CN1371916A - Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use - Google Patents
Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use Download PDFInfo
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Abstract
The present invention relates to a novel sialic acid-binding Ig-like lectin-10 (siglec-10). Said invention provides a polynucleotide for coding said protein molecule and a method for producing said protein molecule by using recombination technology. Said ivnention also discloses the application of polynucleotide coding this novel sialic acid-binding Ig-like lectin-10 molecule. Said invention also verifies the characteristics of siglec-10 which is combined with human erythrocyte in sialic acid dependent mode. Said invention also discloses the strategy for diagnosing and curing diseases, specially for diagnosing and curing cancers by resisting said protein molecule.
Description
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the new coding human cell surface identification and the polynucleotide of signal transduction molecule sigelc-10 polypeptide, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.The sialic acid dependency that the invention still further relates to sigelc-10 is in conjunction with activity.Specifically, polypeptide of the present invention be a kind of new and the identification of cell surface sugar chain, in conjunction with and the relevant cell surface protein of signal conduction.
Sialic acid is an alpha-ketoacid family with nine carbon skeletons, majority is present in the oligosaccharides side chain far-end of cell surface glycoprotein and glycolipid, be exposed in the extracellular environment, in cell and initial contact of multiple cause of disease material as virus, bacterium, protozoon and toxin, thereby the sialic acid on the sugar chain is identified the various follow-up cellular biochemical incidents that cause.Sialic another vital role is the part as various Sugar receptorss.Siglecs (Sialic acid-bindingIg-like lectins, siali acid conjugated immunoglobulin-like agglutinant) is one and is under the jurisdiction of immunoglobulin superfamily (Immunoglobulin superfamily, IgSF) lectin family mediates the interaction of albumen-carbohydrate by the identification sialic acid.
9 member: siglec-1/sialoadhesin, siglec-2/CD22, siglec-3/CD33, siglec-4a/myelin-associated glycoprotein (MAG), siglec-5, siglec-6/OBBP-1, siglec-7/AIRM1, siglec-8/SAF-2 and the siglec-9 of this family have been had been found that up to now.These albumen are the I type membranin of once striding film, structurally comprise extracellular region, stride film district and intracellular region.Extracellular region is made of with the C2-set structural domain of different numbers a N end Ig V-set structural domain.Verified, first V-set Ig spline structure territory is extremely important for the identification carbohydrate, and second Ig spline structure territory helps it in conjunction with activity.Especially the conservative arginine in the V-set structural domain F thigh, concerning the sialic acid dependency of all Siglecs in conjunction with particularly important the activity.Other conserved residues comprises the die aromatischen Aminosaeuren in V-set structural domain A and the G β thigh, N-ethanoyl and the combination of glycerine side group in the N-n acetylneuraminic acid n.In the V-set of all Siglecs and the C2-set structural domain that closes on, conservative halfcystine all occurs and arrange unusually, these halfcystines formed disulfide linkage and V-set structural domain in the lamella of V-set structural domain and the C2-set structural domain that closes between disulfide linkage.
Along with to the structure of Siglecs and going deep into of biological function research, it is found that Siglecs all brings into play critical function in the various biological incident.In the intracellular region of most of Siglecs, all have possible tyrosine phosphorylation site, be positioned at immunity receptor inhibitory motifs based on tyrosine (immunoreceptor tyrosine-basedinhibitory motif, ITIM) in.These tyrosine by phosphorylation after, can by raise with active cells in Phosphoric acid esterase (SHP-1, SHP-2 or SHIP) mediate negative conditioning signal.Can be as siglec-3/CD33 and siglec-7/AIRM1 at the negative conditioning signal of specific antibody crosslinked back conduction; The gene knockout experiment of siglec-2/CD22 also is confirmed that it is the down regulator of B cell activation.
All known Siglecs all are expressed in cell surface a certain or the bone marrow stem cell source that some is specific, be expressed on the monocyte as siglec-1/sialoadhesin, siglec-2/CD22 is expressed on the B cell, siglec-3/CD33 is expressed on the medullary system precursor cell, siglec-7/AIRM1 is expressed on the NK cell, and siglec-8/SAF-2 is expressed on the eosinophilic granulocyte or the like.The specific expressed sign that this limitation expression pattern of Siglecs points out these albumen may become corresponding cell monoid differentiation, and the regulating effect of participation tumor growth and transfer.(monoclonal antibody MoAb) can be widely used in leukemic immunodiagnosis and magnetic target therapy to the monoclonal antibody of anti-Siglecs.Siglec-3/CD33 has become the diagnosing acute marrow series leukemia, and (acute myelogenousleukemia, AML), especially the important symbol of undifferentiated type can be used for distinguishing AML and LL simultaneously.The anti-CD 33 monoclonal antibody has been applied to treat the clinical first phase research of AML, and alternative pernicious hematopoiesis, the answer normal hematopoiesis process removed.
Research shows that the Siglecs family protein is relevant with multiple vital movement.Therefore, significant for the human siali acid conjugated immunoglobulin-like agglutinant sigelc-10 that diagnoses and the therapeutic purpose research and development is new.
The purpose of this invention is to provide a kind of new human siali acid conjugated immunoglobulin-like agglutinant sigelc-10 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.Sigelc-10 albumen of the present invention is a kind of Siglec homolgous molecule.
Another object of the present invention provides the sialic acid dependency of sigelc-10 in conjunction with activity.
