CN1315342A - Novel human As resistance related protein and its coding sequence - Google Patents

Abstract

A new polypeptide-hARRG polypeptide, the polynucleotide for coding it, the process for preparing the said polypeptide by recombination technique, the application of said polypeptide in treating several diseases such as cancer, arsenism, etc., especially treating diseases caused by excessive arsenic in human body, the excitomotors and antagonist against the said polypeptide and their therapeutic action, and the application of said polynucleotide for coding this new-hARRG polypeptide are disclosed.

Description

New human arsenic resistant related gene protein and encoding sequence thereof

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---hARRG (humanarsenic resistant related gene, hARRG) polypeptide, 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.The hARRG polypeptide is a kind of As resistance related protein.

Arsenic is the toxic substance that occurring in nature extensively exists, and long-term exposure can cause acute, arsenicalism in high arsenic environment, and the sickness rate of skin carcinoma, lung cancer is raise.In the process of organic evolution, many biologies (from the bacterium to the Mammals) have all produced the physiological mechanism of antagonism arsenic poison, and the generation of these mechanism is often determined by its genetics basis.

The sixties, Novick (Novick, R.P., Rath, C., Plasmid ¨ Dlinked resistance toinorganic salts in Staphylococcus aureus.J.Bacterial.1968,95 (4): 1335 ¨ D42.) and Hedges (Hedges.R..W.Baumkerg.S., Resisitance to arsenic compounds conferredby a plasmid transmissible between strains of Escherichia coli, J.Bacteriol.1973,115 (1): 459 ¨ D60) reported that at first golden yellow staphylococcus and intestinal bacteria have plasmid-mediated arsenic resistance.

Mobley (Mobley, H.L.T., Rosen, B.P.Energetics of plasmid ¨ Dmediated arsenateresistance in Escherichia coli., Proc.Natl.Acad.Sci, 1982,79,6119 ¨ D6122) and Chen (Chen, C.M., Misra, T.K.Silver.S., et.al, Nucleotide sequence of thestructural genes for an anion pump:the plasmid ¨ Dencoded arsenical resistanceoperon.J.Biol.Chem, 1986,261; 150300 ¨ D38) etc. the people finally from arsenic r plasmid R773, separate and determined anti-arsyl because of operon, it by 5 genomic constitutions is in proper order: arsR, arsD, arsA, arsB, arsC.ArsR and arsD are two transcriptional regulatory genes, and arsA, B, C are that anti-arsenic structure gene encodes respectively that the ATP enzyme is conjugated protein, film oxygen anion channel protein and reductase enzyme protein.

1996, Bobrowicz etc. from the plasmid of eukaryote yeast saccharomyces cerevisiae, found three anti-arsyls because of, ACR1, ACR2, ACR3, and its function studied.ACR1 is a regulatory gene, and the ACR2 arsenic reductase enzyme protein of encoding acts on similar to bacterium arsD.The ACR3 gene is regulated by ACR1 gene positive, the oxygen anion transhipment passage of encoding, effect and the similar (Bobrowicz of bacterium arsB, P., Wysocki, R., Owsianik, G., et.al, Isolation of threecontiguous genes, ACR1, ACR2 and ACR3, involved in resistance to arsenic compoundsin the yeast Saccharomyces cerevisiae, Yest, 1997,13,819-828.).The anti-arsenic mechanism of bacterium and zymic is and will enters into intracellular arsenic, discharges cell by film oxygen anion transhipment passage.Need before this pentavalent arsenic is reduced to trivalent arsenic.

Experiment showed, in the mammalian body also exist can inductive to discharge mechanism (Bencko, the V. of arsenic, Symon, K., The cumulation dynamics in some tissue of hairless mice inhaling arsenic, Atmos.Environ, 1970,4:157 ¨ D161).Wang and Rossman (Wang, Z., Rossman, T.G., Stable andInducible Arsenite Resistance in Chinese Hamster V79 cell.Toxicl.App.Pharmacl.1993,118:80 ¨ D86) but in reported first in 1993 Chinese hamster V79 cell induce the anti-arsenic cell strain As/R28A of the genetic stability of foundation with arsenic.From this cell strain, isolate two the cDNA fragment with anti-arsenic feature: asr1 subsequently, asr2, after these two fragments are each separately transfected into arsenic sensitive cells As/S5 and wild-type V79 cell, all can make two class cells obtain certain arsenic resistance (Rossman, T.G., Wang, Z., Expression cloning for arsenite-resistanceresulted in isolation of tumor-suppressor fau cDNA:possible involvement of theubiquitin system in arsenic carcinogenesis.Carcinogenesis.1999,20 (2): 311 ¨ D316.).Find asr1 and hamster tumor suppressor gene fau height homology through dna sequence analysis.The asr2 gene is not found and known any dna homolog.This full length gene 1041bp, open reading frame 678bp, 225 the amino acid whose albumen of encoding.

Human anti-arsyl is not delivered because of seeing the research article in the world as yet, has announced two gene orders in the gene database (GeneBank), because of with asr2 homology height be speculated as human anti-arsyl because of.The Hu Gengxi of Shanghai cell institute has logined a human asr2 homologous gene (searching number af082871) total length 1423bp in March, 1999,425 the amino acid whose albumen of encoding.Soon German scholar Wambutt subsequently, D etc. have logined a longer human asr2 homologous gene (searching number hsm800627), total length 2983bp, 791 the amino acid whose albumen of encoding June in the same year.Sequential analysis finds that these two genes are actually same gene, and just the latter is longer than the former, and frame is more complete.

