CN1251861A - Human T cell activating protein gene and its coded polypeptide, preparing process and application - Google Patents

Abstract

The present invention relates to a new T-cell activating protein (TAP) in Ly-6 antigen gene family. The cDNA coding sequence of said new human TAP, the polypeptide coded with said sequence, the process for preparing said human TAP protein by recombination technique, and the application of said new human TAP gene and protein are disclosed.

Description

Human T cell activating protein gene, its encoded polypeptides and method for making and purposes

The present invention relates to a kind of new polynucleotide, by the polypeptide of this polynucleotide encoding, the application of these polynucleotide and polypeptide, and the production of described polynucleotide and described polypeptide.More particularly, the present invention relates to a kind of new human T cell activating protein, this albumen is a newcomer of Ly-6 antigen family.

T cell activating protein (T-cell-activating protein, be called for short " TAP ") is a kind ofly to be anchored to the glycoprotein of cell surface by phosphatidylinositols, and its participates in the process (Cell 47:365-370,1986) of cell activation.TAP does not express in all T cells, expresses TAP (Proc.Natl.Acad.Sci.83:7424-7428,1986) in about 10% tool cortisone resistance, sophisticated thymocyte and most static periphery T cell.The assignment of genes gene mapping of encoding murine TAP is in the Ly-6 locus, the T cytodifferentiation antigen (Proc.Natl.Acad.Sci.83:2954-2958,1986) that the genes encoding one class homology on this locus is higher.

1986, Rock group and Reiser group were separated to TAP albumen (the Cell 47:365-370 of mouse in mouse T-T hybridoma; J.Exp.Med.163 (2): 315-333,1986).1988, people such as Reiser cloned the cDNA sequence (Proc.Natl.Acad.Sci.85:2255-2259,1988) of mouse TAP.

Yet up to now, still nobody disclosed the people's who relates among the application TAP sequence.

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding Ly-6 antigen gene family, the new people Ly-6 antigen of the present invention is named as human T cell activating protein, is called for short " TAP ".

Another object of the present invention provides a kind of new Ly-6 antigen protein family member, and albumen of the present invention is named as people TAP albumen.

A further object of the present invention provides a kind of recombinant technology that utilizes and produces the described new antigenic method of people Ly-6.

The invention still further relates to the application of this people TAP antigen gene sequences and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people TAP protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 25-429 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 25-429 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 25-429 position.

In another aspect of this invention, provide a kind of isolating people TAP protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.

In another aspect of this invention, provide a kind of described carrier transformed host cells.

In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people TAP protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people TAP protein-active operationally is connected in expression regulation sequence, form people TAP protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 25-429 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people TAP;

(c) under the condition that is fit to expressing human TAP protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people TAP protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 598 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 25-429 position Nucleotide.

In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, term " people TAP albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people TAP protein-active is as 25-429 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 25-429 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 25-429 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably, under the height stringent condition, with among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 25-429 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 25-429 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.3 with people TAP identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " people TAP protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people TAP protein-active.This term also comprises having and variant form people TAP identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people TAP and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people TAP DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people TAP polypeptide to obtain.The present invention also provides other polypeptide, as comprises people TAP polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of people TAP polypeptide.Usually, this fragment have people TAP peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of people TAP albumen or polypeptide.The difference of these analogues and natural human TAP polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue can also comprise having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, " people TAP conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.

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; Nor-leucine	Leu
			Leu(L)	Nor-leucine; 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; Nor-leucine	Leu

The present invention also comprises people TAP polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people TAP in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of people TAP nucleotide sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people TAP.

The present invention also comprises the method that detects people TAP nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of people TAP polypeptide.Primer length is generally 20-50 Nucleotide.Preferably, this primer or probe sequence are corresponding to the part that is different from mouse TAP nucleotide sequence in the people TAP nucleotide sequence.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

On the other hand, the present invention also comprises people TAP DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people TAP gene product or fragment.Preferably, refer to that those can combine with people TAP gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people TAP, comprise that also those do not influence the antibody of people TAP protein function.The present invention also comprise those can with modify or without the people TAP gene product bonded antibody of modified forms.

