombinant human ULBP-4 albumen that obtains among the embodiment 7 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people ULBP-4 protein gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Wu, fine horse
＜120〉tumor markers and uses thereof
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＜213〉homo sapiens (Homo sapiens)
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acagagactc?tgaggaagca?gtcacctcag?gcctctccat?cctagagacg?gcagaaggac 60
aaacatctcc?tgctggcaca?aggaagtcat?aactcatgtg?agtggagcca?tgtgggatta 120
agaagtgata?ggagagcttg?ctgtctgtct?ctgctctcca?ctgtgtgagg?atacaacagg 180
aagacagcca?tctggtgagg?aagagagggc?cctcgccaga?taccggacct?gctgacacct 240
tgatcttgga?cttcccatct?tccaggaagg?cctgacctca?gttgttccag?ggtaaagaat 300
ttgggcagtg?cccacaccca?cgctgttgga?taacatttct?tcaccatacc?agtgagggtg 360
aatgtgtaca?cgcccagctt?cctgcctgtt?actctccacagt?atg?cga?aga?ata 414
Met?Arg?Arg?Ile
1
tcc?ctg?act?tct?agc?cct?gtg?cgc?ctt?ctt?ttg?ttt?ctg?ctg?ttg?cta 462
Ser?Leu?Thr?Ser?Ser?Pro?Val?Arg?Leu?Leu?Leu?Phe?Leu?Leu?Leu?Leu
5 10 15 20
cta?ata?gcc?ttg?gag?atc?atg?gtt?ggt?ggt?cac?tct?ctt?ggc?ttc?aac 510
Leu?Ile?Ala?Leu?Glu?Ile?Met?Val?Gly?Gly?His?Ser?Leu?Gly?Phe?Asn
25 30 35
ttc?act?ata?aaa?tca?ttg?tcc?aga?cct?gga?cag?ccc?tgg?tgt?gaa?gcg 558
Phe?Thr?Ile?Lys?Ser?Leu?Ser?Arg?Pro?Gly?Gln?Pro?Trp?Cys?Glu?Ala
40 45 50
cag?gtc?ttc?ttg?aat?aaa?aat?ctt?ttc?ctt?cag?tac?aac?agt?gac?aac 606
Gln?Val?Phe?Leu?Ash?Lys?Ash?Leu?Phe?Leu?Gln?Tyr?Ash?Ser?Asp?Asn
55 60 65
aac?atg?gtc?aaa?cct?ctg?ggc?ctc?ctg?ggg?aag?aag?gta?aat?gcc?acc 654
Asn?Met?Val?Lys?Pro?Leu?Gly?Leu?Leu?Gly?Lys?Lys?Val?Asn?Ala?Thr
70 75 80
agc?act?tgg?gga?gaa?ttg?acc?caa?acg?ctg?gga?gaa?gtg?ggg?cga?gac 702
Ser?Thr?Trp?Gly?Glu?Leu?Thr?Gln?Thr?Leu?Gly?Glu?Val?Gly?Arg?Asp
85 90 95 100
ctc?agg?atg?ctc?ctt?tgt?gac?atc?aaa?ccc?cag?ata?aag?acc?agt?gat 750
Leu?Arg?Met?Leu?Leu?Cys?Asp?Ile?Lys?Pro?Gln?Ile?Lys?Thr?Ser?Asp
105 110 115
cct?tcc?act?ctg?caa?gtc?gag?atg?ttt?tgt?caa?cgt?gaa?gca?gaa?cgg 798
Pro?Ser?Thr?Leu?Gln?Val?Glu?Met?Phe?Cys?Gln?Arg?Glu?Ala?Glu?Arg
120 125 130
tgc?act?ggt?gca?tcc?tgg?cag?ttc?gcc?acc?aat?gga?gag?aaa?tcc?ctc 846
Cys?Thr?Gly?Ala?Ser?Trp?Gln?Phe?Ala?Thr?Ash?Gly?Glu?Lys?Ser?Leu
135 140 145
ctc?ttt?gac?gca?atg?aac?atg?acc?tgg?aca?gta?att?aat?cat?gaa?gcc 894
Leu?Phe?Asp?Ala?Met?Ash?Met?Thr?Trp?Thr?Val?Ile?Ash?His?Glu?Ala
150 155 160
agt?aag?atc?aag?gag?aca?tgg?aag?aaa?gac?aga?ggg?ctg?gaa?aag?tat 942
Ser?Lys?Ile?Lys?Glu?Thr?Trp?Lys?Lys?Asp?Arg?Gly?Leu?Glu?Lys?Tyr
165 170 175 180
ttc?agg?aag?ctc?tca?aag?gga?gac?tgc?gat?cac?tgg?ctc?agg?gaa?ttc 990
Phe?Arg?Lys?Leu?Ser?Lys?Gly?Asp?Cys?Asp?His?Trp?Leu?Arg?Glu?Phe
185 190 195
tta?ggg?cac?tgg?gag?gca?atg?cca?gaa?ccg?aca?gtg?tca?cca?gta?aat 1038
Leu?Gly?His?Trp?Glu?Ala?Met?Pro?Glu?Pro?Thr?Val?Ser?Pro?Val?Asn
200 205 210
gct?tca?gat?atc?cac?tgg?tct?tct?tct?agt?cta?cca?gat?aga?tgg?atc 1086