Research shows, arsenic content unusual relevant with some diseases, and therefore, to research and develop the anti-arsenic albumen of people significant for therapeutic purpose.Yet, before the application, do not have to disclose or reported hARRG albumen involved in the present invention or its encoding sequence.

An object of the present invention is to provide isolating new polypeptide---hARRG (human arsenic resistantrelated gene, hARRG) polypeptide 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 hARRG polypeptide.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain coding hARRG polypeptide.

Another object of the present invention provides the method for producing the hARRG polypeptide.

Another object of the present invention provides at polypeptide of the present invention---the antibody of hARRG polypeptide.

Another object of the present invention provides the pharmaceutical composition that contains the hARRG polypeptide.

Another object of the present invention has provided at polypeptide of the present invention---the simulated compound of hARRG polypeptide, antagonist, agonist, inhibitor.

Another object of the present invention provides the method for the diagnosis disease relevant unusually with the hARRG polypeptide with treatment.

The inventor has carried out nearly 2 years research to human asr2 homologous gene, be cloned into the human asr2 homologous gene of total length, this gene order has been grown 24bp than the sequence that Wambutt announces at 5 ' end, and is just more complete than it, and sequence of the present invention has bigger different at Liang Chu with it.Analysis revealed, this sequence of the present invention is the different shear-forms of same gene, thereby be named as human anti-arsenic genes involved (human arsenic resistant related gene, hARRG).

The present invention at first provides a kind of isolated polypeptide, and this polypeptide is the people source, and it comprises: polypeptide or its conservative property varient, bioactive fragment or derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQID NO:2 aminoacid sequence.

The present invention also provides a kind of isolating polynucleotide, and it comprises a kind of nucleotide sequence or its variant that is selected from down group:

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

(b) with polynucleotide (a) complementary polynucleotide;

(c) with (a) or polynucleotide sequence (b) have the polynucleotide of at least 99.764% homogeny.

More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 500-2866 position among the SEQ ID NO:1; (b) has the sequence of 1-2999 position among the SEQ ID NO:1.

The present invention also provides a kind of carrier that contains polynucleotide of the present invention, particularly expression vector; The host cell that this carrier of a kind of usefulness is genetically engineered comprises the host cell of conversion, transduction or transfection; A kind of method for preparing polypeptide of the present invention of cultivating described host cell and reclaiming expression product that comprises.

The present invention also provide a kind of can with polypeptid specificity bonded antibody of the present invention.

The present invention also provides a kind of simulation, activation, antagonism of screening or has suppressed the method for the active compound of hARRG polypeptide protein, and it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.

The present invention also provides a kind of vitro detection disease relevant with hARRG polypeptide protein unconventionality expression or the method for disease susceptibility, comprise the sudden change in polypeptide described in the detection of biological sample or its coded polynucleotide sequence, perhaps the amount or the biological activity of polypeptide of the present invention in the detection of biological sample.

The present invention also provides a kind of pharmaceutical composition, and it contains polypeptide of the present invention or its stand-in, activator, antagonist or inhibitor and pharmaceutically acceptable carrier.

The invention still further relates to polypeptide of the present invention and/or the polynucleotide disease that arsenic too much causes in preparation is used for the treatment of because of body, comprise that all kinds of inflammation such as arseniasis, malignant tumour, toxic encephalopthy, acute renal failure, acute hemolytic anemia, cardiovascular disorder, immunological disease, tetter and liver, lung, segmental bronchus, neural system or other are because the unusual purposes that causes the medicine of disease of hARRG expression of polypeptides.

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

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 is hARRG polypeptide of the present invention and the proteic amino acid sequence homology comparison diagram of hsm800627.The top sequence is the hARRG polypeptide, and the below sequence is a hsm800627 albumen.With character " | " expression, represent with ": " or ". " by similar amino acid between two sequences for same amino acid.

Fig. 2 has shown hARRG polypeptide of the present invention and the proteic nucleotide sequence homology comparison diagram of hsm800627.The top sequence is the hARRG gene among the figure, classifies the hsm800627 gene down as.Capitalization is that hARRG gene and hsm800627 gene difference, hsm800627 sequence lack a base " G " at the 1684bp place, causes the easy bit-errors of reading frame.

Fig. 3 has shown the Northern trace express spectra of hARRG in the human body different tissues.Wherein, in histoorgans such as the heart, brain, placenta, liver, skeletal muscle, kidney and pancreas expression is arranged all, express lessly in the lung, pancreas, liver and heart are expressed higher.Except the transcript of 3.0Kb, the transcript of a still visible 5.5Kb in liver and pancreas in addition.

Fig. 4 has shown the electrophorogram of the reorganization hARRG polypeptide of prokaryotic expression and purifying.Wherein, swimming lane M is protein molecular weight Marker; Swimming lane 1 is full bacterium lysate; Swimming lane 2 is primary elutriant; Swimming lane 3 is secondary elutriant; Swimming lane 4 is the refined solution first time of hARRG polypeptide; Swimming lane 5 is the refined solution second time of hARRG polypeptide.

