CN1710069A - Adenylosuccinate synthetase sample molecule and its coding sequence and use - Google Patents

Abstract

This invention offers a kind of (adenylosuccinatesynthetase like 1 ) -ADSSLl albumen , the polynucleotide of the coded ADSSLl albumen and the method for producing this kind of ADSSLl albumen with recombination technology. This invention also discloses the use of coding the said ADSSLl albumen. ADSSLl albumen has the function to catalyse the formation of adenylosuccinic acid.

Description

Adenylosuccinate synthetase sample molecule and encoding sequence thereof and purposes

Technical field

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding human adenylosuccinate synthetase like 1 (adenylosuccinate synthetase like 1 abbreviates " ADSSL1 " as), and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.Polypeptide of the present invention is a kind of new adenylosuccinate synthetase molecule.

Background technology

Enzyme is the biomacromolecule that a class has catalytic activity and special space conformation in the biomass cells, and the function of keeping normal intracellular matter metabolism, adjusting cell is played an important role.Along with human genome mapping and order-checking are near completion, seek and find new enzyme molecule, inquire into its genetic expression and change the relation that takes place with disease, the popular topic that has become that modern biochemistry studies with regulation and control, research the enzyme activity.The active adjusting of enzyme relates to a lot of aspects, and wherein, more naturally occurring enzyme inhibitorss play important effect in the organism.

Adenylosuccinate synthetase (adenylosuccinate synthetase, AdSS, EC 6.3.4.4)) can catalysis GTP, IMP, L-Asp generates adenylosuccinic acid, and this enzyme is a high conservative, the enzyme of different sources comprises from germ and its homology of Mammals very high (40%-60%), there be the isozyme of two kinds of adenylic acid (AMP)s in the vertebrates for the succsinic acid synthetic enzyme, the iso-electric point difference of these two kinds of enzymes, its tissue distribution is also different with kinetic property.Acid type enzyme in liver (AdSS2 also claims non-muscularity) iso-electric point is about 8.9, and is relevant with the de novo synthesis of purine, is first key enzyme in the reaction; Alkali type enzyme (AdSS1 also the claims muscularity) iso-electric point that distributes in muscle tissue (skeletal muscle, cardiac muscle etc.) is about 6, and is relevant with glycolysis-by the purine circulation.

The purine metabolism enzyme that comprises AdSS plays a significant role in cell function and allelotaxis.Have been found that the AdSS1 enzymic activity significantly reduces in the muscle dysfunction disease, comprise congenital myopathy (congenitalmyopathy), not anti-disease of muscular movement (exercise intolerance) and carrying out property system sclerosis (progressive systemic sclerosis) etc., and the AdSS1 enzymic activity obviously increases in myocardial hypertrophy disease.The defective of AdSS and high lithemia state are closely related, and the AdSS of hereditary defect may cause the congenital gout of some type.The immunosuppressor azathioprine that can suppress purine metabolism enzyme such as AdSS can be alleviated the children acute leukemia and prolong the survival of renal transplantation, thereby has been applied to the treatment of pernicious neoplastic hematologic disorder, rheumatosis, solid organ transplantation and inflammatory bowel.AdSS also is the target spot of some microbiotic (comprising hadacidin and alanosine) effect.Utilize alanosine to suppress to bring into play antiviral and antitumor action after the AdSS activity.Come in more to find that these enzymes may make the biomarker of pernicious tumour.Therefore, in enzymic activity and gene regulating level AdSS is studied and to help to be the development of specific tumour chemotherapy means and the development of specificity purine synthetic inhibitor, particularly be applied to the treatment of the prevention of transplant rejection and muscle dysfunction disease, pernicious neoplastic hematologic disorder.

Research shows that AdSS is relevant with multiple vital movement.Therefore, significant for the adenylosuccinate synthetase of diagnosing and the therapeutic purpose research and development is new.

Summary of the invention

The purpose of this invention is to provide a kind of new human adenylate succsinic acid synthetic enzyme, promptly adenylosuccinate synthetase sample molecule (adenylosuccinate synthetase like 1 abbreviates " ADSSL1 " as) 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.

In a first aspect of the present invention, novel isolated ADSSL1 polypeptide is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ IDNO:2 aminoacid sequence.

Preferably, this polypeptide is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have function that the catalysis adenylosuccinic acid forms, by (a) polypeptides derived.

More 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 people ADSSL1 polypeptide 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 60-1430 position among the SEQ ID NO:1; (b) has the sequence of 1-1738 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.

In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people ADSSL1 protein-active, this method comprises: (a) under the proteic condition of suitable expressing human ADSSL1, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people ADSSL1 protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people ADSSL1 polypeptid specificity bonded antibody.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people ADSSL1 polypeptide active is provided, and the compound that suppresses people ADSSL1 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 ADSSL1 polypeptide.