Human siali acid conjugated immunoglobulin-like agglutinant family member sigelc-10 and known family member that the present invention is novel have high homology, entire infrastructure feature with Siglecs family protein, and can have the sialic acid dependency in conjunction with activity in sialic acid dependency mode in conjunction with HRBC.Therefore, can infer the sialic acid on the glycosyl side chain of the same recognizing cells surface glycoprotein of sigelc-10 and glycolipid with other family members, and the ITIM motif that passes through its intracellular region conducts conditioning signal, regulate and control multiple physiology and pathology activity, play a significant role, and may be worth having important development and application aspect the immunodiagnosis in a plurality of fields such as anti-infective, anti-inflammatory response, antitumor and nerve growth reparation, immunologic function adjusting and the immunotherapy.
In a first aspect of the present invention, novel isolated sigelc-10 polypeptide is provided, this polypeptide is the people source, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
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 40% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people Sigelc of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 223-2031 position among the SEQ ID NO:1; (b) has the sequence of 1-3124 position among the SEQ IDNO:1.
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, provide preparation to have the method for the polypeptide of people sigelc-10 protein-active, this method comprises: (a) under the proteic condition of suitable expressing human sigelc-10, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people sigelc-10 protein-active.
In a fifth aspect of the present invention, provide and above-mentioned people sigelc-10 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-3124 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people sigelc-10 polypeptide active is provided, and the compound that suppresses people sigelc-10 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 sigelc-10 polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of sigelc-10 in the test sample, it comprises: sample is contacted with the proteic specific antibody of sigelc-10, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample sigelc-10 albumen.
In a eighth aspect of the present invention, a kind of disease relevant with people sigelc-10 polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.
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 sigelc-10 polypeptide active, and perhaps screening suppresses the antagonist of people sigelc-10 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people sigelc-10 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 people sigelc-10 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as tumour, inflammation, neural system and cardiovascular diseases.
In a eleventh aspect of the present invention, provide the sialic acid dependency red corpuscle of sigelc-10 to experimental results show that in conjunction with active.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 is people sigelc-10 albumen of the present invention and the proteic amino acid sequence homology comparison diagram of other Sigelc family members.The top sequence is people sigelc-10, and the below sequence is other Sigelc family member albumen.Identical amino acid marks with black matrix between a plurality of sequences, and similar amino acid marks with grey body.Amino-acid residue shown in the circle is that sialic acid is in conjunction with necessary residue; Arrow marks the boundary in different structure territory; ITIM motif for inferring in the square frame.
Fig. 2 is people sigelc-10 Argine Monohydrochloride sequence of the present invention and other family members' a domain analyses synoptic diagram.Wherein delta is represented V-set Ig spline structure territory; Border circular areas is represented C2-set Ig spline structure territory; Round dot is represented the tyrosine residues of intracellular region.
Fig. 3 is the hydrophobicity graphic representation of the proteic Kyte-doolitte hydrophobicity analysis of people sigelc-10 of the present invention.
Fig. 4 is a people sigelc-10 RT-PCR expression analysis of the present invention, and prompting sigelc-10 is expressed in the monocyte in some tumour cell and human peripheral source; In the reactivation process that the dendritic cell that the human peripheral blood mononuclear cell originates is stimulated by LPS, has the certain expression pattern.
Fig. 5 is the shown distribution of people sigelc-10 Northern blot hybridization of the present invention.Results suggest sigelc-10 is the molecule that a kind of tool particular organization distributes.
Fig. 6 is that the red corpuscle of people sigelc-10 of the present invention is in conjunction with experiment.Point out it to have sialic acid dependency red corpuscle in conjunction with activity.
In the present invention, term " sigelc-10 albumen ", " sigelc-10 polypeptide " or " sialic acid combination Property immunoglobulin-like agglutinant sigelc-10 " be used interchangeably, all refer to have human siali acid conjugated immune globulin Albumen or the polypeptide of white sample agglutinin sigelc-10 amino acid sequence (SEQ ID NO:2). They comprise contains or not The siali acid conjugated immunoglobulin-like agglutinant sigelc-10 that contains initial methionine.
As used herein, " separation " refer to material from its primal environment, separate (if crude, Primal environment namely is natural surroundings). Not divide such as the polynucleotide under the native state in the active somatic cell and polypeptide From purifying, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, Then for separation and purification.