The following term of using in this specification sheets and claims has following implication, unless stated otherwise:

" nucleotide sequence " is meant oligonucleotide, Nucleotide or polynucleotide and fragment or part, also can refer to genome or synthetic DNA or RNA, and they can be strand or two strands, represent sense strand or antisense strand.Similarly, term " aminoacid sequence " is meant oligopeptides, peptide, polypeptide or protein sequence and fragment or part.When " aminoacid sequence " among the present invention related to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " did not mean that aminoacid sequence are restricted to the complete natural amino acid relevant with described protein molecule.

Protein or polynucleotide " varient " are meant a kind of polynucleotide sequence that has the aminoacid sequence of one or more amino acid or Nucleotide change or encode it.Described change can comprise disappearance, insertion or the replacement of amino acid in aminoacid sequence or the nucleotide sequence or Nucleotide.Varient can have " conservative property " and change, and wherein the amino acid of Ti Huaning has structure or the chemical property similar with original acid, as replacing Isoleucine with leucine.Varient also can have non-conservation and change, as replacing glycine with tryptophane.

" disappearance " is meant the disappearance of in aminoacid sequence or nucleotide sequence one or more amino acid or Nucleotide.

" insertion " or " interpolation " is meant that the change in aminoacid sequence or nucleotide sequence causes comparing the increase of one or more amino acid or Nucleotide with naturally occurring molecule." replacement " is meant by different amino acid or Nucleotide and replaces one or more amino acid or Nucleotide.

" biological activity " is meant the protein of structure, regulation and control or biochemical function with natural molecule.Similarly, term " immunologic competence " be meant natural, reorganization or synthetic protein and fragment thereof in suitable animal or cell, induce specific immune response and with specific antibody bonded ability.

" agonist " is meant when combining with the hARRG polypeptide, thereby a kind of this protein that causes changes the molecule of regulating this protein active.Agonist can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with the molecule of hARRG polypeptide.

" antagonist " or " inhibition " be meant when combining with the hARRG polypeptide, a kind ofly seals or regulate the biologic activity of hARRG polypeptide or the molecule of immunologic competence.Antagonist and inhibition can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with the molecule of hARRG polypeptide.

" adjusting " is meant that the function of hARRG polypeptide changes, and comprises the change of any other biological property, function or the immune property of the change of the rising of protein active or reduction, binding characteristic and hARRG polypeptide.

" pure basically " is meant and 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 hARRG polypeptide of standard.Basically pure hARRG polypeptide can produce single master tape on the irreducibility polyacrylamide gel.The purity available amino end acid sequence of hARRG polypeptide is analyzed.

" complementary " or " complementation " is meant under salt concn that allows and temperature condition by the natural combination of the polynucleotide of base pairing.For example, sequence " C-T-G-A " can combine with complementary sequence " G-A-C-T ".Complementation between two single chain molecules can be part or whole.Complementary degree between the nucleic acid chains has a significant effect for efficient of hybridizing between the nucleic acid chains and intensity.

" homology " is meant the complementary degree, can be portion homologous or complete homology." portion homologous " is meant a kind of part complementary sequence, and it can partly suppress the hybridization of complete complementary sequence and target nucleic acid at least.The inhibition of this hybridization can detect by hybridizing (Southern trace or Northern trace etc.) under the condition that reduces in the severity degree.Basically homologous sequence or hybridization probe can compete and suppress complete homologous sequence and target sequence the condition that reduces of severity degree under combine.This does not mean the conditions permit non-specific binding that the severity degree reduces because the conditional request two sequences that the severity degree reduces mutual be combined into specificity or selectivity interacts.

" similarity " is meant the degree that the identical or conservative property of corresponding position amino-acid residue when arranging contrast between the aminoacid sequence replaces.For example be used for amino acid that conservative property replaces, electronegative amino acid can comprise aspartic acid and L-glutamic acid; Positively charged amino acid can comprise Methionin and arginine; Having uncharged head group has similar hydrophilic amino acid can comprise leucine, Isoleucine and Xie Ansuan; Glycine and L-Ala; L-asparagine and glutamine; Serine and Threonine; Phenylalanine and tyrosine.

" antisense " is meant and specific DNA or RNA sequence complementary nucleotide sequence." antisense strand " is meant and " sense strand " complementary nucleic acid chains.

" derivative " is meant the hARRG polypeptide or encodes its chemical modification object of nucleic acid.This chemical modification object can be with alkyl, acyl group or the amino hydrogen atom of replacing.The nucleic acid derivative codified keeps the polypeptide of the main biological characteristics of natural molecule.

" antibody " is meant complete antibody molecule and fragment thereof, as Fab, F (ab ') ₂And Fv, its energy specificity is in conjunction with the antigenic determinant of hARRG polypeptide.

" humanized antibody " is meant that the aminoacid sequence in non-antigen binding domain territory is replaced and becomes more similar to people's antibody, but still keep original in active antibody.

" isolating " speech refers to material is shifted out among its original environment (for example, if spontaneous its natural surroundings that just refers to).Such as it is exactly not to be separated that spontaneous polynucleotide or polypeptide are present in the Live Animals, but same polynucleotide or polypeptide with some or all in natural system with it the material of coexistence separately be exactly isolating.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition.Since carrier or composition are not the compositions of its natural surroundings, they remain isolating.

