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CN1303925A - Novel polypeptide-arginyl tRNA synthetase 44 and polynucleotide coding said polypeptide - Google Patents

Novel polypeptide-arginyl tRNA synthetase 44 and polynucleotide coding said polypeptide Download PDF

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CN1303925A
CN1303925A CN 99119864 CN99119864A CN1303925A CN 1303925 A CN1303925 A CN 1303925A CN 99119864 CN99119864 CN 99119864 CN 99119864 A CN99119864 A CN 99119864A CN 1303925 A CN1303925 A CN 1303925A
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polypeptide
polynucleotide
synthetic enzyme
arginyl trna
trna synthetic
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毛裕民
谢毅
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BORONG GENE DEVELOPMENT CO LTD SHANGHAI
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BORONG GENE DEVELOPMENT CO LTD SHANGHAI
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Priority to CN 99119864 priority Critical patent/CN1303925A/en
Priority to PCT/CN2000/000373 priority patent/WO2001031022A1/en
Priority to AU12639/01A priority patent/AU1263901A/en
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Abstract

The present invention discloses a novel polypeptide-arginyl tRNA synthetase 44, polynucleotide coding said polypeptide and its method for producing said polypeptide by using DNA recombination technique. Said invention also discloses the method of using said polypeptide to cure several diseases, such as malignant tumor, blood disease, HIV infection, immunological diseases and various inflammation. Said invention also discloses an antagonist for resisting said polypeptide and its therapeutic effect. Said invention also discloses the application of polynucleotide for coding this novel arginyl tRNA synthetase 44.

Description

The polynucleotide of a kind of new polypeptide---arginyl tRNA synthetic enzyme 44 and this peptide species of coding
The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---arginyl tRNA synthetic enzyme 44, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.
It is amino acid, tRNA, ATP that aminoacyl-tRNA synthetase (aaRS) has three substrates.The esterification of aaRS catalytic amino acid makes it to be connected in the 3 ' end of tRNA, and each aaRS is corresponding to a seed amino acid and several isoaccepting tRNAs.ATP provides activated amino acid required energy, and amino acid activation earlier is aminoacyl adenylate, and then generates aminoacyl-tRNA with tRNA.Interact between AaRS and the substrate and relate to identification and bonded problem.
AaRSs can be divided into two types that respectively contain ten members, and arginyl tRNA synthetic enzyme (ArgRS) belongs to aaRS Class1 (Eriani et al., 1990).The aaRS Class1 has following structural domain (Cavarelli Jetal., 1998) usually:
1. amino acid identified region: two high conservatives are only arranged in conjunction with the crack at aaRS Class1 amino acid
Amino-acid residue, one be tyrosine residues in 347 sites, one is that asparagicacid residue is 191
The site.At ArgRS, Tyr347 participates in the η-nitrogen-atoms of identification amino acid substrate, in the aaRS type
Among other member of 1, tyrosine residues is carried out some other function.Aspl91 is at the 5th β of chain
Folding C end, it does not relate to substrate in conjunction with but structural effect being arranged, and it has stablized the 5th β of chain
Fold and the 6th βZhe Die of chain and the interaction of secondary structure on every side.
2.ATP binding site: two special polypeptide structure territory HIGH are connected in ATP with KMSKS and combine the position
The point.The last constant Gly of HIGH has formed a platform so that combination at the N of the 6th α spiral of chain end
ATP, two His and Lys relate to stable that aminoacylation transition state product forms.
3.tRNA identification and binding site: Add-1 and Add-2 structural domain corecognition anticodon and tRNA
Arm, structural domain Ins-1 have hidden one side of amino acid receptor trunk, and Rossmann is folding to have hidden other
On one side.
4.tRNA grappling platform: four members of aaRS Class1 comprise that ArgRS has the 13rd on a chain
The structural domain that the 14th βZhe Die of βZhe Die and chain formed, be positioned at Rossmann folding after, this
Structural domain relates to tRNA and is anchored to the ArgRS platform.
5.ArgRSN the RNA of end is in conjunction with the Add-1 structural domain of territory: ArgRS and the identification of tRNA, with tRNA
The interaction of D puff Lu the closest.The exposed βZhe Die of Add-1 structural domain is tied at many nucleic acid
Occupy vital role on the hop protein.