Ala?Ser?Asp?Ile?His?Trp?Ser?Ser?Ser?Ser?Leu?Pro?Asp?Arg?Trp?Ile
215 220 225
atc?ctg?ggg?gca?ttc?atc?ctg?tta?gtt?tta?atg?gga?att?gtt?ctc?atc 1134
Ile?Leu?Gly?Ala?Phe?Ile?Leu?Leu?Val?Leu?Met?Gly?Ile?Val?Leu?Ile
230 235 240
tgt?gtc?tgg?tgg?caa?aat?ggt?gag?tgg?cag?gct?ggt?ctc?tgg?ccc?ttg 1182
Cys?Val?Trp?Trp?Gln?Asn?Gly?Glu?Trp?Gln?Ala?Gly?Leu?Trp?Pro?Leu
245 250 255 260
agg?acg?tct?tag?tctggtaagg?actcaagaga?ggtgaatcat?gggtctcatc 1234
Arg?Thr?Ser
caagcccccg?ctccctgatc?catctctctc?cacagaaatg?gtgcactgcc?ttagtcttga 1294
gcccccacac?tacacagcag?ggctggcagc?acaatgtgca?gtgagaggag?ggatgaagca 1354
atcatctcct?ctcgaggagg?ggcaaggtca?aagatctgga?acgcctgcct?ggggtgttgt 1414
ctccttgcca?tccggcccag 1434
<210>2
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<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>2
Met?Arg?Arg?Ile?Ser?Leu?Thr?Ser?Ser?Pro?Val?Arg?Leu?Leu?Leu?Phe
1 5 10 15
Leu?Leu?Leu?Leu?Leu?Ile?Ala?Leu?Glu?Ile?Met?Val?Gly?Gly?His?Ser
20 25 30
Leu?Gly?Phe?Asn?Phe?Thr?Ile?Lys?Ser?Leu?Ser?Arg?Pro?Gly?Gln?Pro
35 40 45
Trp?Cys?Glu?Ala?Gln?Val?Phe?Leu?Asn?Lys?Asn?Leu?Phe?Leu?Gln?Tyr
50 55 60
Asn?Ser?Asp?Asn?Asn?Met?Val?Lys?Pro?Leu?Gly?Leu?Leu?Gly?Lys?Lys
65 70 75 80
Val?Ash?Ala?Thr?Ser?Thr?Trp?Gly?Glu?Leu?Thr?Gln?Thr?Leu?Gly?Glu
85 90 95
Val?Gly?Arg?Asp?Leu?Arg?Met?Leu?Leu?Cys?Asp?Ile?Lys?Pro?Gln?Ile
100 105 110
Lys?Thr?Ser?Asp?Pro?Ser?Thr?Leu?Gln?Val?Glu?Met?Phe?Cys?Gln?Arg
115 120 125
Glu?Ala?Glu?Arg?Cys?Thr?Gly?Ala?Ser?Trp?Gln?Phe?Ala?Thr?Asn?Gly
130 135 140
Glu?Lys?Ser?Leu?Leu?Phe?Asp?Ala?Met?Asn?Met?Thr?Trp?Thr?Val?Ile
145 150 155 160
Asn?His?Glu?Ala?Ser?Lys?Ile?Lys?Glu?Thr?Trp?Lys?Lys?Asp?Arg?Gly
165 170 175
Leu?Glu?Lys?Tyr?Phe?Arg?Lys?Leu?Ser?Lys?Gly?Asp?Cys?Asp?His?Trp
180 185 190
Leu?Arg?Glu?Phe?Leu?Gly?His?Trp?Glu?Ala?Met?Pro?Glu?Pro?Thr?Val
195 200 205
Ser?Pro?Val?Asn?Ala?Ser?Asp?Ile?His?Trp?Ser?Ser?Ser?Ser?Leu?Pro
210 215 220
Asp?Arg?Trp?Ile?Ile?Leu?Gly?Ala?Phe?Ile?Leu?Leu?Val?Leu?Met?Gly
225 230 235 240
Ile?Val?Leu?Ile?Cys?Val?Trp?Trp?Gln?Asn?Gly?Glu?Trp?Gln?Ala?Gly
245 250 255
Leu?Trp?Pro?Leu?Arg?Thr?Ser
260
<210>3
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<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>3
atgcgaagaa?tatccctgac?ttctagccct?gtgcaccttc?ttttgtttct?gctgttgcta 60
ctaatagcct?tggagatcat?ggttggtggt?cactctcttt?gcttcaactt?cactataaaa 120
tcattgtcca?gacctggaca?gccctggtgt?gaagcgcagg?tcttcttgaa?taaaaatctt 180
ttccttcagt?acaacagtga?caacaacatg?gtcaaacctc?tgggcctcct?ggggaagagg 240
gtaaatgcca?ccagcacttg?gggagaattg?acccaaacgc?tgggagaagt?ggggcgagac 300
ctcaggatgc?tcctttgtga?catcaaaccc?cagataaaga?ccagtgatcc?ttccactctg 360
caagtcgaga?tgttttgtca?acgtgaagca?gaacggtgca?ctggtgcatc?ctggcagttc 420
accatcaatg?gagagagatc?cctcctcttt?gacgcaatga?acatgacctg?gacagtaatt 480
aatcatgaag?ccagtaagat?caaggagaca?tggaagaaag?acagagggct?ggaaaagtat 540