As used herein, " isolating hARRG polypeptide " is meant that the hARRG 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 hARRG polypeptide of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.

The invention provides a kind of new polypeptide---hARRG polypeptide, it is made up of the aminoacid sequence shown in the SEQ ID NO: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 hARRG polypeptide.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of hARRG polypeptide of the present invention or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue) (ⅰ) are arranged, 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) merge 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).According to the instruction of this paper, these fragments, derivative and analogue belong within the known scope of those skilled in the art.

The present invention also provides isolating polynucleotide, and 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.In one embodiment, polynucleotide of the present invention are isolating from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 2999 bases, its open reading frame (500-2886bp) 788 amino acid of having encoded.Find relatively that according to amino acid sequence homologous the anti-arsyl of this polypeptide and Chinese hamster pneumonocyte has 89.82% homology because of asr2, deducibility goes out this new people hARRG and has the similar 26S Proteasome Structure and Function of asr2 gene.Also has anti-arsenic function from the structure explanation.

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 IDNO: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 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 a kind of 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, the length of " nucleic acid fragment " contain 10 Nucleotide at least, better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, 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 hARRG polypeptide.

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

The special polynucleotide sequence of coding hARRG polypeptide 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., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory.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 polymerase chain reaction technique, 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 mensuration hARRG transcript; (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.Dna probe can carry out mark with radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect hARRG polypeptide gene expressed proteins product and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

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 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 or the nucleotide sequence of its various dna fragmentations, and available 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.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 hARRG polypeptid coding sequence 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 hARRG polypeptide 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 hARRG polypeptide and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring HarborLaboratory.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 PL promotor of lambda particles phage; 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 hARRG polypeptide 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 L-02, CHO, COS-7 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.Another kind method is to use MgCl ₂Handle.If 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.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), utilize polynucleotide sequence of the present invention to can be used to express or produce the hARRG polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people hARRG polypeptide, 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 and prevention.For example, this polypeptide can be treated all kinds of inflammation such as arseniasis, toxic encephalopthy, acute renal failure, acute hemolytic anemia, cardiovascular diseases, immunological disease, tetter and liver, lung, segmental bronchus, neural system etc.The above all kinds of diseases that arsenic causes still do not have the specific treatment medicine at present, can detoxify with intracellular arsenic in the body entering and discharge outside cell and the body with the hARRG polypeptide, the concentration that has promptly reduced arsenic in the cell has reduced the toxicity of arsenic again, reaches the purpose of efficient treatment.

This polypeptide and agonist thereof also can be used for the prevention of arseniasis, give the workman of the high arsenic environment of contact and live in the geographic resident of high arsenic and use and can in time discharge the generation that enters intravital arsenic and avoid arseniasis.

This polypeptide and antagonist thereof, agonist and inhibitor also can be used for the assisting therapy of malignant tumour.At present, existing scholar has also obtained significant curative effect with malignant tumours such as arsenic agent treatment leukemia, malignant lymphomas.But because the part patient influences dosage and result of treatment because of arseniasis takes place.Other has the part patient that arsenical is produced resistance and affects the treatment.That available this polypeptide and agonist thereof improve patient's healthy tissues and reduce the susceptibility of the anti-arsenic increase of tumor tissues to arsenic with its antagonist and inhibitor, thus the effect that arsenic is treated malignant tumour improved.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) hARRG.Agonist improves biological functions such as the anti-arsenic of hARRG normal cell, and antagonist reduces the anti-arsenic ability of various normal or improper cells such as cancer cells etc.For example, can under the environment of different concns arsenic, mammalian cell be cultivated with the medicine of intending screening.Form number or cell survivaling number with cell colony under the environment of cell colony forming method or XTT determination of color different concns arsenic then, thereby analyze the ability that medicine improves or check the effect of this hARRG.

The antagonist of hARRG polypeptide comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of hARRG polypeptide can combine and eliminate its function with the hARRG polypeptide, 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, the hARRG polypeptide can be added during bioanalysis measures, determine to interactional influence between hARRG polypeptide 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 hARRG polypeptide bonded peptide molecule obtains.During screening, generally tackle the hARRG peptide molecule 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 hARRG polypeptide antigen determinant.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 hARRG polypeptide of the production of polyclonal antibody direct injection immune animal (as rabbit, mouse, rat etc.) obtains.There is multiple adjuvant to 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 hARRG polypeptide includes but not limited to: and 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-hARRG polypeptide.

The antibody of anti-hARRG polypeptide can be used in the immunohistochemistry technology, detects the hARRG polypeptide in the biopsy specimen.

With the also available labelled with radioisotope of hARRG polypeptide bonded monoclonal antibody, 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 hARRG polypeptide 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 hARRG polypeptide positive cells.

Antibody among the present invention can be used for assisting therapy has the malignant tumour of tolerance to arsenic arsenic agent treatment method, and the antibody that gives suitable dosage can be blocked the generation or the activity of tumour cell hARRG polypeptide.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization hARRG polypeptide level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The hARRG polypeptide level that is detected in the test can be with laying down a definition the importance of hARRG polypeptide in various diseases and be used to the disease of diagnosing the hARRG polypeptide 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 hARRG polypeptide 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 hARRG polypeptide or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the hARRG polypeptide of expressing variation, to suppress endogenic hARRG polypeptide active.For example, a kind of hARRG polypeptide of variation can be the hARRG polypeptide 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 hARRG expression of polypeptides 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 hARRG polypeptide are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding hARRG polypeptide is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding hARRG polypeptide can be packaged in the liposome and then 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 hARRG polypeptide 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.