6. the anticodon binding site of anticodon binding site: ArgRS is positioned at the left-hand side of C end, chain the
On the amino-acid residue of 21 α spirals and the 22nd α spiral of chain.The exposed β of Add-1 structural domain
The folding anticodon arm of also discerning.
Studies show that nine aaRS (Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met, and Pro) are by a genes encoding (Vellekamp et al., 1985).The cDNA sequence of people ArgRS and other Mammals ArgRS gene have 87% homology, and aminoacid sequence and Chinese mouse ovary ArgRS have 87.7% homology, with intestinal bacteria ArgRS 37.7% homology are arranged.ArgRS is high conservative (Girjes AA et al., 1995) in mammalian cell.
People's arginyl tRNA synthetic enzyme 44 genes of the present invention and zymic ArgRS gene have 41% homology (homologous protein U51032) on protein level, its structural domain is similar in appearance to the characteristic structural domain of ArgRS gene family--and the identification of-amino acid identified region, ATP-binding site, tRNA combines territory, anticodon binding site with the RNA of binding site, tRNA grappling platform, ArgRS N end.Based on above each point, so think that new gene of the present invention is the gene of a coding people ArgRS gene family, names to be people's arginyl tRNA synthetic enzyme 44.And infer that with this its structural domain is similar to ArgRS gene family structural domain, have similar biological function.
ArgRS catalysis arginine isoaccepting tRNA carries arginine, in the presence of the rrna and the relevant factor, finish peptide chain initial, extend and stop.
The polynucleotide of coding people arginyl tRNA synthetic enzyme 44, and coded people's arginyl tRNA synthetic enzyme 44 be found to be the differentiation of research cell under normal and pathological conditions, the physiological and biochemical procedure of propagation provides a kind of method, also comprises that with the disease that the disorder of cytodifferentiation propagation causes cancer provides a kind of new way for diagnosis, treatment.
An object of the present invention is to provide isolating new polypeptide---arginyl tRNA synthetic enzyme 44 with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of this polypeptide of coding.
Another object of the present invention provides the recombinant vectors of the polynucleotide that contain coding arginyl tRNA synthetic enzyme 44.
Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain coding arginyl tRNA synthetic enzyme 44.
Another object of the present invention provides the method for producing arginyl tRNA synthetic enzyme 44.
Another object of the present invention provides at polypeptide of the present invention---the antibody of arginyl tRNA synthetic enzyme 44.
Another object of the present invention has provided at polypeptide of the present invention---simulated compound, antagonist, agonist, the inhibitor of arginyl tRNA synthetic enzyme 44.
Another object of the present invention provides the method for the unusual relevant disease of diagnoses and treatment and arginyl tRNA synthetic enzyme 44.
In a first aspect of the present invention, novel isolated arginyl tRNA synthetic enzyme 44 is provided, this polypeptide is the people source, and it comprises: have the polypeptide of SEQ ID NO:2 aminoacid sequence or its conservative property variation polypeptide or its active fragments or its reactive derivative, analogue.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 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned arginyl tRNA synthetic enzyme 44 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 714-1925 position among the SEQ ID NO:1; (b) has the sequence of 1-1951 position among the SEQ ID NO: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.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
As used herein, " isolating " are meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating arginyl tRNA synthetic enzyme 44 " is meant that arginyl tRNA synthetic enzyme 44 is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying arginyl tRNA synthetic enzyme 44 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of arginyl tRNA synthetic enzyme 44 polypeptide can be used amino acid sequence analysis.
The invention provides a kind of new polypeptide---arginyl tRNA synthetic enzyme 44, it is made up of the aminoacid sequence shown in the SEQ IDNO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analogue of arginyl tRNA synthetic enzyme 44.As used herein, term " fragment ", " derivative " and " analogue " are meant biological function or the active polypeptide that keeps arginyl tRNA synthetic enzyme of the present invention 44 identical basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) is a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.
The invention provides isolating nucleic acid (polynucleotide), substantially the polynucleotide that have a polypeptide of SEQ ID NO:2 aminoacid sequence by coding are formed.Polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 1951 bases, and its open reading frame (714---1925) 403 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide and yeast ArgRS gene have 44% homology, deducibility goes out this arginyl tRNA synthetic enzyme 44 and has the similar 26S Proteasome Structure and Function of yeast ArgRS gene.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has protein or the polypeptide of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence that has only mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be 20-30 Nucleotide at least, be more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.Nucleic acid fragment also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding arginyl tRNA synthetic enzyme 44.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
The special polynucleotide sequence of coding arginyl tRNA synthetic enzyme 44 of the present invention can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.