In a seventh aspect of the present invention, provide and whether had the proteic method of ADSSL1 in the test sample, it comprises: sample is contacted with the proteic specific antibody of ADSSL1, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample ADSSL1 albumen.

In a eighth aspect of the present invention, a kind of disease relevant with people ADSSL1 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 ADSSL1 polypeptide active, and perhaps screening suppresses the antagonist of people ADSSL1 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people ADSSL1 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 composition is provided, it contains ADSSL1 polypeptide of the present invention and the acceptable carrier of 0.001-99.99%.A kind of preferred compositions is a pharmaceutical composition, and it contains people ADSSL1 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 the not anti-disease of congenital myopathy, muscular movement, carrying out property system sclerosis, myocardial hypertrophy disease, high lithemia, muscle dysfunction disease, pernicious neoplastic hematologic disorder and transplant rejection respectively.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Description of drawings

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 has shown the nucleotide sequence (SEQ ID NO:1) of people AdSSL1, and wherein ORF is positioned at the 60-1430 position.

Fig. 2 has shown the aminoacid sequence (SEQ ID NO:2) of people AdSSL1.

Fig. 3 has shown the RT-PCR expression analysis of people AdSSL1 in different cells.Prompting AdSSL1 is expressed in noumenal tumour and the blood tumor cell.Wherein, Marker is the molecular weight standard thing.

Fig. 4 has shown people AdSSL1 Northern engram analysis result in health adult tissue.Results suggest AdSSL1 has predominant expression in muscle tissue such as skeletal muscle, cardiac muscle.

Fig. 5 has shown that the Western that the carrier for expression of eukaryon of people AdSSL1 is expressed analyzes in eukaryotic cell.Wherein, swimming lane 1: the COS-7 cell of the carrier for expression of eukaryon transfection of people AdSSL1; Swimming lane 2: the COS-7 cell of blank carrier transfection.

Fig. 6 has shown that the enzymic activity of people AdSSL1 detects test-results.*P＜0.01

Embodiment

Extensive studies is isolated a kind of novel alkali type adenylosuccinate synthetase to the inventor first from human bone marrow substrate cell cDNA library, AdSSL1 by going deep into.The alkali type AdSS of AdSSL1 and known various source of species has higher homology, and iso-electric point is 8.76, the expression that in muscle tissue, has superiority, and have the AdSS enzymic activity that typical catalysis adenylosuccinic acid forms.This shows that the AdSSL1 molecule may be a kind of novel alkali type adenylosuccinate synthetase, and can be similar with other AdSS, performance important regulating and controlling effect in the growth course of cell and tissue, and may be worth having important development and application aspect the immunodiagnosis in a plurality of fields such as antitumor, resisting transplant rejection, anti-musculature disorder and anti-inflammatory response and the immunotherapy.

In the present invention, term " ADSSL1 albumen ", " ADSSL1 polypeptide " or " adenylosuccinate synthetase sample molecule ADSSL1 " are used interchangeably, all refer to the to have human adenylosuccinate synthetase like 1 ADSSL1 aminoacid sequence albumen or the polypeptide of (SEQ ID NO:2).They comprise the adenylosuccinate synthetase sample molecule ADSSL1 that contains or do not contain initial methionine.

As used herein, " isolating " is 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 ADSSL1 albumen or polypeptide " is meant that the ADSSL1 polypeptide 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 ADSSL1 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of ADSSL1 polypeptide can be used amino acid sequence analysis.

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 the proteic fragment of people ADSSL1, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human ADSSL1 albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

In the present invention, term " people ADSSL1 polypeptide " refers to have the SEQ ID NO:2 polypeptide of sequence of people ADSSL1 protein-active.This term also comprises having and variant form people ADSSL1 albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people ADSSL1 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people ADSSL1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people ADSSL1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people ADSSL1 polypeptide or its segmental fusion rotein (as the fusion rotein that forms with GST).Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people ADSSL1 polypeptide.Usually, this fragment have people ADSSL1 peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of people ADSSL1 albumen or polypeptide.The difference of these analogues and natural human ADSSL1 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

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

In the present invention, " people ADSSL1 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

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

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 " is meant that in the present invention coding has the protein of SEQID 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 of an encoding 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 " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, 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 above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.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 is added with 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 90%, be more preferably 95% 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 above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding ADSSL1.

Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.

People ADSSL1 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 ADSSL1 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 ADSSL1 polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people ADSSL1 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 ADSSL1 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 ADSSL1 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory 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; Lambda particles phage PL promotor; 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 ADSSL1 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 ADSSL1 protein function as pharmacological agent ADSSL1 protein function.The peptide molecule that can suppress or stimulate people ADSSL1 protein function that can be used for seeking therapeutic value with the recombinant human ADSSL1 protein screening peptide library of expressing.