The polynucleotide of coding hARRG polypeptide can be used for diagnosing the disease relevant with the hARRG polypeptide.The unconventionality expression of the expression that the polynucleotide of coding hARRG polypeptide can be used for detecting hARRG hARRG whether or under morbid state.As the dna sequence dna of the hARRG polypeptide of encoding can be used for biopsy specimen is hybridized to judge the expression situation of hARRG.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 hARRG with the special primer of hARRG.

The sudden change that detects the hARRG polypeptide gene also can be used for the disease of diagnosing the hARRG polypeptide relevant.The form of hARRG sudden change comprises that the point mutation compared with normal wild type hARRG polypeptid DNA 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).Yet, have only chromosomal marker thing seldom to can be used for the marker chromosomes position now 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 Manual of BasicTechniques, 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, Mendelian Inheritance 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.The hARRG polypeptide 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 hARRG polypeptide will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

The constitutional features of hARRG gene order of the present invention: " AAG " 3 bases have been Duoed than the Hsm800627 sequence in hARRG sequence 1410bp place, are " tactccccccag " and lacked 12 its sequences of base at the 2574bp place.Process shows that with the homology analysis that the middle EST fragment of gene pool (GenBank) is carried out these two kinds of variable shear-forms also have existence in the EST that has recorded, and this has just got rid of the wrong possibility of order-checking.Analyze above two variable shear-forms and find that they meet that the shearing site feature of eukaryote intron---end all has " AG ".

HARRG of the present invention has the function of sequence: with the height homologous sequence of hARRG gene, be isolated gene with certain anti-arsenic effect from the anti-arsenic cell strain of Chinese hamster.We studies show that, people's anti-arsyl is especially expressed the abundantest in liver, pancreas because of distribution is all arranged in the great majority tissue.In higher organism, the phenomenon ubiquity of alternative splicing.In different developmental phases and cell type, alternative splicing can make same gene produce the albumen of difference in functionality, thereby regulate cells whose development and physiological function as a kind of gene expression regulation mode.All in open reading frame, can translate becomes different protein to two kinds of different splicing forms of this of this gene, thereby exercises different functions.Because this gene is widely distributed in vivo,, may be the necessary gene of cell metabolism so under normal circumstances except that anti-arsenic effect, necessarily possess some other physiological function.

Studies show that arsenic is a kind of protoplasmic poison material, and can benumb capillary vessel, and make the change of liver fat, central necrosis of hepatic lobule, the heart, liver, kidney, enteremia, epithelium is carefully benumbed the necrosis of blood capillary test-tube baby, telangiectasis.Arsenic can combine the activity that suppresses these enzymes with all enzymes that contains sulfydryl in cell, thereby causes the serious disorder and the obstacle of cellular metabolism.The dysfunction of blood vessel that distinctive neural system and internal organs occur, vascular tone descend, and perviousness increases, and the angioparalysis of brain and internal organs causes the moving obstacle of maincenter and peripheral nervous system and internal organs.Acute poisoning shows as that abdomen strand is painful, diarrhoea, blood urine, shock, central nervous system paralysis, even dead.Also can show as the dysfunction and the inflammation of acute hemolytic anemia and liver, kidney, brain, spinal cord.Chronic poisoning mainly shows as skin hyperkeratosis, vascular lesion, and with the infringement of segmental bronchus, lung liver, intestines.Arsenic also has mutagenesis, teratogenesis, carcinogenesis.International cancer research institution (IARC) confirms that long-term contact arsenic can cause skin carcinoma, lung cancer.The gravid woman contacts arsenic can cause fetal anomaly.Arsenic is chromosomal strong clastogen, can cause that chromosome aberration, reorganization, fracture and micronuclear rates increase.This shows that arsenic is a kind of poisonous element of serious harm human health.Thereby anti-arsyl because of expression product can will enter intracellular arsenic by metabolism detoxifcation and it is discharged extracellular guarantee that cell and body avoid the harm of arsenic.The variable shearing of hARRG will influence the unlatching of the anti-arsenic effect of this albumen and the power of effect.Because arsenic is a kind of carcinogenic substance, the power of anti-arsenic effect directly has influence on arsenic in intracellular concentration, and then influences the time and intensity of arsenic to gene action, thereby might be to transgenation, the certain influence of carcinogenic generation.

As mentioned above, because the hARRG polypeptide protein plays an important role in body critical functions such as antitoxin and metabolism, being separated into of therefore new hARRG polypeptide protein encoding gene determined the effect of this albumen under healthy and morbid state and provided the foundation.

In addition, because people hARRG of the present invention has the natural acid sequence that is derived from the people, therefore, compare, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour with the albumen of the same clan that derives from other species.