Sequence dna fragment of the present invention also can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.
In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.
Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) level of the transcript of mensuration arginyl tRNA synthetic enzyme 44; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.
In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 10 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene order information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.
In (4) kind method, detect the protein product of arginyl tRNA synthetic enzyme 44 genetic expressions and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.
Use method (Saiki, the et al.Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, 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 polynucleotide 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 gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can with ordinary method such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467) measure.This class polynucleotide sequence is measured also available commercial sequencing kit etc.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or directly with the host cell of arginyl tRNA synthetic enzyme 44 encoding sequences through the genetically engineered generation, and the method that produces polypeptide of the present invention through recombinant technology.
Among the present invention, the polynucleotide sequence of coding arginyl tRNA synthetic enzyme 44 can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " 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 carrier.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on the T7 promotor 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 may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.
Method well-known to those having ordinary skill in the art can be used to make up the dna sequence dna that contains coding arginyl tRNA synthetic enzyme 44 and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, aLaboratory Manual, cold Spring 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; The P of lambda particles phage LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte 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 that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, 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.
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 etc.
Persons skilled in the art all know how to select appropriate carriers/transcriptional regulatory element (as promotor, enhanser etc.) and selected marker.
Among the present invention, the polynucleotide of coding arginyl tRNA synthetic enzyme 44 or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contain the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to 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; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.
Can carry out with routine techniques well known to those skilled in the art with dna sequence dna of the present invention or the recombinant vectors transformed host cell that contains described dna sequence dna.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.Alternative 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, perhaps conventional mechanical method such as microinjection, electroporation, liposome packing etc.
By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce arginyl tRNA synthetic enzyme 44 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people arginyl tRNA synthetic enzyme 44, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). in suitable medium, cultivate host cell;
(3). separation, protein purification from substratum or cell.
In step (2), 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.
In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular 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.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, 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 antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat malignant tumour, adrenal gland defect, tetter, all kinds of inflammation, HIV infection and immunological disease etc.
Because ArgRS catalysis arginine isoaccepting tRNA carries arginine, in the presence that rrna and the relevant factor are finished, finish peptide chain initial, extend and stop, finish proteinic biosynthesizing.The abnormal expression of people's arginyl tRNA synthetic enzyme 44 of the present invention will produce proteinic biosynthesizing obstacle, and produce various diseases thus.These diseases include but not limited to:
Grow disorders: spina bifida, the cranium fissure, anencephalia, the brain bulging, the hole deformity of brain, congenital hydrocephalus, the aqueduct deformity, achondroplastic dwarf's disease, spondyloepiphyseal dysplasia disease, the Langer-Giedion syndromes, hypogenitalism, epispadia, latent, with malformation syndrome of short and small stature such as Conradi syndromes and Danbolt-Closs syndromes, congenital glaucoma or cataract, congenital little fissura palpebrae, retinal development is unusual, atrophia nervi optici congenita, ulcuscuris, monster, the Williams syndromes, the Alagille syndromes, shellfish syndrome Weis two
Metabolism and nutritive disease: atrophy, hyperlipidaemia and hyperlipoproteinemia, obesity, deficiency disease
Inflammation: atopic reaction, adult respiratory distress syndrome, lung eosinophilia, rheumatoid arthritis, similar rheumatism sample sacroiliitis, cholecystitis, glomerulonephritis, dermatomyositis, polymyositis, Addison's disease
Various tumours: substrate epithelial tumor, squama shape epithelial tumor, mucinous tumors, fibroma, lipoma, chondroma, vascular tumor, lymphoma, hemopoietic tissue tumour, neuroma, adenoma
The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) arginyl tRNA synthetic enzyme 44.Agonist improves arginyl tRNA synthetic enzyme 44 biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can in the presence of medicine, the film preparation of mammalian cell or expression arginyl tRNA synthetic enzyme 44 be cultivated with the arginyl tRNA synthetic enzyme 44 of mark.Measure the medicine raising then or check this interactional ability.