On the other hand, the present invention also comprises people ADSSL1DNA 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 ADSSL1 gene product or fragment.Preferably, refer to that those can combine with people ADSSL1 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 ADSSL1, comprise that also those do not influence the antibody of people ADSSL1 protein function.The present invention also comprise those can with modify or without the people ADSSL1 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 ADSSL1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human ADSSL1 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 prepare (see people such as Kohler, Nature256; 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 Mon degree centigrade of lonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people ADSSL1 protein function and the antibody that does not influence people ADSSL1 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people ADSSL1 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 ADSSL1 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 ADSSL1 can be used in the immunohistochemistry technology, detects the people ADSSL1 albumen in the biopsy specimen.

Antibody among the present invention can be used for treating or prevention and the relevant disease of people ADSSL1 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people ADSSL1 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 ADSSL1 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 of people ADSSL1 protein positive.

The production of polyclonal antibody can choose ADSSL1 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 ADSSL1 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.

Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of muscle dysfunction disease, pernicious neoplastic hematologic disorder and transplant rejection aspect.When using ADSSL1 albumen of the present invention, also can use the other treatment agent simultaneously, as TNF etc.

The present invention also provides a kind of pharmaceutical composition, and it contains ADSSL1 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount (as 0.01-99.99%).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 ADSSL1 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 ADSSL1 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 ADSSL1 of the proteic nothing expression of ADSSL1 or unusual/non-activity.The ADSSL1 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic ADSSL1 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the ADSSL1 transgenosis to cell.The method that structure carries the recombinant viral vector of ADSSL1 gene is found in existing document (Sambrook, et al.).Recombinant human ADSSL1 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 ADSSL1 mRNA 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 ADSSL1 obtains.During screening, must carry out mark to people ADSSL1 protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people ADSSL1 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people ADSSL1 protein level that is detected in the test can be with laying down a definition the importance of people ADSSL1 albumen in various diseases and be used to the disease of diagnosing ADSSL1 albumen to work.

Whether having the proteic method of ADSSL1 in a kind of detection test sample is to utilize the proteic specific antibody of ADSSL1 to detect, and it comprises: sample is contacted with the ADSSL1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample ADSSL1 albumen.

The proteic polynucleotide of ADSSL1 can be used for the diagnosis and the treatment of ADSSL1 protein related diseases.Aspect diagnosis, the proteic polynucleotide of ADSSL1 can be used for detecting the proteic expression of ADSSL1 ADSSL1 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of ADSSL1 as the ADSSL1 dna sequence dna.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 ADSSL1 albumen and also can detect the proteic transcription product of ADSSL1.

The sudden change that detects the ADSSL1 gene also can be used for the disease of diagnosing ADSSL1 albumen relevant.The form of ADSSL1 protein mutation comprises that the point mutation compared with normal wild type ADSSL1DNA 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 ADSSL1 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 Inheritancein 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.

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 bone marrow substrate cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1738 bases, and its open reading frame is positioned at the 60-1430 position, and the coding total length is 457 amino acid whose people AdSSL1 albumen (SEQID NO:2).This AdSSL1 albumen belongs to adenylosuccinate synthetase, and adenylosuccinate synthetase is the important enzyme in the purine metabolism, plays important effect in cell function, tissue development and homeostasis, thereby has 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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the clone of people AdSSL1 cDNA

Extract the total RNA of human bone marrow substrate cell with Trizol reagent (Life Technologies company).Then, from total RNA, separate poly (A) mRNA.With poly (A) mRNA after reverse transcription forms cDNA, clone test kit (Life Technologies) with SuperScriptII cDNA fragment orientation is inserted on the multiple clone site of carrier, transform bacillus coli DH 5 alpha bacterium commonly used 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 (as SEQ ID NO:1 and shown in Figure 1), the new protein (as SEQ ID NO:2 and shown in Figure 2) of encoding.This protein is named as human adenylosuccinate synthetase like 1, its encoding gene called after human adenylosuccinate synthetase like 1 gene.

Sequence SEQ ID NO:1 total length is 1738bp, comprises 3 ' end non-coding region of 5 of 59bp ' end non-coding region and 305bp, and coding contains 457 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 50.2kD.Structural analysis shows that the proteic 32-455 of AdSSL1 position comprises an AdSS architecture signals (AdSS signature), in the 42-48 position and the 362-365 position comprise GTP binding domains (GTP-bindingdomain), the prompting AdSSL1 belong to adenylosuccinate synthetase.

They are different with known for the BLAST analysis revealed, with mouse adenylosuccinate synthetase 1 apparent altitude homology.In addition, AdSSL1 and other AdSS molecules such as people AdSS, mouse AdSS2, rat AdSS and yeast AdSS albumen also show the homology of certain level.

Embodiment 2: carry out the cell expressing analysis of people AdSSL1 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 g Oligo-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.Pcr amplification AdSSL1 primer is as follows: adopted primer 5 '-ATGTCGGGGACCCGAGCC-3 ' (SEQID NO:3) is arranged, antisense primer 5 '-CTAAAACAGCTGGATCAT-3 ' (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, 0.2mMdNTP and 1U rTaq archaeal dna polymerase (Takara), 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 60-1430 shown in the SEQ ID NO:1 are identical.