As used herein, " sigelc-10 albumen or the polypeptide of separation " refers to that the sigelc-10 polypeptide is not basically Contain natural relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use standard Purified technology of protein purifying sigelc-10 albumen. Basically pure polypeptide is at non-reduced polyacrylamide gel Upper energy produces single master tape. The purity of sigelc-10 can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide. The present invention Polypeptide can be the product of natural purifying, or the product of chemical synthesis, or use recombinant technique from protokaryon or true Produce among the nuclear host (for example, bacterium, yeast, higher plant, insect and mammalian cell). Give birth to according to restructuring The host that the product scheme is used, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated. The present invention Polypeptide also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analog of people sigelc-10 albumen. As used herein, term " fragment ", " derivative " and " analog " refer to basically keep natural human sigelc-10 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) One or more conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue) are arranged, And the amino acid residue of such replacement can be can be not yet by the genetic code coding, or (ii) one or The polypeptide that has substituted radical in a plurality of amino acid residues, or (iii) mature polypeptide and another compound (such as prolonging The compound of long polypeptide half-life, for example polyethylene glycol) merge formed polypeptide, or (iv) additional amino acid order Row be fused to this peptide sequence and the polypeptide that forms (such as targeting sequencing or secretion sequence or be used for the sequence of this polypeptide of purifying Or the proteinogen sequence, or with the fusion of the formation of antigen I gG fragment). 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 sigelc-10 polypeptide " refers to have the SEQ ID of people sigelc-10 protein active The polypeptide of NO.2 sequence. This term also comprise have with people sigelc-10 albumen identical function, SEQ ID NO.2 The variant form of sequence. These variant forms comprise (but being not limited to): several (be generally 1-50, better Ground 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and Add one or several (being generally in 20, preferably is in 10, more in that C end and/or N are terminal Be in 5 goodly) amino acid. For example, in the art, the amino acid close or similar with performance replaces The time, the common function that can not change protein. Again such as, add one or several ammonia in that C end and/or N are terminal Base acid also can not change the function of protein usually. This term also comprise people sigelc-10 albumen active fragment and Reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, The induced mutation body, can be coded with the DNA of people sigelc-10 DNA hybridization under high or low stringency condition Albumen and the polypeptide or the albumen that utilize the antiserum acquisition of anti-people sigelc-10 polypeptide. The present invention also provides Other polypeptide, as comprise the fusion of people sigelc-10 polypeptide or its fragment. Except the polypeptide of total length almost, The present invention has also comprised the soluble fragments of people sigelc-10 polypeptide. Usually, this fragment has people sigelc-10 Peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 Individual continuous amino acid is more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analog of people sigelc-10 albumen or polypeptide. These analogs and natural human sigelc-10 The difference of polypeptide can be the difference on the amino acid sequence, also can be the difference that does not affect on the modified forms of sequence, Perhaps have both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can be by various Technology obtains, as by radiation or be exposed to mutagens and produce random mutagenesis, and also can be by direct mutagenesis method or its The biological technology of his known molecular. Analog also comprises having and is different from the amino acid whose residue of natural L-(such as D-ammonia Base acid) analog, and have the similar of that non-natural exists or synthetic amino acid (such as β, gamma-amino acid) Thing. Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(usually the not changing primary structure) form of modification comprises: chemically derived form such as the second of the polypeptide that body is interior or external Acidylate or carboxylated. Modify and also to comprise glycosylation, such as those in the synthetic and processing of polypeptide or further add work step Carry out glycosylation modified in rapid and polypeptide that produce. This modification can by polypeptide is exposed to carry out glycosylated Enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finishing. Modified forms also comprises having phosphorylation amino The sequence of acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine). Thereby also comprising being modified improves Its anti-proteolysis performance or optimized the polypeptide of solubility property.
In the present invention, " people sigelc-10 albumen conservative variation polypeptide " refers to the amino acid with SEQ ID NO:2 Sequence is compared, and has 10 at the most, and preferably at the most 8, more preferably at the most 5,3 amino acid quilts at the most best Similar performance or close amino acid are replaced and are formed polypeptide. These conservative variation polypeptide preferably carry out according to table 1 Amino acid substitution and producing.
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, genomic DNA Or artificial synthetic DNA. DNA can be strand or double-stranded. DNA can be coding strand or noncoding strand. The coding region sequence of encoding mature polypeptide can with the identical or degeneracy of coding region sequence shown in the SEQ ID NO:1 Variant. As used herein, " variant of degeneracy " refers to that in the present invention coding has SEQ ID NO:2 Protein, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotides of the mature polypeptide of coding SEQ ID NO:2 comprise: the coded sequence of an encoding mature polypeptide; The coded sequence of mature polypeptide and various additional code sequence; The coded sequence of mature polypeptide is (with optional additional code Sequence) and non-coding sequence.
Term " polynucleotides of coded polypeptide " can be the polynucleotides that comprise this polypeptide of encoding, and also can be also The polynucleotides that 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 The fragment of polypeptide or polypeptide, analog and derivative. The variant of these polynucleotides can be the equipotential of natural generation The variant that variant or non-natural take place. These nucleotide diversity bodies comprise replace variant, deletion mutation body and Insert variant. As known in the art, allelic variant is the replacement form of polynucleotides, and it may be The replacement of one or more nucleotides, disappearance or insertion, but can be from the function of the polypeptide that changes in fact its coding.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotides of at least 80% homogeny more preferably. The present invention be more particularly directed under stringent condition and institute of the present invention State the interfertile polynucleotides of polynucleotides. In the present invention, " stringent condition " refers to: (1) is strong at low ion Hybridization and wash-out under degree and the higher temperature, such as 0.2 * SSC, 0.1%SDS, 60 ℃; Or in (2) when hybridization, be added with change The property agent, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃ etc.; Or (3) are only two orders Homogeny between the row is at least more than 90%, is more preferably 95% and just hybridizes when above. And, interfertile many The polypeptide of nucleotide coding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization. As used herein, the length of " nucleic acid fragment " Degree contains 15 nucleotides at least, better is at least 30 nucleotides, is more preferably at least 50 nucleotides, preferably More than at least 100 nucleotides. Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid to determine and/or to separate The polynucleotide of coding sigelc-10 albumen.
Polypeptide among the present invention preferably provides with the form of separating with polynucleotides, more preferably is purified to homogeneous.
People sigelc-10 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, or derivatives thereof) 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.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), 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 sigelc-10 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the sigelc-10 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people sigelc-10 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 sigelc-10 polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.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 sigelc-10 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, coldSpring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P
LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.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, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
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 sigelc-10 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism sigelc-10 protein function as pharmacological agent sigelc-10 protein function.The peptide molecule that can suppress or stimulate people sigelc-10 protein function that can be used for seeking therapeutic value with the recombinant human sigelc-10 protein screening peptide library of expressing.