The antagonist of arginyl tRNA synthetic enzyme 44 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of arginyl tRNA synthetic enzyme 44 can combine and eliminate its function with arginyl tRNA synthetic enzyme 44, or suppresses the generation of this polypeptide, or combines with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.
In screening during as the compound of antagonist, arginyl tRNA synthetic enzyme 44 can be added during bioanalysiss measure, determine to interactional influence between arginyl tRNA synthetic enzyme 44 and its acceptor whether compound is antagonist by measuring compound.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with arginyl tRNA synthetic enzyme 44 bonded peptide molecules obtains.During screening, generally tackle arginyl tRNA synthetic enzyme 44 molecules and carry out mark.
The invention provides and use polypeptide, and fragment, derivative, analogue or their cell are as the method for antigen with production antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody at arginyl tRNA synthetic enzyme 44 antigenic determinants.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.
The method of the available arginyl tRNA of the production of polyclonal antibody synthetic enzyme 44 direct injection immune animals (as rabbit, mouse, rat etc.) obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The technology of the monoclonal antibody of preparation arginyl tRNA synthetic enzyme 44 include but not limited to hybridoma technology (Kohler and Milstein.Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.Pat No.4946778) also can be used for producing the single-chain antibody of anti-arginyl tRNA synthetic enzyme 44.
The antibody of anti-arginyl tRNA synthetic enzyme 44 can be used in the immunohistochemistry technology, detects the arginyl tRNA synthetic enzyme 44 in the biopsy specimen.
With the also available labelled with radioisotope of arginyl tRNA synthetic enzyme 44 bonded monoclonal antibodies, 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 also can be used for designing the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of arginyl tRNA synthetic enzyme 44 high-affinities 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 arginyl tRNA synthetic enzyme 44 positive cells.
The disease that antibody among the present invention can be used for treating or prevention and arginyl tRNA synthetic enzyme 44 are relevant.The antibody that gives suitable dosage can stimulate or block the generation or the activity of arginyl tRNA synthetic enzyme 44.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization arginyl tRNA synthetic enzyme 44 levels.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.Arginyl tRNA synthetic enzyme 44 levels that detected in the test can be with laying down a definition the importance of arginyl tRNA synthetic enzyme 44 in various diseases and be used to the disease of diagnosing arginyl tRNA synthetic enzyme 44 to work.
Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carries out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension, be more preferably and carry out mass spectroscopy.
The polynucleotide of coding arginyl tRNA synthetic enzyme 44 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 nothing expression of arginyl tRNA synthetic enzyme 44 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the arginyl tRNA synthetic enzyme 44 of expressing variation, to suppress endogenic arginyl tRNA synthetic enzyme 44 activity.For example, a kind of arginyl tRNA synthetic enzyme 44 of variation can be the arginyl tRNA synthetic enzyme 44 that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of 44 expression of arginyl tRNA synthetic enzyme or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the polynucleotide of coding arginyl tRNA synthetic enzyme 44 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding arginyl tRNA synthetic enzyme 44 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding arginyl tRNA synthetic enzyme 44 can be packaged in the liposome and be transferred in the cell in addition.
Polynucleotide import 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.
Suppress the oligonucleotide (comprising sense-rna and DNA) of arginyl tRNA synthetic enzyme 44mRNA 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.
The polynucleotide of coding arginyl tRNA synthetic enzyme 44 can be used for the diagnosis with the relative disease of arginyl tRNA synthetic enzyme 44.The unconventionality expression of the expression that the polynucleotide of coding arginyl tRNA synthetic enzyme 44 can be used for detecting arginyl tRNA synthetic enzyme 44 arginyl tRNA synthetic enzyme 44 whether or under morbid state.As the dna sequence dna of the arginyl tRNA synthetic enzyme 44 of encoding can be used for biopsy specimen is hybridized to judge the expression situation of arginyl tRNA synthetic enzyme 44.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 the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect arginyl tRNA synthetic enzyme 44 with arginyl tRNA synthetic enzyme 44 special primers.
The sudden change that detects arginyl tRNA synthetic enzyme 44 genes also can be used for diagnosing the relevant disease of arginyl tRNA synthetic enzyme 44.The form of arginyl tRNA synthetic enzyme 44 sudden change comprises that the point mutation compared with normal wild type arginyl tRNA synthetic enzyme 44DNA 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).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.
In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.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.
The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).
The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manualof Basic Techniques, Pergamon Press, New York (1988).