RT-PCR result as shown in Figure 3, AdSSL1 mRNA all sees Table in all solid tumor cells that detect and blood tumor cell system and reaches, wherein high expression level in tumour cells such as KG-1, SMMC7721, MCF-7 and CaoV-3.

The Northern engram analysis of embodiment 3 people AdSSL1

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 is as shown in Figure 4: the Northern trace is presented at that people AdSSL1 mRNA is a 2.2kb band in the health adult tissue, and high expression level in muscle tissue such as skeletal muscle, heart is expressed very low in its hetero-organization.This show people AdSSL1 be a kind of in muscle tissue the molecule of predominant expression.

Embodiment 4 people AdSSL1 are recombinant expressed

In this embodiment, the human bone marrow substrate cell cDNA of reverse transcription is a template, and 5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people AdSSL1 DNA as inserting fragment.

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

5’-CAGGATCCATGTCGGGGACCCGAGCC-3’(SEQ?ID?NO：5)

This primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence of translation initiation and people AdSSL1 after this restriction enzyme site;

3 ' end primer sequence is:

5’-GTGAATTC?CTAAAACAGCTGGATCAT-3’(SEQ?ID?NO：6)

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people AdSSL1 of EcoR I restriction enzyme.

With the PCR product purification that obtains after BamHI-EcoR I enzyme cut and recombinate according to a conventional method with plasmid pGEM-3ZF (available from Promega company) 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 AdSSL1 cDNA BamHI-EcoR I endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T (Pharmacia company), forms carrier pGEX-2T-AdSSL1, then transformed into escherichia coli BL21.Positive colony is cut evaluation with the BamHI-EcoRI enzyme, the capable 0.8% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted designed AdSSL1 encoding sequence.

Choosing the positive e. coli bl21 clone who expresses AdSSL1 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 degrees centigrade 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 ₄PH 7.3) resuspended, add 20%Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, 4 ℃ of centrifugal 10min of 12000g then, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, add 500ul gsh elution buffer (10mM gsh, 50mM Tris-HCl, pH 8.0) room temperature leaves standstill after 30 minutes and collects elutriant, repeats wash-out 2-3 time, obtains people AdSSL1-GST fusion rotein.The molecular weight of fusion rotein conforms to predictor.

Embodiment 5: anti-people AdSSL1 production of antibodies

The recombinant human AdSSL1 fusion rotein 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 AdSSL1 gene translation product with it.

Found that antibody can combine with albumen of the present invention specifically.

Embodiment 6: people AdSSL1 Construction of eukaryotic and gene transfection

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 AdSSL1 full length coding region DNA as inserting fragment.The PCR reaction parameter be 95 ℃ 15 seconds, 58 ℃ 30 seconds, 72 ℃ 30 seconds, 20 circulations were extended 10 minutes for back 72 ℃,

Upstream primer is 5 '-(5 ' end contains EcoR I site to AC GAA TTC ATGTCGGGGACCCGAGCC-3 ', and the initial code of people AdSSL1 coding region) (SEQ ID NO:7), downstream primer be 5 '-T GAA GCT TAA CAGCTG GAT CAT-3 ' (5 ' end contains Hind III site) (SEQ ID NO:8).

Behind the PCR product purification directly and pGEM-T carrier (Promega company) recombinate according to a conventional method and be converted into the competence bacillus coli DH 5 alpha.Picking white clone identifies, also order-checking (sequencing primer is T7 and SP6) of purifying.With the purified rear clone of EcoR I-Hind III endonuclease bamhi of correct sequence to expression vector pcDNA3.1/Myc-His (-) B (Invitrogen company).After enzyme was cut the evaluation positive colony, recon was designated as pcDNA-people AdSSL1.Confirm through order-checking, inserted designed people AdSSL1 encoding sequence.

People AdSSL1 eukaryotic expression plasmid DNA and control plasmid pcDNA3.1/Myc-His (-) B with LipofectAMINE reagent (Invitrogen company) cotransfection COS-7 African green monkey kidney cell, were detected after the transfection in 48 hours.