On the other hand, the present invention also comprises people sigelc-10 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 sigelc-10 gene product or fragment.Preferably, refer to that those can combine with people sigelc-10 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 sigelc-10, comprise that also those do not influence the antibody of people sigelc-10 protein function.The present invention also comprise those can with modify or without the people sigelc-10 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 sigelc-10 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human sigelc-10 albumen 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 sigelc-10 protein function and the antibody that does not influence people sigelc-10 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people sigelc-10 gene product, obtains by the routine immunization technology.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 sigelc-10 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).
The proteic antibody of anti-people sigelc-10 can be used in the immunohistochemistry technology, detects the people sigelc-10 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people sigelc-10, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.
Antibody among the present invention can be used for treating or prevention and the relevant disease of people sigelc-10 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people sigelc-10 or activity.
Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people sigelc-10 albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual 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 (for example lymph-node cell etc.) of people sigelc-10 protein positive.
The production of polyclonal antibody can choose sigelc-10 albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Utilize albumen of the present invention,, can filter out with sigelc-10 albumen interactional material takes place, as part, 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.
Polypeptide of the present invention can be directly used in disease treatment, for example, is used for tumour, inflammation, the treatment of neural system and cardiovascular diseases aspect.When using sigelc-10 albumen of the present invention, also can use the other treatment agent simultaneously, as IFN-α, IFN-β, TNF-α, TNF-β etc.
The present invention also provides a kind of pharmaceutical composition, and it contains sigelc-10 polypeptide of the present invention or its agonist, antagonist 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 is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the sigelc-10 albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The proteic polynucleotide of people sigelc-10 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of sigelc-10 of the proteic nothing expression of sigelc-10 or unusual/non-activity.The sigelc-10 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic sigelc-10 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the sigelc-10 transgenosis to cell.The method that structure carries the recombinant viral vector of sigelc-10 gene is found in existing document (Sambrook, et al.).Recombinant human sigelc-10 gene can be packaged in the liposome in addition, and then is transferred in the cell.
Suppress the oligonucleotide (comprising sense-rna and DNA) of people sigelc-10mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people sigelc-10 obtains.During screening, must carry out mark to people sigelc-10 protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people sigelc-10 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people sigelc-10 protein level that is detected in the test can be with laying down a definition the importance of people sigelc-10 albumen in various diseases and be used to the disease of diagnosing sigelc-10 albumen to work.
Whether having the proteic method of sigelc-10 in a kind of detection test sample is to utilize the proteic specific antibody of sigelc-10 to detect, and it comprises: sample is contacted with the sigelc-10 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample sigelc-10 albumen.
The proteic polynucleotide of sigelc-10 can be used for the diagnosis and the treatment of sigelc-10 protein related diseases.Aspect diagnosis, the proteic polynucleotide of sigelc-10 can be used for detecting the proteic expression of sigelc-10 sigelc-10 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of sigelc-10 as the sigelc-10DNA sequence.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), 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 sigelc-10 albumen and also can detect the proteic transcription product of sigelc-10.
The sudden change that detects the sigelc-10 gene also can be used for the disease of diagnosing sigelc-10 albumen relevant.The form of sigelc-10 protein mutation comprises that the point mutation compared with normal wild type sigelc-10DNA sequence, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of sigelc-10 prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritance inMan (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 3124 bases, and its open reading frame is positioned at the 223-2031 position, and the coding total length is 602 amino acid whose people sigelc-10 albumen (SEQ ID NO:2).This sigelc-10 albumen belongs to the Siglec family molecule, with siglec-1/sialoadhesin, siglec-2/CD22, siglec-3/CD33, siglec-5, siglec-6/OBBP-1, siglec-7/AIRM1, siglec-8/SAF-2, and siglec-9 aminoacid sequence height homology, consistence can be up to more than 50% in Ig spline structure territory, and similarity then can reach 60%.Sigelc-10 albumen structurally has the constitutional features of Siglec family, comprise and have a V-set Ig spline structure territory and three C2-set structural domains, contain conservative arginine and die aromatischen Aminosaeuren in the structural domain, specific conservative halfcystine pattern of rows and columns, and the ITIM motif of intracellular region etc.In addition, albumen also comprises four N-glycosylation sites (100-103,355-358,364-367 residue), immunoglobulin (Ig) and main histocompatibility and meets thing albumen identifier (423-429 residue) and tyrosine phosphorylation site (314-321 residue).RT-PCR analysis revealed sigelc-10 has expression in some tumour cell, have expression in various degree simultaneously in peripheral blood lymphocytes and in the dendritic cell in different time LPS stimulated peripheral mononuclear cells source.The Northern engram analysis shows in tissues such as peripheral blood, spleen and liver to have particular expression.Red corpuscle can be in conjunction with HRBC in conjunction with experiment confirm sigelc-10, and thisly has the sialic acid dependency in conjunction with activity.The research prompting of having carried out, sigelc-10 may the same recognizing cells surface glycoprotein with other family members and the glycosyl side chain of glycolipid on sialic acid, and the ITIM motif that passes through its intracellular region conducts conditioning signal, regulate and control multiple physiology and pathology activity, the tool potential is antitumor, anti-inflammatory effect.Therefore, sigelc-10 albumen or its relevant antagonist, agonist etc. can be diseases such as treatment tumour, inflammation, neural system and cardiovascular diseases new immunodiagnosis and targeted therapy approach are provided, thereby have great application prospect.
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, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: the clone of people sigelc-10 cDNA
Extract the total RNA of human dendritic cell with Trizol reagent (Life Technologies company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is cloned test kit (available from Gibco) with SuperScriptII cDNA fragment orientation is inserted on the multiple clone site of carrier, transform DH5 α bacterium and form the cDNA plasmid library.Measure the sequence of random choose clone's 5 ' end with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a cDNA clone's dna sequence dna is new full-length cDNA.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (shown in SEQ ID NO:1), the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as human siali acid conjugated immunoglobulin-like agglutinant sigelc-10, its encoding gene called after human siali acid conjugated immunoglobulin-like agglutinant sigelc-10 gene.