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, MendelianInheritance in Man (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.
Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.
Polypeptide of the present invention, polynucleotide and stand-in thereof, agonist, antagonist and inhibitor and suitable pharmaceutical carrier combination back can be used.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.
The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.
Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.Arginyl tRNA synthetic enzyme 44 comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's arginyl tRNA synthetic enzyme 44 will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.
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 the amino acid sequence homology comparison diagram of arginyl tRNA synthetic enzyme 44 of the present invention and yeast ArgRS gene.The top sequence is an arginyl tRNA synthetic enzyme 44, and the below sequence is a yeast ArgRS gene.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".
Fig. 2 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating arginyl tRNA synthetic enzyme 44.44kDa is proteinic molecular weight.The arrow indication is isolated protein band.
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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: the clone of arginyl tRNA synthetic enzyme 44
Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 0539g02 is new DNA.By synthetic a series of primers the contained insertion cDNA fragment of this clone is carried out two-way mensuration.The result shows, the contained full-length cDNA of 0539g02 clone is 1951bp (shown in Seq ID NO:1), from 714bp to 1925bp the open reading frame (ORF) of a 1212bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after pBS-0539g02, encoded protein matter called after arginyl tRNA synthetic enzyme 44.
Embodiment 2:cDNA clone's homology retrieval
With the sequence and the encoded protein sequence thereof of arginyl tRNA synthetic enzyme 44 of the present invention, with Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.The gene the highest with arginyl tRNA synthetic enzyme 44 homologys of the present invention is a kind of known yeast ArgRS gene, and its encoded protein number is U51032 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 44%.Embodiment 3: with the gene of RT-PCR method clones coding arginyl tRNA synthetic enzyme 44
Total RNA is a template with fetus brain cell, is that primer carries out the synthetic cDNA of reverse transcription reaction with oligo-dT, with behind the test kit purifying of Qiagene, carries out pcr amplification with following primer:
Primer1:5’-GGCGGCGCACTTCCGTAGAGGTG-3’(SEQ?ID?NO:3)
Primer2:5’-CATTTTAAAAGCCATTTTAATGG-3’(SEQ?ID?NO:4)
Primer1 is the forward sequence that begins of 1bp that is positioned at the 5 ' end of SEQ ID NO:1;
Primer2 be SEQ ID NO:1 in 3 ' end reverse sequence.
The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/L MgCl 2, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-1951bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting is analyzed arginyl tRNA synthetic enzyme 44 expression of gene:
Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ g RNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With α- 32P dATP prepares by random priming 32The dna probe of P-mark.Used dna probe is arginyl tRNA synthetic enzyme 44 coding region sequences (714bp to 1926bp) of pcr amplification shown in Figure 1.Probe (about 2 * 10 with the 32P-mark 6Cpm/ml) spend the night in 42 ℃ of hybridization in a solution with the nitrocellulose filter that has shifted RNA, this solution comprises 50% methane amide-25mM KH 2PO 4(pH7.4)-5 * SSC-5 * Denhardt ' s solution and 200 μ g/ml salmon sperm DNAs.After the hybridization, filter membrane is washed 30min in 55 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with Phosphor Imager.Embodiment 5: vivoexpression, separation and the purifying of reorganization arginyl tRNA synthetic enzyme 44
According to SEQ ID NO:1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:
Primer3:5’-CCCCATATGATGCAGTTTGGTCTTCTGGGAACTG-3’ (Seq?ID?No:5)
Primer4:5’-CCCGGATCCTTACATCCTACATACAGGTGTTATTCC-3’(Seq?ID?No:6)
5 ' end of these two sections primers contains Nde I and BamH I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, Nde I and BamH I restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-0539g02 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0539g02 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with Nde I and BamH I, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0539g02) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-0539g02) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), has obtained the target protein arginyl tRNA synthetic enzyme 44 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 44kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.Embodiment 6 anti-arginyl tRNA synthetic enzyme 44 production of antibodies
Synthesize following arginyl tRNA synthetic enzyme 44 specific polypeptide with Peptide synthesizer (PE company product):
NH 2-Met-Gln-Phe-Gly-Leu-Leu-Gly-Thr-Gly-Phe-Gln-Leu-Phe-Gly-Tyr-COOH(SEQ?ID?NO:7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with arginyl tRNA synthetic enzyme 44 specifically.