Embodiment 7: the Western of people AdSSL1 eukaryotic expression product detects

After the transfection 48 hours, collecting cell (5 * 10 ⁶Cell/sample), wash one time with PBS after, extract reagent (Pierce company) with the T-PER tissue protein and extract whole protein, concrete operations are undertaken by the test kit explanation.Use BCA method (Pierce company) to measure protein concentration afterwards, after being adjusted to unanimity, the per 20 μ l of sample and 6 times of sample-loading buffer (100mmol/L Tris-HCL of 4 μ l, 200mmol/L DTT, 4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine, PH6.8) mixing, after 100 ℃ of water-baths were boiled 5 minutes, row 12%SDS-PAGE electrophoresis moved on to the protein transduction in the polyacrylamide on the nitrocellulose filter in 4 ℃ with the 100V constant voltage subsequently, ponceau dyeing and label orientation mark the Marker position.4 hours (the TBST solution of 10% skim-milk) of room temperature blocking-up, the anti-His tag antibody in mouse source was diluted in the skim-milk solution by 1: 1000, after 2 hours, wash 3 times each 10 minutes in room temperature reaction with TBST (the TBS solution of 0.05%Tween20) with cellulose nitrate film.In addition corresponding two of horseradish peroxidase (HRP) mark anti-(dilutions in 1: 2000) then, incubated at room 60 minutes, TBST gives a baby a bath on the third day after its birth inferior, each 10 minutes, A liquid in the Western trace luciferase assay reagent (Cell Signaling company) and B liquid is mixed with equal-volume, and incubated at room was after 1 minute, dry, press mold, exposure is developed.

Result such as Fig. 5 show.Can detect the people AdSSL1 Recombinant Protein Expression of band His label in the COS-7 African green monkey kidney cell of people AdSSL1 carrier for expression of eukaryon transfection, molecular weight is about 52Kd, conforms to the calculating molecular weight of people AdSSL1.And in control plasmid pcDNA3.1/Myc-His (-) B cells transfected, do not detect expression.

Embodiment 8: the active detection of the adenylosuccinate synthetase of people AdSSL1

Utilize people AdSSL1 total length recombinant protein recombinant expressed among the embodiment 4, set up AdSS enzymic activity detection architecture, comprise 10mM MgCI in the detection damping fluid (20mM Hepes, pH 8.0) ₂, 60 μ M GTP, 150 μ MIMP, 25mM L-Asp.Begin reaction after adding reorganization AdSSL1 albumen or GST albumen in contrast, under 25 ℃, 280nm condition, by detecting the formation (ε of people AdSSL1 albumen catalysis adenylosuccinic acid ₂₈₀11.7mM ^-1.cm ^-1) reflect the adenylosuccinate synthetase activity.

Result such as Fig. 6 show.Recombinant full-lenght people AdSSL1 albumen has the adenylosuccinate synthetase activity of medium level, is 0.2 μ mol/min/mg albumen, and contrast GST albumen does not have this enzymic activity.Prompter AdSSL1 albumen has typical adenylosuccinate synthetase activity.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Inst. of Immunology, Zhejiang Univ.

＜120〉adenylosuccinate synthetase sample molecule and encoding sequence thereof and purposes

<130>042151

<160>8

<170>PatentIn?version?3.1

<210>1

<211>1738

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(60)..(1430)