Sequence SEQ ID NO:1 total length is 3124bp, comprises 5 ' the end non-coding region of 222bp and 3 ' the end non-coding region of 1093bp, and coding contains 602 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 68..6 kD.
The proteic full length amino acid sequence of people sigelc-10 of the present invention is as follows:
Wherein residue is the N-glycosylation site in the square frame; The two-wire district is that immunoglobulin (Ig) and main histocompatibility meet thing albumen identifier; The shadow zone is the tyrosine phosphorylation site.
They are different with known for the BLAST analysis revealed, with human siali acid conjugated immunoglobulin-like agglutinant sigelc-5 in Ig spline structure territory inner amino acid array height homology, consistence reaches 50%, similarity reaches 60% (Fig. 1), belongs to siali acid conjugated immunoglobulin-like agglutinant sigelc family molecule.In addition, in sigelc-10 albumen, also comprise immunoglobulin (Ig) (Ig) and main histocompatibility meets thing (major histocompatibilitycomplex, MHC) albumen identifier (428-434 residue), this also further points out its contactin (IgSF) member.
Domain analyses shows that sigelc-10 albumen belongs to siali acid conjugated immunoglobulin-like agglutinant sigelc (Fig. 2).
The Kyte-doolitte hydrophobicity analysis shows that sigelc-10 albumen is I type transmembrane protein (Fig. 3).Embodiment 2: carry out the cell expressing analysis of people sigelc-10 with the RT-PCR method
Extract the total RNA of dendritic cell that is in the corresponding clone of logarithmic phase, human peripheral blood mononuclear cell and stimulates the human peripheral blood mononuclear cell source of different time with Trizol reagent, get 5 μ g cell total rnas and 1 μ gOligo-dT through LPS
12-
18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ l ddH after finishing
2O dilutes.The used primer of pcr amplification siglec-10 is as follows: adopted primer 5 ' TGGCTCAGAAGCGGAATC (SEQ IDNO:3) is arranged, antisense primer 5 ' CCTCATTGGAACTTGACTTCTGC (SEQ ID NO:4), simultaneously with β-actin as positive control.The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.5mM primer, 0.2mM dNTP and 1U rTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 95 ℃ 15 seconds, 57 ℃ 30 seconds, 72 ℃ 30 seconds, capable 1.5% agarose gel electrophoresis of PCR product is tentatively confirmed after 28 circulations.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 1756-2033 shown in the SEQ ID NO:1 are identical.
The prompting of people sigelc-10 RT-PCR expression analysis, sigelc-10 is expressed in the monocyte in some tumour cell and human peripheral source; In the reactivation process that the dendritic cell that the human peripheral blood mononuclear cell originates is stimulated by LPS, has certain expression pattern (Fig. 4).
In addition, by above-mentioned identical RT-PCT condition, adopt primer tcctatgcgg agATGCTACT GCCAC (SEQID NO:7) and AAGAGACCCT CATTGGAACT TGACT (SEQ ID NO:8), amplification obtains sigelc-10 coding region amplified production.The dna sequence analysis result shows the DNA sequences encoding of this PCR product identical with shown in the SEQ ID NO:1.The Northern engram analysis of embodiment 3 people sigelc-10
Carry out the Northern trace by following ordinary method: filter membrane to be checked places the hybridization solution of 10ml through 68 ℃ of preheatings, in hybrid heater (Bellco) in 68 ℃ of prehybridizations 30 minutes; The cDNA probe that mark is good was in 95～100 ℃ of sex change 2～5 minutes, and (cDNA probe final concentration is 2～10ng/ml or 1～2 * 10 to put rapid cooling back adding hybridization solution on ice
6Cpm/ml), fully mixing was hybridized 2 hours in 68 ℃.After hybridization finishes, filter membrane with 2 * SSC, the drip washing of 0.05%SDS room temperature for several times, the washing lotion several is changed in continuing vibration flushing 30～40 minutes therebetween.Use 0.1 * SSC, 0.1%SDS in 50 ℃ of vibration flushings 20～40 minutes subsequently.Last filter membrane wrapped up with plastic fresh-keeping membrane, in-70 ℃ of exposure X line films 24～48 hours.
Northern blot hybridization result shows: in peripheral blood, ovary, spleen (3.3kb), and have in various degree in the liver, lungs, thymus gland many healthy tissuess such as (1.8kb) and express, this shows that sigelc-10 albumen is a kind of albumen (Fig. 5) of particular expression.Embodiment 4 people sigelc-10 are recombinant expressed
In this embodiment, be template with the total length plasmid DNA among the embodiment 1,5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people sigelc-10DNA as inserting fragment.
5 ' the end Oligonucleolide primers sequence of using in the PCR reaction is:
5’-ACGAATTCGCGCCTCCTATGCGGAGATG-3’(SEQ?ID?NO：5)
This primer contains the restriction enzyme site of EcoR I restriction enzyme, is the part encoding sequence of people sigelc-10 after this restriction enzyme site;
3 ' end primer sequence is:
5’-ACGAATTCCGTTGGAGAATGCCGTTGAG-3’(SEQ?ID?NO：6)
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people sigelc-10 of EcoR I restriction enzyme.