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 (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people TAP gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human TAP or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people TAP function and the antibody that does not influence people TAP function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people TAP gene product, obtains by immunological technique.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people TAP gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

People TAP nucleotide sequence 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 SEQ ID NO.3 sequence disclosed according to the present invention, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of pressing the currently known methods preparation 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.Certainly, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

In one embodiment of the invention, polynucleotide total length of the present invention is 598 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 25-429 position Nucleotide.These polynucleotide are so to obtain, with human brain λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer A1:5 '-TCTGAGAGGAACCCTTCTCTGAG-3 ' and reverse primer B1:5 '-ATTAGAGCACCTACCTACCCAGC-3 ' carry out PCR, obtain the purpose fragment of 598bp.Obtain the full length cDNA sequence of SEQ IDNO.3 after the order-checking.

According to homology result relatively, nucleotide sequence of the present invention and encoded protein matter sequence thereof and mouse TAP gene have shown high homology, and therefore, both functions must have close ties.

T cell surface protein TAP can be by not relying on antigenic mode activated T cell (J.Immunol.138:91-97,1987).The TAP gene of mouse is located in Ly-6 locus (Immunogenetics 20:47-56,1984), and its albumen contains 10 cysteine residues (Proc.Natl.Acad.Sci.85:2255-2259,1988).Glycosyl on the TAP albumen may be by O-mode of connection link to each other with albumen (Proc.Natl.Acad.Sci.84:3370-3374,1987).Ubiquitous hydrophobic region in the albumen that the proteic C end of TAP has with phosphatidylinositols links to each other, thereby can be anchored to cell surface (Proc.Natl.Acad.Sci.85:2255-2259,1988) by phosphatidylinositols.The albumen that links to each other with phosphatidylinositols on the T cell surface has the ability (Cell 47:365-370,1986) that stimulates the T cell mostly.

The T cell activation process that TAP participates in may comprise following two steps: the antibody induction T cell activation of at first anti-TAP, and the stimulation of promotion TXi Baoshouti; Then, TAP is at nearly all L3T4 ⁺Express on the periphery T cell (Proc.Natl.Acad.Sci.83:2255-2259,1988).

The expression of TAP in ontogeny shows that TAP promptly begins to express (J.Immunol.137:1232-1238,1986) in early days what fetal thymus was grown.The T cell that is expressed in of TAP is subjected to the significantly rising of mitogenetic stimulation back, but also is subjected to the rise effect (Immunology 64:267-271,1988) of external source IFN-.When TAP expresses defective, can cause producing some immune deficiencies, as prematurity T cell do not reply and the lpr/lpr disease in unusual lymphocyte.

In addition, reply at paroxysmal nocturnal hemoglobinuria disease medium size lymphocyte that weaken may relevant with the formation defective of phosphatidylinositols mixture (Cell 52:665-674,1988).The Ly-6 locus at TAP place has expression in spleen, kidney, lung, low slightly expression is also arranged in the heart, brain.Gene on the Ly-6 locus can activity be transcribed in many non-Lymphoid tissues, and this points out them may have some special functions (Proc.Natl.Acad.Sci.85:2255-2259,1988) in these tissues.

In the accompanying drawings,

Fig. 1 is the homology comparison diagram of the nucleotide sequence of people TAP of the present invention and mouse TAP.Wherein, identical Nucleotide marks with " | ".

Fig. 2 is the homology comparison diagram of the aminoacid sequence of people TAP of the present invention and mouse TAP.Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with ". ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone and the mensuration of the cDNA sequence of people TAP

1. primer amplification

With human brain λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-TCTGAGAGGAACCCTTCTCTGAG-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B1:5 '-ATTAGAGCACCTACCTACCCAGC-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR fragment that electrophoresis detection obtains is about the purpose fragment of 600bp.

2. the order-checking of PCR product

With pcr amplification product and the pGEM-T that as above obtains ^Carrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 598bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 25-429 position Nucleotide.