<223>

<400>1

cggacgccgg?cggcggcggg?ctcctggccg?ggccagcgca?gcggaagagc?caagccagc????59

atg?tcg?ggg?acc?cga?gcc?tcc?aac?gac?cgg?ccc?ccc?ggc?gca?ggc?ggc????107

Met?Ser?Gly?Thr?Arg?Ala?Ser?Asn?Asp?Arg?Pro?Pro?Gly?Ala?Gly?Gly

1???????????????5???????????????????10??????????????????15

gtc?aag?cgg?ggg?cgg?ctg?cag?cag?gag?gcg?gcg?gcg?acc?ggc?tcc?cgc????155

Val?Lys?Arg?Gly?Arg?Leu?Gln?Gln?Glu?Ala?Ala?Ala?Thr?Gly?Ser?Arg

20??????????????????25??????????????????30

gtg?acg?gtg?gtg?ctg?ggc?gcg?cag?tgg?ggg?gac?gag?ggc?aaa?ggc?aag????203

Val?Thr?Val?Val?Leu?Gly?Ala?Gln?Trp?Gly?Asp?Glu?Gly?Lys?Gly?Lys

35??????????????????40??????????????????45

gtg?gtg?gac?ctg?ctg?gcc?acg?gac?gcc?gac?atc?atc?agc?cgc?tgc?cag????251

Val?Val?Asp?Leu?Leu?Ala?Thr?Asp?Ala?Asp?Ile?Ile?Ser?Arg?Cys?Gln

50??????????????????55??????????????????60

ggg?ggc?aac?aac?gcc?ggc?cac?acg?gtg?gtg?gtg?gat?ggg?aaa?gag?tac????299

Gly?Gly?Asn?Asn?Ala?Gly?His?Thr?Val?Val?Val?Asp?Gly?Lys?Glu?Tyr

65??????????????????70??????????????????75??????????????????80

gac?ttc?cac?ctg?ctg?ccc?agc?ggc?atc?atc?aac?acc?aag?gcc?gtg?tcc????347

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

85??????????????????90??????????????????95

ttc?att?ggc?aac?ggg?gtg?gtc?atc?cac?ttg?cca?ggc?ttg?ttt?gag?gaa????395

Phe?Ile?Gly?Asn?Gly?Val?Val?Ile?His?Leu?Pro?Gly?Leu?Phe?Glu?Glu

100?????????????????105?????????????????110

gca?gag?aag?aat?gaa?aag?aaa?ggc?ctg?aag?gac?tgg?gag?aag?agg?ctc????443

Ala?Glu?Lys?Asn?Glu?Lys?Lys?Gly?Leu?Lys?Asp?Trp?Glu?Lys?Arg?Leu

115?????????????????120?????????????????125

atc?atc?tct?gac?aga?gcc?cac?ctt?gtg?ttt?gat?ttt?cac?cag?gct?gtc????491

Ile?Ile?Ser?Asp?Arg?Ala?His?Leu?Val?Phe?Asp?Phe?His?Gln?Ala?Val

130?????????????????135?????????????????140

gac?gga?ctt?cag?gaa?gtg?cag?cgc?cag?gca?caa?gag?ggg?aag?aat?ata????539

Asp?Gly?Leu?Gln?Glu?Val?Gln?Arg?Gln?Ala?Gln?Glu?Gly?Lys?Asn?Ile

145?????????????????150?????????????????155?????????????????160

ggc?acc?acc?aag?aag?gga?atc?gga?cca?acc?tac?tct?tcc?aaa?gct?gcc????587

Gly?Thr?Thr?Lys?Lys?Gly?Ile?Gly?Pro?Thr?Tyr?Ser?Ser?Lys?Ala?Ala

165?????????????????170?????????????????175

cgg?aca?ggc?ctc?cgc?atc?tgc?gac?ctc?ctg?tca?gat?ttt?gat?gag?ttt????635

Arg?Thr?Gly?Leu?Arg?Ile?Cys?Asp?Leu?Leu?Ser?Asp?Phe?Asp?Glu?Phe

180?????????????????185?????????????????190

tcc?tcc?aga?ttc?aag?aac?ctg?gcc?cac?cag?cac?cag?tcg?atg?ttc?ccc????683

Ser?Ser?Arg?Phe?Lys?Asn?Leu?Ala?His?Gln?His?Gln?Ser?Met?Phe?Pro

195?????????????????200?????????????????205

acc?ctg?gaa?ata?gac?att?gaa?ggc?caa?ctc?aaa?agg?ctc?aag?ggc?ttt????731

Thr?Leu?Glu?Ile?Asp?Ile?Glu?Gly?Gln?Leu?Lys?Arg?Leu?Lys?Gly?Phe

210?????????????????215?????????????????220

gct?gag?cgg?atc?aga?ccc?atg?gtc?cga?gat?ggt?gtt?tac?ttt?atg?tat????779

Ala?Glu?Arg?Ile?Arg?Pro?Met?Val?Arg?Asp?Gly?Val?Tyr?Phe?Met?Tyr

225?????????????????230?????????????????235?????????????????240

gag?gca?ctc?cac?ggc?ccc?ccc?aag?aag?atc?ctg?gtg?gag?ggt?gcc?aac????827