Embodiment 1: the clone of human anti-arsenic genes involved (hARRG) cDNA

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 Quick mRNA separating kit (Qiagene 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 and form the cDNA library.Measure the sequence of all clones' 5 ' and 3 ' end with Dye Terminate Cycle Reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencings views (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genbank) measured are compared, found that the cDNA sequence of one of them clone 0233H10 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 0233H10 clone is 2999bp (shown in SEQ ID NO:1), from 500bp to 2866bp the open reading frame (ORF) of a 2364bp is arranged, new 788 amino acid protein of (not comprising terminator codon) (shown in SEQ ID NO:2) that contains of encoding.This clone is named as pBS-233H10, and coded protein is named as the hARRG polypeptide.

Embodiment 2:hARRG cDNA clone's homology retrieval is with sequence and the encoded protein sequence thereof of hARRG of the present invention, with Blast program (Basic local Alignment searchtool) [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 hARRG homology of the present invention is the anti-arsenic protein gene of a kind of known person, and its encoded protein number is hsm800627 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 98.858%; Similarity is 99.112%.Nucleotide homology relatively is shown in Fig. 2, and wherein capitalization is hARRG gene and hsm800627 gene difference, and the hsm800627 sequence lacks a base " G " at the 1684bp place, causes the easy bit-errors of reading frame (homogeny of two sequences is 99.764%).

Embodiment 3: with RT-PCR method clone hARRG gene

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:

Primer 1:5 '-GGCCATTATGGCCGGGGGGAGGCG-3 ' (SEQ ID NO:3)

Primer 2: 5 '-CCCTGTGGAAGTGCTTTTATTAGCAG-3 ' (SEQ IDNO.4)

The forward sequence that primer 1 begins for the 5 ' 1bp that holds that is positioned at SEQ ID NO:1; Primer 2 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/LTris-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 beta-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 1-2999bp shown in the SEQ ID NO:1 are identical.

Embodiment 4:Northern blotting is analyzed the expression of hARRG:

Extract the total RNA[Anal.Biochem 1987,162 of different tissues, 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 shallow lake 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-1mMEDTA-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 the hARRG coding region sequence (500bp to 2866bp among the SEQID NO:1) of pcr amplification.Will ³²The probe of P-mark (about 2 * 10 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.

Found that hARRG all has distribution in the majority tissue, promptly in histoorgans such as the heart, brain, placenta, liver, skeletal muscle, kidney and pancreas expression is arranged all, and expression is less in the lung, pancreas, liver and heart are expressed higher.Except the transcript of 3.0Kb, the transcript (see figure 3) of still visible-5.5Kb in liver and pancreas in addition.

Embodiment 5: vivoexpression, separation and the purifying of reorganization hARRG

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:

Primer 3:5 '-cgcgcatatgagcacagctctgacc-3 ' (SEQ ID NO5)

Primer 4:5 '-cgcgaattccggctcaaaagaaatcaac-3 ' (SEQ ID NO6)

5 ' end of these two sections primers contains Nde I and EcoR I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, Nde I and EcoR 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-0097d10 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: the RT-PCR amplified production that contains embodiment 3 among the cumulative volume 50 μ l is respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l as template, primer-3 and primer-4.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 EcoR 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-0097d10) 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-0097d10) 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 hARRG polypeptide of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 4) at the 89.2kDa 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-hARRG production of antibodies

Synthesize the specific polypeptide of following hARRG with Peptide synthesizer (PE company product):

NH2-Met-Ser-Thr-Ala-Leu-Thr-His-Thr-Thr-Val-Ala-Met-Arg-Cys-Pro(SEQ?IDNO: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 be specifically and the hARRG protein binding.

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

Sequence table (1) general information: (ⅱ) denomination of invention: new human arsenic resistant related gene protein and encoding sequence thereof (ⅲ) sequence number: the information of 7 (2) SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 2999bp