With the PCR product purification that obtains after EcoR I enzyme cut and recombinate according to a conventional method with plasmid pGEM-3ZF again and be converted into the competence e. coli bl21, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The people sigelc-10 cDNA EcoRI endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T (Pharmacia company), forms carrier pGEX-2T-people sigelc-10, then transformed into escherichia coli BL21.Positive colony is cut evaluation with EcoR I enzyme, and the forward clone cuts evaluation with the BamHI enzyme, and the capable 0..8% agarose gel electrophoresis of product is analyzed.Confirm through order-checking, inserted designed sigelc-10 encoding sequence.
Choosing the positive BL21 clone who expresses sigelc-10 is inoculated in 100ml 2 * YTA substratum, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 100mM IPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mM Na
2HPO
4, 1.8mM KH
2PO
4PH7.3) resuspended, add 20% Triton X-100 to 1% jog 30min after ultrasonic (B.BraunLabsonic U) fragmentation again, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, (pH8.0) room temperature leaves standstill after 30 minutes and collects elutriant for 10mM gsh, 50mM Tris-HCl to add 500ul gsh elution buffer, repeat wash-out 2-3 time, obtain people sigelc-10 albumen.
Carry out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 10 amino-acid residues of N-end shown in 10 amino acid of N-end and the SEQ ID NO:2 are identical as a result.Embodiment 5: anti-people sigelc-10 production of antibodies
The recombinant protein people sigelc-10 that obtains among the embodiment 4 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 sigelc-10 gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.
Embodiment 6:sigelc-10 relies on the activity detection of mode in conjunction with HRBC with sialic acid
With the total length siglec-10 plasmid DNA among the embodiment 1 with LipofectAMINE reagent (LifeTechnologies company) transfection COS-7 cell, with siglec-5 as positive control.Carried out red corpuscle after the transfection in 48 hours in conjunction with experiment: the HRBC in normal people's peripheral blood source is given a baby a bath on the third day after its birth time with PBA (containing 0.25% BSA among the PBS), is resuspended in the DMEM substratum and adds among 0.25% BSA.Each transfection hole adds 1ml red cell suspension, 37 ℃ hatch 30 minutes after, the unconjugated cell of flush away is fixed with 0.25% Paraformaldehyde 96.With sialidase COS-7 cell or red corpuscle are digested (0.1U/ml was hatched 2-3 hour for 37 ℃) before the transfection.Be combined with more than 10 erythrocytic COS-7 cell in 20 visuals field of counting and take pictures under mirror, the result is expressed as and is combined with the per-cent (Fig. 6) that erythrocytic COS-7 cell accounts for total cell count.
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 appended book of the application institute restricted portion equally.
Sequence table
(1) general information:
(i) denomination of invention: human siali acid conjugated immunoglobulin-like agglutinant, its encoding sequence and purposes
(ii) sequence number: 8
(2) information of SEQ ID NO:1:
(i) sequence signature:
(A) length: 3124bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ii) molecule type: cDNA
( iii ) ：SEQ ID NO：1：gcccccagga gacccagagg acaactgggc aaggtgggcc ggagagtgtg ggggaaggca 60aaggagttct gtgagctcag cgtctgaagc tcatttcatg catcaggccc cagggctcag 120cttccgcctt cggcttcccc ttctgccaag agccctgagc cactcacagc acgaccagag 180aacaggcctg tctcaggcag gccctgcgcc tcctatgcgg agATGCTACT GCCACTGCTG 240CTGTCCTCGC TGCTGGGCGG GTCCCAGGCT ATGGATGGGA GATTCTGGAT ACGAGTGCAG 300GAGTCAGTGA TGGTGCCGGA GGGCCTGTGC ATCTCTGTGC CCTGCTCTTT CTCCTACCCC 360CGACAAGACT GGACAGGGTC TACCCCAGCT TATGGCTACT GGTTCAAAGC AGTGACTGAG 420ACAACCAAGG GTGCTCCTGT GGCCACAAAC CACCAGAGTC GAGAGGTGGA AATGAGCACC 480CGGGGCCGAT TCCAGCTCAC TGGGGATCCC GCCAAGGGGA ACTGCTCCTT GGTGATCAGA 540GACGCGCAGA TGCAGGATGA GTCACAGTAC TTCTTTCGGG TGGAGAGAGG AAGCTATGTG 600AGATATAATT TCATGAACGA TGGGTTCTTT CTAAAAGTAA CAGCCCTGAC TCAGAAGCCT 660GATGTCTACA TCCCCGAGAC CCTGGAGCCC GGGCAGCCGG TGACGGTCAT CTGTGTGTTT 720AACTGGGCCT TTGAGGAATG TCCACCCCCT TCTTTCTCCT GGACGGGGGC TGCCCTCTCC 780TCCCAAGGAA CCAAACCAAC GACCTCCCAC TTCTCAGTGC TCAGCTTCAC GCCCAGACCC 840CAGGACCACA ACACCGACCT CACCTGCCAT GTGGACTTCT CCAGAAAGGG TGTGAGCGTA 900CAGAGGACCG TCCGACTCCG TGTGGCCTAT GCCCCCAGAG ACCTTGTTAT CAGCATTTCA 960CGTGACAACA CGCCAGCCCT GGAGCCCCAG CCCCAGGGAA ATGTCCCATA CCTGGAAGCC 1020CAAAAAGGCC AGTTCCTGCG GCTCCTCTGT GCTGCTGACA GCCAGCCCCC TGCCACACTG 1080AGCTGGGTCC TGCAGAACAG