Derive the aminoacid sequence of people TAP according to the full length cDNA sequence that obtains, totally 134 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.

Embodiment 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with people TAP of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+Swiss Prot+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that, it and mouse TAP gene (gi|198921|gb|M18184|MUSLY62[198921]) have shown high homology, as use the PCGENE software analysis, it and mouse TAP gene have 97.7% identity (Fig. 1) on nucleic acid level, on protein level (Fig. 2) then in full accord, therefore can affirm that albumen of the present invention is people's TAP albumen, and have identical or close with mouse TAP function.

Studies show that the T cell activation can be realized (Ann.Rev.Immunol.3:237-261,1985) by the antigen that the identification of the acceptor on the T cell is presented by main histocompatibility complex on the helper.And T cell surface protein TAP can be by not relying on antigenic mode activating T cell (J.Immunol.138:91-97,1987).The TAP gene of mouse is scheduled the Ly-6 locus, these locus coding many hematopoietic cell surface proteins of being regulated in growth course (Immunogenetics 20:47-56,1984).

TAP is the T cell surface glycoprotein, its molecular weight under non-reduced condition between 10-12KD, under reductive condition between 15-17KD (Proc.Natl.Acad.Sci.83:7424-7428,1986).The SDS-PAGE gel electrophoresis shows that the proteic molecular weight of TAP increases in reducing environment, show that its albumen contains disulfide linkage (Cell47:365-370,1986), the cDNA sequential analysis of TAP shows and contains 10 cysteine residues (Proc.Natl.Acad.Sci.85:2255-2259 in the TAP albumen, 1988), correspond among the SEQ ID NO.4 the 29th among the present invention, the 32nd, the 41st, the 46th, the 53rd, the 74th, the 78th, the 98th, the 99th and the 104th 's halfcystine (the 10th is the amino acid of signal peptide sequence with the 20th halfcystine).

The aminoacid sequence of TAP does not contain the N-glycosylation site, and the glycosyl on its albumen may be by O-mode of connection link to each other with albumen (Proc.Natl.Acad.Sci.84:3370-3374,1987).TAP albumen does not contain typically wears the membrane structure territory, C end has the ubiquitous hydrophobic region of albumen that links to each other with phosphatidylinositols (C holds and is rich in leucine and Xie Ansuan, Isoleucine, phenylalanine etc.), can be anchored to cell surface (Proc.Natl.Acad.Sci.85:2255-2259,1988) by phosphatidylinositols.Experiment shows that the proteic internalization that links to each other with phosphatidylinositols does not rely on (Eur.J.Immunol.22:15-21,1992) that clothing depression mode (coated pit-independent pathway) is carried out on the T cell surface.These albumen that link to each other with phosphatidylinositols have the ability that stimulates the T cell mostly, and prompting phosphatidylinositols mode of connection may have been represented a kind of signal transduction path (Cell47:365-370,1986).

The monoclonal antibody of anti-TAP can be induced the propagation of ripe thymocyte, and the irritant reaction of its intensity and concanavalin A is (J.Immunol.137:1232-1238,1986) quite.The T cell activation process that TAP participates in may comprise following two steps: the antibody induction T cell activation of at first anti-TAP, and the stimulation of promotion TXi Baoshouti; Then, TAP is at nearly all L3T4 ⁺Express on the periphery T cell.At thymic tissue, the expression of TAP indicates immunocompetent acquisition (Proc.Natl.Acad.Sci.85:2255-2259,1988).The expression of TAP in ontogeny shows that TAP expresses promptly beginning in early days of growing of fetal thymus, and at this time the marker molecule of other most of T cells also occurs.These early stage lymphocytes can be activated also explanation by the TAP molecule, and there are dependency (J.Immunol.137:1232-1238,1986) in the expression of TAP and immunocompetent acquisition.The T cell that is expressed in of TAP is subjected to the significantly rising of mitogenetic stimulation back, also is subjected to the rise effect of external source IFN-.Known T cell can be secreted IFN-after activation, show that TAP expresses rising in the activated T cell be the autocrine effect (Immunology 64:267-271,1988) that is subjected to endogenous IFN-.When TAP expresses defective, can cause producing some immune deficiencies, as prematurity T cell do not reply and the lpr/lpr disease in unusual lymphocyte.In addition, reply at paroxysmal nocturnal hemoglobinuria disease medium size lymphocyte and weaken and to form defective relevant (Cell 52:665-674,1988) with the phosphatidylinositols mixture.Northern Blot experiment shows that the Ly-6 locus at TAP place has expression in spleen, kidney, lung, and low slightly expression is also arranged in the heart, brain.Gene on the Ly-6 locus has activity to transcribe in many non-Lymphoid tissues, and this points out them may have some special functions (Proc.Natl.Acad.Sci.85:2255-2259,1988) in these tissues.