ttcaggaagc?tctcaaaggg?agactgcgat?cactggctca?gggaattctt?agggcactgg 600
gaggcaatgc?cagaaccgac?agtgtcacca?gtaaatgctt?cagatatcca?ctggtcttct 660
tctagtctac?cagacagatg?gatcatcctg?ggggcattca?tcctgttact?tttaatggga 720
attgttctca?tctgtgtctg?gtggcaaaat?ggtgagtggc?aggctggtct?ctggcccttg 780
aggacgtctt?ag 792
<210>4
<211>263
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>4
Met?Arg?Arg?Ile?Ser?Leu?Thr?Ser?Ser?Pro?Val?His?Leu?Leu?Leu?Phe
1 5 10 15
Leu?Leu?Leu?Leu?Leu?Ile?Ala?Leu?Glu?Ile?Met?Val?Gly?Gly?His?Ser
20 25 30
Leu?Cys?Phe?Asn?Phe?Thr?Ile?Lys?Ser?Leu?Ser?Arg?Pro?Gly?Gln?Pro
35 40 45
Trp?Cys?Glu?Ala?Gln?Val?Phe?Leu?Asn?Lys?Asn?Leu?Phe?Leu?Gln?Tyr
50 55 60
Ash?Ser?Asp?Asn?Asn?Met?Val?Lys?Pro?Leu?Gly?Leu?Leu?Gly?Lys?Arg
65 70 75 80
Val?Asn?Ala?Thr?Ser?Thr?Trp?Gly?Glu?Leu?Thr?Gln?Thr?Leu?Gly?Glu
85 90 95
Val?Gly?Arg?Asp?Leu?Arg?Met?Leu?Leu?Cys?Asp?Ile?Lys?Pro?Gln?Ile
100 105 110
Lys?Thr?Ser?Asp?Pro?Ser?Thr?Leu?Gln?Val?Glu?Met?Phe?Cys?Gln?Arg
115 120 125
Glu?Ala?Glu?Arg?Cys?Thr?Gly?Ala?Ser?Trp?Gln?Phe?Thr?Ile?Asn?Gly
130 135 140
Glu?Arg?Ser?Leu?Leu?Phe?Asp?Ala?Met?Asn?Met?Thr?Trp?Thr?Val?Ile
145 150 155 160
Asn?His?Glu?Ala?Ser?Lys?Ile?Lys?Glu?Thr?Trp?Lys?Lys?Asp?Arg?Gly
165 170 175
Leu?Glu?Lys?Tyr?Phe?Arg?Lys?Leu?Ser?Lys?Gly?Asp?Cys?Asp?His?Trp
180 185 190
Leu?Arg?Glu?Phe?Leu?Gly?His?Trp?Glu?Ala?Met?Pro?Glu?Pro?Thr?Val
195 200 205
Ser?Pro?Val?Asn?Ala?Ser?Asp?Ile?His?Trp?Ser?Ser?Ser?Ser?Leu?Pro
210 215 220
Asp?Arg?Trp?Ile?Ile?Leu?Gly?Ala?Phe?Ile?Leu?Leu?Leu?Leu?Met?Gly
225 230 235 240
Ile?Val?Leu?Ile?Cys?Val?Trp?Trp?Gln?Asn?Gly?Glu?Trp?Gln?Ala?Gly
245 250 255
Leu?Trp?Pro?Leu?Arg?Thr?Ser
260
<210>5
<211>34
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>5
cgggatccat?gcgaagaata?tccctgactt?ctag 34
<210>6
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>6
gccaagctta?gacgtcctca?agggccagag?ac 32
<210>7
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>7
atgcgaagaa?tatccctgac?ttctagc 27
<210>8
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>8
attttgccac?cagacacaga?tgagaa 26
<210>9
<211>34
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>9
ccgggatcct?tggagatcat?ggttggtggt?cact 34
<210>10
<211>35
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>10
ggccaagctt?ccatctatct?ggtagactag?aagaa 35

Claims (10)

1. an isolating people ULBP-4 polypeptide is characterized in that, this polypeptide is selected from down a kind of of group:
(a) polypeptide of aminoacid sequence shown in 1-263 position in SEQ ID NO:2 or 4;
(b) polypeptide of aminoacid sequence shown in 24-263 position in SEQ ID NO:2 or 4;
(c) replacement, disappearance or the interpolation through 1-10 amino-acid residue of 1-263 position in SEQ ID NO:2 or 4 or 24-263 amino acids sequence formed, and have with NKG2D receptors bind function by (a) or (b) polypeptides derived.