Sequence table
(1) general information:
(ⅱ) denomination of invention: arginyl tRNA synthetic enzyme 44 and encoding sequence thereof
(ⅲ) sequence number: 7
(2) information of SEQ ID NO:1:
(ⅰ) sequence signature:
(A) length: 1951bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
( ⅹⅰ ) ：SEQ ID NO:1： 1 GGCGGCGCACTTCCGTAGAGGTGGACATGGCGTGCGGCTTTCGCCGCGCTATTGCTTGCC 61 AGCTTTCCAGAGTGTTGAATCTTCCACCAGAAAACTTGATCACATCAATATCTGCAGTTC121 CAATTTCCCAAAAAGAAGAAGTAGCTGATTTTCAGCTTTCTGTGGATTCTTTATTGGAAA181 AAGACAATGACCATTCAAGACCAGATATTCAAGTTCAAGCCAAGAGACTAGCAGAGAAGC241 TAAGATGTGATACAGTGGTGAGTGAAATCAGTACTGGTCAAAGGACTGTAAATTTCAAAA301 TAAACAGAGAGCTCTTAACAAAGACAGTGCTACAACAAGTAATTGAAGATGGCTCAAAAT361 ATGGATTAAAAAGTGAACTTTTCTCTGGACTTCCCCAGAAGAAGATTGTGGTTGAATTCA421 GGGTAGCAAGACCTCTTTACAAAGGTACTCAAGGCTCTAAGATAGTGAAATCATCTAGTC481 TTGAAACAGGCACAGCATCATTTTTGTCACTTTCTGCTGATGAAAGCAAGTCACAAGATC541 ACCCCAGATTCAAGGGAAGAAAAATAGAGTACATCTCTTAATGTTCACCTAATGTTGCCA601 AAAAATTTCATGTTGGACATTTGCGTTCTACCATCATAGGAAATTTTATAGCAAATCTCA661 AAGAAGCTTTAGGACATCAAGTAATAAGAATAAATTACCTTGGCGATTGGGGCATGCAGT721 TTGGTCTTCTGGGAACTGGCTTCCAGCTGTTTGGCTATGAGGAAAAACTGCAGTCCAATC781 CTCTACAGCATCTCTTTGAAGTTTATGTACAAGTTAATAAAGAAGCAGCAGATGATAAAA841 GTGTAGCAAAAGCAGCACAGGAGTTCTTCCAACGATTGGAACTGGGCGATGTGCAAGCAC901 TTTCACTGTGGCAAAAATTTCGGGACTTGAGCATTGAAGAGTACATTCGGGTTTACAAGC 961 GTCTGGGAGTATATTTTGATGAATATTCAGGAGAATCATCTTATCGTGAAAAATCTCAAG1021 AGGTCTTAAAGTTGCTGGAGAGTAAAGGACTCCTACTGAAAACAATAAAAGGAACGGCTG1081 TAGTAGATCTCTCTGGGAATGGCGACCCCTCCTCAATTTGTACTGTAATGCGAAGTGATG1141 GGACTTCTCTCTATGCAACCAGAGATCTTGCAGCTGCTATAGATCGAATGGACAAGTATA1201 ATTTTGATACAATGATATATGTGACAGATAAAGGACAAAAAAAGCATTTTCAGCAAGTAT1261 TCCAAATGCTGAAGATCATGGGATATGACTGGGCAGAAAGGTGCCAGCACGTGCCCTTTG1321 GAGTAGTACAGGGAATGAAGACTCGAAGAGGAGATGTCACTTTCCTGGAAGATGTTTTAA1381 ATGAGATTCAATTAAGGATGCTACAGAACATGGCTTCAATTAAGACAACTAAAGAACTCA1441 AGAACCCACAAGAGACTGCAGAGAGGGTCGGGCTCGCAGCACTCATTATTCAGGACTTCA1501 AAGGTTTACTCTTATCTGACTACAAGTTCAGCTGGGATCGTGTTTTCCAGAGTCGCGGGG1561 ACACAGGAGTCTTCCTACAGTACACACACGCCCGCCTCCACAGTTTGGAAGAGACTTTTG1621 GATGTGGGTACCTGAATGACTTCAACACTGCTTGTTTACAAGAGCCACAGTCTGTTTCAA1681 TTCTTCAGCATCTTCTCAGGTTCGACGAGGTGCTTTATAAATCATCTCAGGACTTTCAAC1741 CCAGGCATATCGTCAGTTACCTTCTAACTTTAAGTCATCTTGCAGCTGTGGCACACAAAA1801 CACTACAAATAAAAGATAGTCCTCCTGAAGTGGCTGGGGCCAGACTTCATCTTTTCAAAG1861 CTGTCCGTTCTGTCCTAGCCAATGGAATGAAACTTCTTGGAATAACACCTGTATGTAGGA1921 TGTAATTTCCATTAAAATGGCTTTTAAAATG
(3) information of SEQ ID NO:2:
(ⅰ) sequence signature:
(A) length: 403 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: polypeptide