People TAP of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor TAP can also merge with other members of this family or exchange fragment, to produce new albumen.

At the antibody of inventor TAP, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family, as the TAP of mouse).

For example, inventor TAP nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people TAP or the overexpression that suppresses people TAP.People TAP albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people TAP disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3

The expression of people TAP in intestinal bacteria

The cDNA sequence of coding people TAP is used corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, is that template increases with the amplified production among human brain λ gt11cDNA library (available from Clontech company) or the embodiment 1, with synthetic insertion fragment.

5 ' Oligonucleolide primers sequence is

5′-TTGCGGATCCATGGACACTTCTCACACTA-3′(SEQ?ID?NO.5)，

This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is 19 Nucleotide of the people TAP encoding sequence that begun by initiator codon;

3 ' Oligonucleolide primers sequence is

5’-GTTCGTCGACTCAGAGCAAGGTCTGCAGG-3’(SEQ?ID?NO.6)，

This primer contains the restriction enzyme site of SalI restriction enzyme, the encoding sequence of translation termination and people TAP.

The restriction enzyme site of restriction enzyme is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp ^r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With BamHI and SalI digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).The extracting plasmid is cut with the PstI enzyme and identify to be inserted clip size and direction, and sequence verification result shows that the cDNA of people TAP inserts the fragment carrier of correctly packing into.

Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people TAP from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people TAP from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.At first, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein can be incorporated in second post from nickel-chelate column, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 14KDa.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 4

The expression of people TAP in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, the cDNA sequence of coding people TAP is used corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, with the amplified production among human brain λ gt11cDNA library (available from Clontech company) or the embodiment is that template increases, with the synthetic fragment of inserting.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5′-TTGCAAGCTTATGGACACTTCTCACACTA-3′(SEQ?ID?NO.7)，

This primer contains the restriction enzyme site of HindIII restriction enzyme, and what connect is 19 Nucleotide of the people TAP encoding sequence that begun by initiator codon;

3 ' end primer sequence is:

5’-GTTCGGATCCTCAGAGCAAGGTCTGCAGG-3’(SEQ?ID?NO.8)

This primer contains the encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and people TAP.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII, BamHI digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid is cut with the PstI enzyme and identify to be inserted clip size and direction, and sequence verification result shows that the cDNA of people TAP inserts the fragment carrier of correctly packing into.

Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mM TrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 14KDa.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 5

Preparation antibody

The recombinant protein that as above obtains is used for immune animal to produce antibody, specific 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 TAP gene translation product with it.Found that antibody can precipitate 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 (1) general information: (i) applicant: Fudan University is denomination of invention (ii): human T cell activating protein gene, its encoded polypeptides and method for making and purposes be the sequence number (iii): information (i) sequence signature of 8 (2) SEQ ID NO.1

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.1:TCTGAGAGGA ACCCTTCTCT GAG 23 (2) SEQ ID NO.2

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:2ATTAGAGCAC CTACCTACCC AGC 23 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 598bp