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ⅱ ) ：cDNA ( ⅹⅰ ) ：SEQ ID NO:1:GGCCATTATG GCCGGGGGGA GGCGTGGGTG ACGCAGGCGC AGCGCGGGCT GCGCGCGCTA 60CTGCCCATCC CCGGTTGTCC CACTTTTGTT CGCCTCTCTT CGGTCCTCTA CTCAAGAGCT 120CCGTCTCCGT CTCGCCCTCC TCGAAGTCCT CGTCGCGCGC CCGCGACCCA GGTCGCCCTG 180AAATCTAGCC CGTCCGAGCG CGAGTCCAAC GGCCGCGGCC GCACCAAGGC CCCCTCAGAC 240CGTGCCATGG GTGACAGTGA TGACGAGTAC GATCGAAGGC GCAGGGACAA GTTCAGAAGA 300GAGCGCAGCG ACTACGACCG TTCCCGCGAG AGAGATGAAA GACGTCGAGG GGACGATTGG 360AATGACAGAG AGTGGGACCG TGGCCGTGAG CGCCGTAGTC GGGGTGAATA TCGGGACTAT 420GACCGGAATC GGCGAGAGCG CTTCTCGCCA CCTCGCCACG AACTCAGCCC GCCACAGAAG 480CGCATGAGGA GAGACTGGGA TGAGCACAGC TCTGACCCAT ACCACAGTGG CTATGAGATG 540CCCTATGCTG GGGGGGGGTG GGGGCCCAAC TTATGGCCCC CCTCAGCCCT GGGGCCACCC 600TGAAGTCCAC ATCATGCAGC ACCATGTCCT GCCTATCCAG GCCAGGCTGG GCAGCATTGC 660AGAGATTGAC CTGGGTGTGC CGCCGCCCGT GATGAAGACC TTCAAGGAGT TTCTCCTCTC 720CCTGGATGAC TCGGTGGATG AGACGGAGGC CGTCAAGCGC TATAATGACT ACAAGCTGGA 780TTTCCGGAGG CAACAGATGC AGGATTTCTT CCTGGCGCAC AAAGATGAGG AGTGGTTTCG 840GTCTAAGTAC CACCCAGATG AGGTGGGGAA GCGTCGGCAG GAGGCCCGGG GGGCCCTGCA 900AAACCGACTG AGGGTCTTCC TGTCCCTCAT GGAGACTGGC TGGTTTGATA ACCTTCTCCT 960GGACATAGAC AAAGCTGATG CCATTGTCAA GATGCTGGAT GCAGCCGTGA TTAAGATGGA 1020AGGAGGCACG GAGAATGATC TCCGCATCCT GGAGCAGGAG GAGGAGGAGG AGCAGGCAGG 1080AAAGCCTGGG GAGCCCAGCA AGAAAGAAGA AGGACGGGCT GGAGCAGGCC TAGGGGACGG 1140GGAGCGCAAA ACCTACGACA AGGATGAGAA GAAGGAAGAC GGCAAGCAGG CTGAGAATGA 1200CAGTTCTAAT GATGACAAAA CAAAGAAGTC GGAGGGTGAT GGGGACAAGG AAGAGAAGAA 1260AGAAGACTCC GAGAAGGAAG CCAAAAAGAG TAGCAAGAAG CGGAACCGGA AGCACAGTGG 1320TGACGACAGC TTTGACGAGG GCAGCGTGTC AGAGTCTGAG TCGGAGTCAG AGAGCGGCCA 1380GGCTGAGGAG GAGAAGGAGG AGGCCGAAGA AGCGCTCAAG GAGAAGGAGA AGCCCAAGGA 1440AGAAGAATGG GAGAAGCCCA AGGACGCCGC GGGGCTGGAG TGCAAGCCGC GGCCGCTGCA 1500TAAGACCTGC TCCCTCTTCA TGCGCAACAT CGCGCCCAAC ATCTCCCGGG CCGAGATCAT 1560CTCCCTTTGT AAAAGGTACC CAGGCTTTAT GCGGGTGGCG CTCTCAGAGC CCCAGCCAGA 1620GAGGAGGTTT TTCCGTCGTG GCTGGGTGAC CTTCGACCGC AGTGTTAACA TTAAAGAGAT 1680CTGTTGGAAC CTGCAGAACA TCCGTCTCCG GGAGTGTGAG CTGAGCCCTG GTGTGAACAG 1740GGACCTGACC CGGCGCGTTC GCAACATCAA CGGCATCACC CAGCACAAGC AGATTGTGCG 1800CAACGACATC AAGCTGGCGG CCAAGCTGAT CCACACGCTG GATGACAGGA CACAGCTTTG 1860GGCCTCAGAA CCAGGGACGC CTCCCCTGCC CACGAGCCTG CCCTCGCAAA ACCCGATCTT 1920GAAGAATATC ACCGACTACC TGATCGAGGA AGTAAGCGCC GAGGAGGAGG AGCTGCTGGG 1980GAGCAGCGGG GGCGCTCCTC CTGAGGAGCC TCCTAAGGAA GGGAACCCGG CAGAGATCAA 2040CGTGGAGCGG GATGAGAAGT TGATTAAGGT CTTGGACAAG CTCCTCCTTT ACCTGCGCAT 2100CGTGCATTCC TTGGATTATT ACAACACCTG TGAGTACCCC AACGAGGACG AGATGCCCAA 2160TCGCTGTGGG ATCATCCACG TTCGGGGGCC CATGCCACCC AACCGCATCA GTCACGGGGA 2220AGTGCTGGAG TGGCAGAAGA CTTTTGAGGA GAAGCTCACG CCGTTGCTGA GTGTGCGGGA 2280GTCACTCTCA GAGGAAGAGG CCCAGAAGAT GGGGCGCAAA GACCCAGAGC AGGAAGTGGA 2340GAAGTTCGTC ACCTCCAACA CGCAGGAACT GGGCAAGGAT AAGTGGCTGT GTCCTCTCAG 2400TGGCAAGAAA TTCAAGGGTC CTGAGTTTGT GCGCAAACAT ATCTTCAACA AGCATGCAGA 2460GAAAATTGAG GAAGTGAAAA AGGAAGTCGC GTTTTTTAAC AACTTCCTCA CTGATGCTAA 2520GCGCCCAGCT CTGCCTGAGA TCAAGCCAGC CCAGCCACCT GGCCCCGCCC AGAGTTTGAC 2580CCCAGGACTC CCCTACCCAC ACCAGACTCC CCAGGGCCTG ATGCCCTATG GTCAGCCCCG 2640GCCCCCGATC TTGGGCTATG GAGCTGGTGC TGTCCGCCCT GCAGTCCCCA CAGGAGGCCC 2700TCCATACCCC CATGCCCCGT ATGGTGCTGG TCGAGGGAAC TATGATGCCT TCCGAGGCCA 2760GGGAGGTTAT CCTGGGAAAC CTCGCAACAG GATGGTTCGT GGAGACCCAA GGGCCATTGT 2820GGAATATCGG GACCTGGATG CCCCAGACGA TGTTGATTTC TTTTGAGCCG TCCCCCGTTC 2880CTCAGTCCTG TATCATCCAT ACTTGTACTA CCTTGTCCTA TGAAGCTCTG AGAATTTTTT 2940GTACGATCAG CCTTACTGCT AATAAAAGCA CTTCCACAGG GAAAAAAAAA AAAAAAAAA 2999 ( 2 ) SEQ ID NO:2： ( ⅰ ) ：