AGTCCTCTCC TCGTCCCATC CCTGGGGCCC TAGACCCCTG 1140GGGCTGGAGC TGCCCGGGGT GAAGGCTGGG GATTCAGGGC GCTACACCTG CCGAGCGGAG 1200AACAGGCTTG GCTCCCAGCA GCGAGCCCTG GACCTCTCTG TGCAGTATCC TTCAGAGAAC 1260CTGAGAGTGA TGGTTTCCCA AGCAAACAGG ACAGTCCTGG AAAACCTTGG GAACGGCACG 1320TCTCTCCCAG TACTGGAGGG CCAAAGCCTG TGCCTGGTCT GTGTCACACA CAGCAGCCCC 1380CCAGCCAGGC TGAGCTGGAC CCAGAGGGGA CAGGTTCTGA GCCCCTCCCA GCCCTCAGAC 1440CCCGGGGTCC TGGAGCTGCC TCGGGTTCAA GTGGAGCACG AAGGAGAGTT CACCTGCCAC 1500GCTCGGCACC CACTGGGCTC CCAGCACGTC TCTCTCAGCC TCTCCGTGCA CTATAAGAAG 1560GGACTCATCT CAACGGCATT CTCCAACGGA GCGTTTCTGG GAATCGGCAT CACGGCTCTT 1620CTTTTCCTCT GCCTGGCCCT GATCATCATG AAGATTCTAC CGAAGAGACG GACTCAGACA 1680GAAACCCCGA GGCCCAAGTT CTCCCGGCAC AGCACGATCC TGGATTACAT CAATGTGGTC 1740CCGACGGCTG GCCCCCTGGC TCAGAAGCGG AATCAGAAAG CCACACCAAA CAGTCCTCGG 1800ACCCCTCTTT CACCAGGTGC TCCCTCCCCA GAATCAAAGA AGAACCAGAA AAAGCAGTAT 1860CAGTTGCCCA GTTTCCCAGA ACCCAAATCA TCCACTCAAG CCCCAGAATC CCAGGAGAGC 1920CAAGAGGAGC TCCATTATGC CACGCTCAAC TTCCCAGGCG TCAGACCCAG GCCTGAGGCC 1980CGGATGCCCA AGGGCACCCA GGCGGATTAT GCAGAAGTCA AGTTCCAATG Agggtctctt 2040aggctttagg actgggactt cggctaggga ggaaggtaga gtaagaggtt gaagataaca 2100gagtgcaaag tttccttctc tccctctctc tctctctttc tctctctctc tctctttctc 2160tctctgttaa aaaaacatct ggccagggca cagtggctta cgcctgtaat cccagcactt 2220tgggaggttg aggtgggcag atcgcctgag gtcgggagtt cgagaccagc ctggccaact 2280tggtgaaacc ccgtctctac taaaaataca aaaattagct gggcatggtg gcaggcgcct 2340gtaatcctac ctacttggga agctgaggca ggagaatcac ttgaacctgg gagacggagg 2400ttgcagtgag ccaagatcac accattgcac gccagcctgg gcaacaaagc gagactccat 2460ctcaaaaaaa aaatcctcca aatgggttgg gtgtctgtaa tcccagcact ttgggaggct 2520aaggtgggtg gattgcttga gcccaggagt tcgagaccag cctgggcaac atggtgaaac 2580cccatctcta caaaaaatac aaaacatagc tgggcttggt ggtgtgtgcc tgtagtccca 2640gctgtcagac atttaaacca gagcaactcc atctggaata ggagctgaat aaaatgaggt 2700tgagacctac tgggctgcat tctcagacag tggaggcatt ctaagtcaca ggatgagaca 2760ggaggtccgt acaagataca ggtcataaag actttgcatg ataaaacaga ttgcagtaaa 2820gaagccaacc aaatcccacc aaaaccaagt tggccacgag agtgacctct ggtcgtcctc 2880actgctacac tcctgacagc accatgacag tttacaaatg ccatggcaac atcaggaagt 2940tacccgatat gtcccaaaag ggggaggaat gaataatcca ccccttgttt agcaaataag 3000caagaaataa ccataaaagt gggcaaccag cagctctagg cgctgctctt gtctatggag 3060tagccattct tttgttcctt tactttctta ataaacttgc tttcacctta aaaaaaaaaa 3120aaaa 3124
(2) information of SEQ ID NO:2:
(i) sequence signature:
(A) length: 602 amino acid
(B) type: amino acid
(C) topological framework: linearity
(ii) molecule type: polypeptide
( iii ) ：SEQ ID NO：2：MLLPLLLSSL LGGSQAMDGR FWIRVQESVM VPEGLCISVP CSFSYPRQDW 50TGSTPAYGYW FKAVTETTKG APVATNHQSR EVEMSTRGRF QLTGDPAKGN 100CSLVIRDAQM QDESQYFFRV ERGSYVRYNF MNDGFFLKVT ALTQKPDVYI 150PETLEPGQPV TVICVFNWAF EECPPPSFSW TGAALSSQGT KPTTSHFSVL 200SFTPRPQDHN TDLTCHVDFS RKGVSVQRTV RLRVAYAPRD LVISISRDNT 250PALEPQPQGN VPYLEAQKGQ FLRLLCAADS QPPATLSWVL QNRVLSSSHP 300WGPRPLGLEL PGVKAGDSGR YTCRAENRLG SQQRALDLSV QYPSENLRVM 350VSQANRTVLE NLGNGTSLPV LEGQSLCLVC VTHSSPPARL SWTQRGQVLS 400PSQPSDPGVL ELPRVQVEHE GEFTCHARHP LGSQHVSLSL SVHYKKGLIS 450TAFSNGAFLG IGITALLFLC LALIIMKILP KRRTQTETPR PKFSRHSTIL 500DYINVVPTAG PLAQKRNQKA TPNSPRTPLS PGAPSPESKK NQKKQYQLPS 550FPEPKSSTQA PESQESQEEL HYATLNFPGV RPRPEARMPK GTQADYAEVK 600FQ 602
(2) information of SEQ ID NO:3
(i) sequence signature
(A) length: 18 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO:3
TGGCTCAGAA?GCGGAATC 18
(2) information of SEQ ID NO:4
(i) sequence signature
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO:4
CCTCATTGGA?ACTTGACTTC?TGC 23
(2) information of SEQ ID NO:5
(i) sequence signature
(A) length: 28 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO:5
ACGAATTCGC?GCCTCCTATG?CGGAGATG 28
(2) information of SEQ ID NO:6
(i) sequence signature
(A) length: 28 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO:6
ACGAATTCCG?TTGGAGAATG?CCGTTGAG 28
(2) information of SEQ ID NO:7
(i) sequence signature
(A) length: 25 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO:7
TCCTATGCGG?AGATGCTACT?GCCAC 25
(2) information of SEQ ID NO:8
(i) sequence signature
(A) length: 25 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO:8
AAGAGACCCT?CATTGGAACT?TGACT 25
Claims (14)
1. an isolating people siglec-10 polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence has at least 40% homogeny with a kind of nucleotide sequence catalysis territory that is selected from down group:
(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
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) has the sequence of 223-2031 position among the SEQ ID NO:1;
(b) has the sequence of 1-3124 position among the SEQ ID NO:1.