( ⅹⅰ ) ：SEQ ID NO:2： 1 Met Gln Phe Gly Leu Leu Gly Thr Gly Phe Gln Leu Phe Gly Tyr16 Glu Glu Lys Leu Gln Ser Asn Pro Leu Gln His Leu Phe Glu Val31 Tyr Val Gln Val Asn Lys Glu Ala Ala Asp Asp Lys Ser Val Ala46 Lys Ala Ala Gln Glu Phe Phe Gln Arg Leu Glu Leu Gly Asp Val61 Gln Ala Leu Ser Leu Trp Gln Lys Phe Arg Asp Leu Ser Ile Glu76 Glu Tyr Ile Arg Val Tyr Lys Arg Leu Gly Val Tyr Phe Asp Glu91 Tyr Ser Gly Glu Ser Ser Tyr Arg Glu Lys Ser Gln Glu Val Leu106 Lys Leu Leu Glu Ser Lys Gly Leu Leu Leu Lys Thr Ile Lys Gly121 Thr Ala Val Val Asp Leu Ser Gly Asn Gly Asp Pro Ser Ser Ile136 Cys Thr Val Met Arg Ser Asp Gly Thr Ser Leu Tyr Ala Thr Arg151 Asp Leu Ala Ala Ala Ile Asp Arg Met Asp Lys Tyr Asn Phe Asp166 Thr Met Ile Tyr Val Thr Asp Lys Gly Gln Lys Lys His Phe Gln181 Gln Val Phe Gln Met Leu Lys Ile Met Gly Tyr Asp Trp Ala Glu196 Arg Cys Gln His Val Pro Phe Gly Val Val Gln Gly Met Lys Thr211 Arg Arg Gly Asp Val Thr Phe Leu Glu Asp Val Leu Asn Glu Ile226 Gln Leu Arg Met Leu Gln Asn Met Ala Ser Ile Lys Thr Thr Lys241 Glu Leu Lys Asn Pro Gln Glu Thr Ala Glu Arg Val Gly Leu Ala256 Ala Leu Ile Ile Gln Asp Phe Lys Gly Leu Leu Leu Ser Asp Tyr271 Lys Phe Ser Trp Asp Arg Val Phe Gln Ser Arg Gly Asp Thr Gly286 Val Phe Leu Gln Tyr Thr His Ala Arg Leu His Ser Leu Glu Glu301 Thr Phe Gly Cys Gly Tyr Leu Asn Asp Phe Asn Thr Ala Cys Leu316 Gln Glu Pro Gln Ser Val Ser Ile Leu Gln His Leu Leu Arg Phe331 Asp Glu Val Leu Tyr Lys Ser Ser Gln Asp Phe Gln Pro Arg His346 Ile Val Ser Tyr Leu Leu Thr Leu Ser His Leu Ala Ala Val Ala361 His Lys Thr Leu Gln Ile Lys Asp Ser Pro Pro Glu Val Ala Gly376 Ala Arg Leu His Leu Phe Lys Ala Val Arg Ser Val Leu Ala Asn391 Gly Met Lys Leu Leu Gly Ile Thr Pro Val Cys Arg Met
(4) information of SEQ ID NO:3
(ⅰ) sequence signature
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:3:GGCGGCGCACTTCCGTAGAGGTG 23
(5) 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
(ⅹ ⅰ) sequence description: SEQ ID NO:4:CATTTTAAAAGCCATTTTAATGG 23
(6) information of SEQ ID NO:5
(ⅰ) sequence signature
(A) length: 34 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:5:CCCCATATGATGCAGTTTGGTCTTCTGGGAACTG 34
(7) information of SEQ ID NO:6
(ⅰ) sequence signature
(A) length: 35 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:6:CCCGGATCCTTACATCCTACATACAGGTGTTATTCC 35
(8) information of SEQ ID NO:7:
(ⅰ) sequence signature:
(A) length: 15 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: polypeptide
(ⅹ ⅰ) sequence description: SEQ ID NO:7:Met-Gln-Phe-Gly-Leu-Leu-Gly-Thr-Gly-Phe-Gln-Leu-Phe-Gly-Tyr 15

Claims (18)

1, a kind of isolated polypeptide-arginyl tRNA synthetic enzyme 44 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.