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

( xi ) ：SEQ ID NO.3TCTGAGAGGA ACCCTTCTCT GAGGATGGAC ACTTCTCACA CTACAAAGTC CTGTTTGCTG 60ATTCTTCTTG TGGCCCTACT GTGTGCAGAA AGAGCTCAGG GACTGGAGTG TTACCAGTGC 120TATGGAGTCC CATTTGAGAC TTCTTGCCCA TCAATTACCT GCCCCTACCC TGATGGAGTC 180TGTGTTACTC AGGAGGCAGC AGTTATTGTG GATTCTCAAA CAAGGAAAGT AAAGAACAAT 240CTTTGCTTAC CCATCTGCCC TCCTAATATT GAAAGTATGG AGATCCTGGG TACTAAGGTC 300AACGTGAAGA CTTCCTGTTG CCAGGAAGAC CTCTGCAATG TAGCAGTTCC CAATGGAGGC 360AGCACCTGGA CCATGGCAGG GGTGCTTCTG TTCAGCCTGA GCTCAGTCCT CCTGCAGACC 420TTGCTCTGAT GGTCCTCCCA ATGACCTCCA CCCTTGTCCT TTTATCCTCA TGTGCAACAA 480TTCTTCCTGG AGCCCTCTAG TGATGAATTA TGAGTTATAG AAGCTCCAAG GTGGGAGTAG 540TGTGTGAAAT ACCATGTTTT GCCTTTATAG CCCCTGCTGG GTAGGTAGGT GCTCTAAT 598

(2) information of SEQ ID NO.4:

(i) sequence signature:

(A) length: 134 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO.4

Met??Asp??Thr??Ser??His??Thr??Thr??Lys??Ser??Cys??Leu??Leu??Ile??Leu??Leu???15

Val??Ala??Leu??Leu??Cys??Ala??Glu??Arg??Ala??Gln??Gly??Leu??Glu??Cys??Tyr???30

Gln??Cys??Tyr??Gly??Val??Pro??Phe??Glu??Thr??Ser??Cys??Pro??Ser??Ile??Thr???45

Cys??Pro??Tyr??Pro??Asp??Gly??Val??Cys??Val??Thr??Gln??Glu??Ala??Ala??Val???60

Ile??Val??Asp??Ser??Gln??Thr??Arg??Lys??Val??Lys??Asn??Asn??Leu??Cys??Leu???75

Pro??Ile??Cys??Pro??Pro??Asn??Ile??Glu??Ser??Met??Glu??Ile??Leu??Gly??Thr???90

Lys??Val??Asn??Val??Lys??Thr??Ser??Cys??Cys??Gln??Glu??Asp??Leu??Cys??Asn???105

Information (i) sequence signature of Val Ala Val Pro Asn Gly Gly Ser Thr Trp Thr Met Ala Gly Val 120 Leu Leu Phe Ser Leu Ser Ser Val Leu Leu Gln Thr Leu Leu 134 (2) SEQ ID NO.5

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.5:TTGCGGATCC ATGGACACTT CTCACACTA 29 (2) SEQ ID NO.6

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.6:GTTCGTCGAC TCAGAGCAAG GTCTGCAGG 29 (2) SEQ ID NO.7

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.7:TTGCAAGCTT ATGGACACTT CTCACACTA 29 (2) SEQ ID NO.8

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.8:GTTCGGATCC TCAGAGCAAG GTCTGCAGG 29

Claims (12)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people TAP protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 25-429 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 25-429 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.4.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 25-429 position.

4. isolating people TAP protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

6. a carrier is characterized in that, it contains the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6.

8. a generation has the method for the polypeptide of people TAP protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people TAP protein-active operationally is connected in expression regulation sequence, form people TAP protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 25-429 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people TAP;

(c) under the condition that is fit to expressing human TAP protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people TAP protein-active.

9. method as claimed in claim 8 is characterized in that, this sequence is from Nucleotide 25-429 position among the SEQ ID NO.3.

10. energy and the described people TAP of claim 4 protein polypeptide specificity bonded antibody.

11. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

12. a probe molecule is characterized in that, it contains about 8-100 continuous nucleotide in the described dna molecular of claim 1.