(A) length: 788 amino acid (

(B) type: amino acid

( D ) ： ( ⅱ ) ： ( ⅹⅰ ) ：SEQ ID NO:2:MSTALTHTTV AMRCPMLGGG GGPTYGPPQP WGHPEVHIMQ HHVLPIQARL 50GSIAEIDLGV PPPVMKTFKE FLLSLDDSVD ETEAVKRYND YKLDFRRQQM 100QDFFLAHKDE EWFRSKYHPD EVGKRRQEAR GALQNRLRVF LSLMETGWFD 150NLLLDIDKAD AIVKMLDAAV IKMEGGTEND LRILEQEEEE EQAGKPGEPS 200KKEEGRAGAG LGDGERKTYD KDEKKEDGKQ AENDSSNDDK TKKSEGDGDK 250EEKKEDSEKE AKKSSKKRNR KHSGDDSFDE GSVSESESES ESGQAEEEKE 300EAEEALKEKE KPKEEEWEKP KDAAGLECKP RPLHKTCSLF MRNIAPNISR 350AEIISLCKRY PGFMRVALSE PQPERRFFRR GWVTFDRSVN IKEICWNLQN 400IRLRECELSP GVNRDLTRRV RNINGITQHK QIVRNDIKLA AKLIHTLDDR 450TQLWASEPGT PPLPTSLPSQ NPILKNITDY LIEEVSAEEE ELLGSSGGAP 500PEEPPKEGNP AEINVERDEK LIKVLDKLLL YLRIVHSLDY YNTCEYPNED 550EMPNRCGIIH VRGPMPPNRI SHGEVLEWQK TFEEKLTPLL SVRESLSEEE 600AQKMGRKDPE QEVEKFVTSN TQELGKDKWL CPLSGKKFKG PEFVRKHIFN 650KHAEKIEEVK KEVAFFNNFL TDAKRPALPE IKPAQPPGPA QSLTPGLPYP 700HQTPQGLMPY GQPRPPILGY GAGAVRPAVP TGGPPYPHAP YGAGRGNYDA 750FRGQGGYPGK PRNRMVRGDP RAIVEYRDLD APDDVDFF 788 ( 2 ) SEQ ID NO:3 ( ⅰ )

(A) length: 24 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:3:GGCCATTATG GCCGGGGGGA GGCG 24 (2) SEQ ID NO:4

(A) length: 26 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:4:CCCTGTGGAA GTGCTTTTAT TAGCAG 26 (2) SEQ ID NO:5

(A) length: 25 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅱ) sequence signature of SEQ ID NO:5:CGCGCATATG AGCACAGCTC TGACC 25 (2) SEQ ID NO:6

(A) length: 28 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:6:CGCGAATTCC GGCTCAAAAG AAATCAAC 28 (2) SEQ ID NO:7

(A) length: 15 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: polypeptide (ⅹ ⅰ) sequence description: SEQ ID NO:7:Met Ser Thr Ala Leu Thr His Thr Thr Val Ala Met Arg Cys Pro 15

Claims (11)

1, a kind of isolated polypeptide-hARRG polypeptide is characterized in that it comprises: contain the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or fragment, analogue or the derivative of its polypeptide.

2, polypeptide as claimed in claim 1 is characterized in that, described amino acid, analogue or derivative have the similarity with the aminoacid sequence at least 98.858% shown in the SEQ ID NO:2.

3, polypeptide as claimed in claim 2 is characterized in that, it is the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.

4, a kind of isolating polynucleotide is characterized in that, described polynucleotide comprise the Nucleotide that is selected from down group:

(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 99.764% homogeny (b) are arranged.

5, polynucleotide as claimed in claim 4 is characterized in that, described polynucleotide comprise that coding has the polynucleotide of aminoacid sequence shown in the SEQ ID NO:2.

6, polynucleotide as claimed in claim 4 is characterized in that, the sequence of described polynucleotide includes the sequence of 1-2999 position among the sequence of 500-2866 position among the SEQID NO:1 or the SEQ ID NO:1.

7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that, it contains the described number Nucleotide of claim 4.

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;

(b) host cell that requires 4 described polynucleotide to transform or transduce with claim.

9, a kind of preparation method with polypeptide of hARRG polypeptide active is characterized in that, described method comprises:

(a) expressing under the hARRG polypeptide condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate polypeptide with hARRG polypeptide active.

10, a kind of can with polypeptide bonded antibody, it is characterized in that, described antibody be can with hARRG polypeptid specificity bonded antibody.

11, a kind of pharmaceutical composition is characterized in that, it contains described hARRG polypeptide of claim 1 and pharmaceutically acceptable carrier.

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