2. polypeptide as claimed in claim 1 is characterized in that this amino acid sequence of polypeptide is shown in 1-263 position in SEQ IDNO:2 or 4.
3. isolating polynucleotide is characterized in that, described polynucleotide are selected from down a kind of of group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with the complete complementary polynucleotide of polynucleotide (a).
4. polynucleotide as claimed in claim 3 is characterized in that, 1-263 position or 24-263 amino acids polypeptide of sequence among this polynucleotide encoding SEQ ID NO:2 or 4.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:
(a) nucleotide sequence of 403-1194 position among the SEQ ID NO:1;
(b) nucleotide sequence of 1-1434 position among the SEQ ID NO:1;
(c) nucleotide sequence of 472-1194 position among the SEQ ID NO:1;
(d) nucleotide sequence of 1-792 position among the SEQ ID NO:3;
(e) nucleotide sequence of 70-792 position among the SEQ ID NO:3.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. the preparation method of the described ULBP-4 polypeptide of claim 1 is characterized in that, this method comprises:
(a) under conditions suitable for the expression, cultivate the described host cell of claim 7;
(b) from culture, isolate the ULBP-4 polypeptide.
9. energy and the described ULBP-4 polypeptid specificity of claim 1 bonded antibody.
10. a test kit that detects tumour is characterized in that, described test kit contains the described ULBP-4 specific antibody of claim 9.
CNB028284321A 2002-03-04 2002-03-04 Tumor tag and the use thereof Expired - Fee Related CN100358917C (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2002/000137 WO2003074556A1 (en) 2002-03-04 2002-03-04 Tumor tag and the use thereof

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WO (1) WO2003074556A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE434624T1 (en) 2001-10-04 2009-07-15 Immunex Corp UL16 BINDING PROTEIN 4

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001083545A1 (en) * 2000-04-27 2001-11-08 Takeda Chemical Industries, Ltd. Novel proetin and use thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2275141A1 (en) * 1996-10-29 1998-05-07 Thomas Spies Cell stress regulated human mhc class i gene

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001083545A1 (en) * 2000-04-27 2001-11-08 Takeda Chemical Industries, Ltd. Novel proetin and use thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A cluster of ten novel MHC class I related genes on human chromosome 6q24.2-q25.3. Radosavljevic M et al.Genomics,Vol.79 No.1. 2002 *
Lanier L L et al.GenBank AY054974. 2001 *
ULBPs, noval MHC class I-realted molecules,bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Cosman D et al.Immunity,Vol.14 No.2. 2001 *

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