Glu?Ala?Leu?His?Gly?Pro?Pro?Lys?Lys?Ile?Leu?Val?Glu?Gly?Ala?Asn

245?????????????????250?????????????????255

gcc?gcc?ctc?ctc?gac?att?gac?ttc?ggg?acc?tac?ccc?ttt?gtg?act?tca????875

Ala?Ala?Leu?Leu?Asp?Ile?Asp?Phe?Gly?Thr?Tyr?Pro?Phe?Val?Thr?Ser

260?????????????????265?????????????????270

tcc?aac?tgc?acc?gtg?ggc?ggt?gtg?tgc?acg?ggc?ctg?ggc?atc?ccc?ccg????923

Ser?Asn?Cys?Thr?Val?Gly?Gly?Val?Cys?Thr?Gly?Leu?Gly?Ile?Pro?Pro

275?????????????????280?????????????????285

cag?aac?ata?ggt?gac?gtg?tat?ggc?gtg?gtg?aaa?gcc?tat?acc?aca?cgt????971

Gln?Asn?Ile?Gly?Asp?Val?Tyr?Gly?Val?Val?Lys?Ala?Tyr?Thr?Thr?Arg

290?????????????????295?????????????????300

gtg?ggc?atc?ggg?gcc?ttc?ccc?acc?gag?cag?atc?aac?gag?att?gga?ggc????1019

Val?Gly?Ile?Gly?Ala?Phe?Pro?Thr?Glu?Gln?Ile?Asn?Glu?Ile?Gly?Gly

305?????????????????310?????????????????315?????????????????320

ctg?ctg?cag?acc?cgc?ggc?cac?gag?tgg?gga?gtg?acc?aca?ggc?agg?aag????1067

Leu?Leu?Gln?Thr?Arg?Gly?His?Glu?Trp?Gly?Val?Thr?Thr?Gly?Arg?Lys

325?????????????????330?????????????????335

agg?cgc?tgc?ggc?tgg?ctc?gac?ctg?atg?att?cta?aga?tat?gct?cac?atg????1115

Arg?Arg?Cys?Gly?Trp?Leu?Asp?Leu?Met?Ile?Leu?Arg?Tyr?Ala?His?Met

340?????????????????345?????????????????350

gtc?aac?gga?ttc?act?gcg?ctg?gcc?ctg?acg?aag?ctg?gac?atc?ctg?gac????1163

Val?Asn?Gly?Phe?Thr?Ala?Leu?Ala?Leu?Thr?Lys?Leu?Asp?Ile?Leu?Asp

355?????????????????360?????????????????365

gta?ctg?ggt?gag?gtt?aaa?gtc?ggt?gtc?tca?tac?aag?ctg?aac?ggg?aaa????1211

Val?Leu?Gly?Glu?Val?Lys?Val?Gly?Val?Ser?Tyr?Lys?Leu?Asn?Gly?Lys

370?????????????????375?????????????????380

agg?att?ccc?tat?ttc?cca?gct?aac?cag?gag?atg?ctt?cag?aag?gtc?gaa????1259

Arg?Ile?Pro?Tyr?Phe?Pro?Ala?Asn?Gln?Glu?Met?Leu?Gln?Lys?Val?Glu

385?????????????????390?????????????????395?????????????????400

gtt?gag?tat?gaa?acg?ctg?cct?ggg?tgg?aaa?gca?gac?acc?aca?ggc?gcc????1307

Val?Glu?Tyr?Glu?Thr?Leu?Pro?Gly?Trp?Lys?Ala?Asp?Thr?Thr?Gly?Ala

405?????????????????410?????????????????415

agg?agg?tgg?gag?gac?ctg?ccc?cca?cag?gcc?cag?aac?tac?atc?cgc?ttt????1355

Arg?Arg?Trp?Glu?Asp?Leu?Pro?Pro?Gln?Ala?Gln?Asn?Tyr?Ile?Arg?Phe

420?????????????????425?????????????????430

gtg?gag?aat?cac?gtg?gga?gtc?gca?gtc?aaa?tgg?gtt?ggt?gtt?ggc?aag????1403

Val?Glu?Asn?His?Val?Gly?Val?Ala?Val?Lys?Trp?Val?Gly?Val?Gly?Lys

435?????????????????440?????????????????445

tca?aga?gag?tcg?atg?atc?cag?ctg?ttt?tagtcgcaga?ctgagctgat??????????1450

Ser?Arg?Glu?Ser?Met?Ile?Gln?Leu?Phe

450?????????????????455

cccaacaggc?cctggcagcg?tctggacttg?tgtaaacagc?agcagtcacg?ttcctcggcc??1510

gccacaacca?acaccaaagc?aggaaaacca?ttttctgtac?ttttatattt?ctgttcaacc??1570

tgttggtttt?tacaatgatt?ttaaacattg?gaaagccagc?cttgtgtata?tttttaaaaa??1630

ttatattcaa?aatgagccaa?agtgctcaga?gaccttctat?gacacattag?tgtcacatgg??1690

ttgcgtgtcc?agccgaagca?gtgtaataaa?catctccaat?ggcccctg???????????????1738

<210>2

<211>457

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met?Ser?Gly?Thr?Arg?Ala?Ser?Asn?Asp?Arg?Pro?Pro?Gly?Ala?Gly?Gly