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. preparation method with polypeptide of people siglec-10 polypeptide active is characterized in that this method comprises:
(a) under the condition that is fit to expressing human siglec-10 polypeptide, cultivate the described host cell of claim 7;
(b) from culture, isolate polypeptide with people siglec-10 polypeptide active.
9. energy and the described people siglec-10 of claim 1 polypeptid specificity bonded antibody.
10. nucleic acid molecule, it contains a successive 10-3124 Nucleotide in the described polynucleotide of claim 3.
11. whether there is the method for siglec-10 polypeptide in the test sample, it is characterized in that, comprising:
The described antibody of sample and claim 9 is contacted,
Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample siglec-10 polypeptide.
12. the purposes of polypeptide as claimed in claim 1 is characterized in that, it is used to screen the agonist that promotes people siglec-10 polypeptide active, and perhaps screening suppresses the antagonist of people siglec-10 polypeptide active or is used to the peptide finger print identification.
13. the purposes of a nucleic acid molecule as claimed in claim 10 is characterized in that, it is used as primer and is used for pcr amplification reaction, perhaps is used for hybridization as probe, perhaps is used to make gene chip or microarray.
14. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01105501 CN1212334C (en) | 2001-02-28 | 2001-02-28 | Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01105501 CN1212334C (en) | 2001-02-28 | 2001-02-28 | Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1371916A true CN1371916A (en) | 2002-10-02 |
| CN1212334C CN1212334C (en) | 2005-07-27 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01105501 Expired - Lifetime CN1212334C (en) | 2001-02-28 | 2001-02-28 | Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1212334C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007081608A2 (en) * | 2005-11-21 | 2007-07-19 | Genentech, Inc. | Novel gene disruptions, compositions and methods relating thereto |
| CN103237813A (en) * | 2010-10-05 | 2013-08-07 | 第一三共株式会社 | Antibody targeting osteoclast-elated protein Siglec-15 |
| CN105367658A (en) * | 2015-12-02 | 2016-03-02 | 朱进 | Human-derived anti-sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) antibody IgG and application thereof |
| CN108130374A (en) * | 2018-03-22 | 2018-06-08 | 北京泱深生物信息技术有限公司 | The diagnostic products of kidney and medicine composition |
| CN108603883A (en) * | 2016-01-27 | 2018-09-28 | 富士胶片和光纯药株式会社 | The determination method of prostate cancer |
| CN114591434A (en) * | 2021-04-30 | 2022-06-07 | 杭州邦顺制药有限公司 | anti-Siglec 15 antibody and preparation method and application thereof |
-
2001
- 2001-02-28 CN CN 01105501 patent/CN1212334C/en not_active Expired - Lifetime
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007081608A2 (en) * | 2005-11-21 | 2007-07-19 | Genentech, Inc. | Novel gene disruptions, compositions and methods relating thereto |
| WO2007081608A3 (en) * | 2005-11-21 | 2008-01-10 | Genentech Inc | Novel gene disruptions, compositions and methods relating thereto |
| EP2002714A1 (en) * | 2005-11-21 | 2008-12-17 | Genentech, Inc. | Novel gene disruptions, compositions and methods relating thereto |
| CN103237813A (en) * | 2010-10-05 | 2013-08-07 | 第一三共株式会社 | Antibody targeting osteoclast-elated protein Siglec-15 |
| CN105367658A (en) * | 2015-12-02 | 2016-03-02 | 朱进 | Human-derived anti-sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) antibody IgG and application thereof |
| CN105367658B (en) * | 2015-12-02 | 2020-06-16 | 朱进 | Human anti-sialic acid binding immunoglobulin-like lectin-9 antibody IgG and application thereof |
| CN108603883A (en) * | 2016-01-27 | 2018-09-28 | 富士胶片和光纯药株式会社 | The determination method of prostate cancer |
| CN108130374A (en) * | 2018-03-22 | 2018-06-08 | 北京泱深生物信息技术有限公司 | The diagnostic products of kidney and medicine composition |
| CN114591434A (en) * | 2021-04-30 | 2022-06-07 | 杭州邦顺制药有限公司 | anti-Siglec 15 antibody and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1212334C (en) | 2005-07-27 |
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