2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in the SEQ ID NO:2.
3, polypeptide as claimed in claim 2 is characterized in that it comprises the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.
4, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:
(a) coding has the polynucleotide of the polypeptide of aminoacid sequence shown in the SEQ ID NO:2 or its fragment, analogue, derivative;
(b) with polynucleotide (a) complementary polynucleotide; Or
(c) with (a) or the polynucleotide of at least 70% homogeny (b) are arranged.
5, polynucleotide as claimed in claim 4 is characterized in that described polynucleotide comprise the polynucleotide that coding has aminoacid sequence shown in the SEQID NO:2.
6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-1951 position among the sequence of 714-1925 position among the SEQ IDNO:1 or the SEQ ID NO:1.
7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is the recombinant vectors that is formed by the described polynucleotide of arbitrary claim among the claim 4-6 and plasmid, virus or vehicle expression vector establishment.
8, a kind of genetically engineered host cell that contains exogenous polynucleotide is characterized in that it is to be selected from following a kind of host cell:
(a) host cell that transforms or transduce with the described recombinant vectors of claim 7; Or
(b) host cell that transforms or transduce with the described polynucleotide of the arbitrary claim among the claim 4-6.
9, a kind of preparation method with arginyl tRNA synthetic enzyme 44 active polypeptide is characterized in that described method comprises:
(a) expressing under arginyl tRNA synthetic enzyme 44 conditions, cultivate the described through engineering approaches host cell of claim 8;
(b) from culture, isolate and have arginyl tRNA synthetic enzyme 44 active polypeptide.
10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with arginyl tRNA synthetic enzyme 44 specificity bonded antibody.
11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or the active compound that suppresses arginyl tRNA synthetic enzyme 44.
12, compound as claimed in claim 11 is characterized in that it is the polynucleotide sequence shown in the SEQ ID NO:1 or its segmental antisense sequences.
13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used to regulate arginyl tRNA synthetic enzyme 44 in vivo, the method for external activity.
14, a kind of disease relevant or method of disease susceptibility of detecting with the described polypeptide of arbitrary claim among the claim 1-3, it is characterized in that it comprises the described polypeptide expression amount that detects, perhaps detect the activity of described polypeptide, perhaps detect and cause described expression of polypeptides amount or active unusual nucleotide diversity in the polynucleotide.
15, as the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that it is applied to screen stand-in, the agonist of arginyl tRNA synthetic enzyme 44, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.
16, as the application of the described nucleic acid molecule of arbitrary claim among the claim 4-6, it is characterized in that it is used for nucleic acid amplification reaction as primer, perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.
17,, it is characterized in that forming pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as the relevant unusually disease of diagnosis or treatment and arginyl tRNA synthetic enzyme 44 with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.
18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that being used for the treatment of as malignant tumour with described polypeptide, polynucleotide or compound, hemopathy, the medicine of HIV infection and immunological disease and all kinds of inflammation.
CN 99119864 1999-10-27 1999-10-27 Novel polypeptide-arginyl tRNA synthetase 44 and polynucleotide coding said polypeptide Pending CN1303925A (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN 99119864 CN1303925A (en) 1999-10-27 1999-10-27 Novel polypeptide-arginyl tRNA synthetase 44 and polynucleotide coding said polypeptide
PCT/CN2000/000373 WO2001031022A1 (en) 1999-10-27 2000-10-27 A NOVEL POLYPEPTIDE, AN ARGINYL tRNA SYNTHETASE 44 AND THE POLYNUCLEOTIDE ENCODING THE POLYPEPTIDE
AU12639/01A AU1263901A (en) 1999-10-27 2000-10-27 A novel polypeptide, an arginyl trna synthetase 44 and the polynucleotide encoding the polypeptide

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