1???????????????5???????????????????10??????????????????15

Val?Lys?Arg?Gly?Arg?Leu?Gln?Gln?Glu?Ala?Ala?Ala?Thr?Gly?Ser?Arg

20??????????????????25??????????????????30

Val?Thr?Val?Val?Leu?Gly?Ala?Gln?Trp?Gly?Asp?Glu?Gly?Lys?Gly?Lys

35??????????????????40??????????????????45

Val?Val?Asp?Leu?Leu?Ala?Thr?Asp?Ala?Asp?Ile?Ile?Ser?Arg?Cys?Gln

50??????????????????55??????????????????60

Gly?Gly?Asn?Asn?Ala?Gly?His?Thr?Val?Val?Val?Asp?Gly?Lys?Glu?Tyr

65??????????????????70??????????????????75??????????????????80

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

85??????????????????90??????????????????95

Phe?Ile?Gly?Asn?Gly?Val?Val?Ile?His?Leu?Pro?Gly?Leu?Phe?Glu?Glu

100?????????????????105?????????????????110

Ala?Glu?Lys?Asn?Glu?Lys?Lys?Gly?Leu?Lys?Asp?Trp?Glu?Lys?Arg?Leu

115?????????????????120?????????????????125

lle?Ile?Ser?Asp?Arg?Ala?His?Leu?Val?Phe?Asp?Phe?His?Gln?Ala?Val

130?????????????????135?????????????????140

Asp?Gly?Leu?Gln?Glu?Val?Gln?Arg?Gln?Ala?Gln?Glu?Gly?Lys?Asn?Ile

145?????????????????150?????????????????155?????????????????160

Gly?Thr?Thr?Lys?Lys?Gly?Ile?Gly?Pro?Thr?Tyr?Ser?Ser?Lys?Ala?Ala

165?????????????????170?????????????????175

Arg?Thr?Gly?Leu?Arg?Ile?Cys?Asp?Leu?Leu?Ser?Asp?Phe?Asp?Glu?Phe

180?????????????????185?????????????????190

Ser?Ser?Arg?Phe?Lys?Asn?Leu?Ala?His?Gln?His?Gln?Ser?Met?Phe?Pro

195?????????????????200?????????????????205

Thr?Leu?Glu?Ile?Asp?Ile?Glu?Gly?Gln?Leu?Lys?Arg?Leu?Lys?Gly?Phe

210?????????????????215?????????????????220

Ala?Glu?Arg?Ile?Arg?Pro?Met?Val?Arg?Asp?Gly?Val?Tyr?Phe?Met?Tyr

225?????????????????230?????????????????235?????????????????240

Glu?Ala?Leu?His?Gly?Pro?Pro?Lys?Lys?Ile?Leu?Val?Glu?Gly?Ala?Asn

245?????????????????250?????????????????255

Ala?Ala?Leu?Leu?Asp?Ile?Asp?Phe?Gly?Thr?Tyr?Pro?Phe?Val?Thr?Ser

260?????????????????265?????????????????270

Ser?Asn?Cys?Thr?Val?Gly?Gly?Val?Cys?Thr?Gly?Leu?Gly?Ile?Pro?Pro

275?????????????????280?????????????????285

Gln?Asn?Ile?Gly?Asp?Val?Tyr?Gly?Val?Val?Lys?Ala?Tyr?Thr?Thr?Arg

290?????????????????295?????????????????300

Val?Gly?Ile?Gly?Ala?Phe?Pro?Thr?Glu?Gln?Ile?Asn?Glu?Ile?Gly?Gly

305?????????????????310?????????????????315?????????????????320

Leu?Leu?Gln?Thr?Arg?Gly?His?Glu?Trp?Gly?Val?Thr?Thr?Gly?Arg?Lys

325?????????????????330?????????????????335

Arg?Arg?Cys?Gly?Trp?Leu?Asp?Leu?Met?Ile?Leu?Arg?Tyr?Ala?His?Met

340?????????????????345?????????????????350

Val?Asn?Gly?Phe?Thr?Ala?Leu?Ala?Leu?Thr?Lys?Leu?Asp?Ile?Leu?Asp

355?????????????????360?????????????????365

Val?Leu?Gly?Glu?Val?Lys?Val?Gly?Val?Ser?Tyr?Lys?Leu?Asn?Gly?Lys

370?????????????????375?????????????????380

Arg?Ile?Pro?Tyr?Phe?Pro?Ala?Asn?Gln?Glu?Met?Leu?Gln?Lys?Val?Glu

385?????????????????390?????????????????395?????????????????400

Val?Glu?Tyr?Glu?Thr?Leu?Pro?Gly?Trp?Lys?Ala?Asp?Thr?Thr?Gly?Ala

405?????????????????410?????????????????415

Arg?Arg?Trp?Glu?Asp?Leu?Pro?Pro?Gln?Ala?Gln?Asn?Tyr?Ile?Arg?Phe

420?????????????????425?????????????????430

Val?Glu?Asn?His?Val?Gly?Val?Ala?Val?Lys?Trp?Val?Gly?Val?Gly?Lys

435?????????????????440?????????????????445

Ser?Arg?Glu?Ser?Met?Ile?Gln?Leu?Phe

450?????????????????455

<210>3

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

atgtcgggga?cccgagcc????????????????????????????????????????????????18

<210>4

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

ctaaaacagc?tggatcat????????????????????????????????????????????????18

<210>5

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>5

caggatccat?gtcggggacc?cgagcc???????????????????????????????????????26

<210>6

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>6

gtgaattcct?aaaacagctg?gatcat???????????????????????????????????????26

<210>7

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>7

acgaattcat?gtcggggacc?cgagcc???????????????????????????????????????26

<210>8

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>8

tgaagcttaa?cagctggatc?at???????????????????????????????????????????22

Claims (10)

1. isolating people ADSSL1 polypeptide is characterized in that this polypeptide is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have function that the catalysis adenylosuccinic acid forms, by (a) polypeptides derived.

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 is selected from down group:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(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 60-1430 position among the SEQ ID NO:1;

(b) has the sequence of 1-1738 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. the preparation method of a peptide species is characterized in that, this method comprises:

(a) under conditions suitable for the expression, cultivate the described host cell of claim 7;

(b) from culture, isolate people ADSSL1 protein polypeptide.

9. energy and the described people ADSSL1 of claim 1 polypeptid specificity bonded antibody.

10. a composition is characterized in that, it contains described polypeptide of claim 1 and the acceptable carrier of 0.001-99.99%.

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