CN1380407A - New human-phosphoguanosine reductase, its coding sequence and application - Google Patents
New human-phosphoguanosine reductase, its coding sequence and application Download PDFInfo
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Abstract
The present invention relates to a novel human guanosine monophosphate reductase GMPR2. Said invention provides the polynucleotide for coding said protein molecule and method for producing this protein molecule by means of DNA recombination technology. It also discloses the application of the polynuleotide coding this novel guanosine monophosphate reductase, dand the zymological acitivity and the relationship of it and tumour cell multiplication and cell differentiation are verified. Said invention also discloses the strategy of resisting said protein molecule for diagnosing and curing diseases, speciallyf ro diagnosing and curing the diseases of tumour, etc.
Description
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding people guanosine monophosphate(GMP) reductase enzyme (guanosine monophosphate reductase 2 abbreviates " GMPR2 " as) polypeptide, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.The invention still further relates to the enzyme activity of GMPR2 and the relation of breaking up with tumor cell proliferation.Specifically, polypeptide of the present invention is a kind of new and cell proliferation, differentiation, apoptosis, and the relevant intracellular protein of inflammatory reaction.
Enzyme is the biomacromolecule material that a class has catalytic activity and special space conformation in the human body cell, and the function of keeping normal intracellular matter metabolism, adjusting cell is played an important role.Separation and purification, the biologic activity of enzyme, the space conformation of enzyme and the important component part that the relation between the enzyme activity is the traditional biological chemistry always of research enzyme.In recent years, 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.
People's guanosine monophosphate(GMP) reductase enzyme (Guanosine monophosphate reductase, GMPR) (EC1.6.6.8) the catalysis guanosine monophosphate(GMP) is reduced to inosinic acid, this reaction process is irreversible, needs nicotinamide-adenine Nucleotide as coenzyme.This reaction product inosinic acid can further be converted into adenine nucleotide or synthetic guanylic acid, be to connect the key substance that transforms mutually between adenine nucleotide and the guanylic acid, bringing into play important effect keeping in the cell balance between the adenine nucleotide and guanylic acid.
GMPR finds that also purifying comes out at first in intestinal bacteria, have only endoerythrocytic GMPR activity once by the further investigation report in human body.The gene of coding GMPR also at first in the intestinal bacteria clonal expression come out.The gene of people's GMPR is also come out by people's clonal expressions such as Kanno, but the understanding of this gene is one section zigzag distance is arranged.Final result of study thinks that the gene of coding GMPR is to be positioned on No. 6 karyomit(e) one independently gene.
Because nucleic acid is the main component of forming cell and genetic material, the transmission of the propagation of its metabolism and cell, differentiation, signal transduction and genetic information is all closely related, therefore, GMPR is used as antitumor or anti-infectious chemotherapeutical target material always and is furtherd investigate, as effect and the anti-Li Siman protozoon effect etc. of GMPR in the methotrexate antineoplaston.Have increasing of the multinomial GMPR of discovering closely related with differentiation with the propagation of leukemia cell HL-60, may be the GMPR that increases reduced Triphosaden (GTP) level in the cell or disturbed the signal transduction relevant with GTP and suppressed cell propagation, promoted the differentiation of tumour cell.
Studies show that GMPR may play a significant role in pathologic processes such as infection, tumour, and may have important development and application in a plurality of fields such as anti-infective, antitumor and be worth.Therefore, significant for the new people's guanosine monophosphate(GMP) reductase enzyme of therapeutic purpose research and development.Yet, before the present invention, people's guanosine monophosphate(GMP) reductase enzyme of other types was not also disclosed.
The purpose of this invention is to provide a kind of new people's guanosine monophosphate(GMP) reductase enzyme GMPR2 albumen with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.GMPR2 albumen of the present invention is a kind of GMPR homolgous molecule.
Another object of the present invention provides the influence to the HL-60 cytodifferentiation of the enzyme activity of GMPR2 and GMPR2 high expression level.
In a first aspect of the present invention, novel isolated GMPR2 polypeptide is provided, this polypeptide is the people source, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people GMPR 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 348-1394 position among the SEQ ID NO:1; (b) has the sequence of 1-1875 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 GMPR2 protein-active, this method comprises: (a) under the proteic condition of suitable expressing human GMPR2, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people GMPR2 protein-active.
In a fifth aspect of the present invention, provide and above-mentioned people GMPR2 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-1875 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people GMPR2 polypeptide active is provided, and the compound that suppresses people GMPR2 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 GMPR2 polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of GMPR2 in the test sample, it comprises: sample is contacted with the proteic specific antibody of GMPR2, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample GMPR2 albumen.
In a eighth aspect of the present invention, a kind of disease relevant with people GMPR2 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 GMPR2 polypeptide active, and perhaps screening suppresses the antagonist of people GMPR2 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people GMPR2 of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.
In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people GMPR2 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as tumour, infection.
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 is defined.
Fig. 1 is the cDNA sequence of people GMPR2 of the present invention, and wherein non-coding sequence is represented with bold-faced letter, and encoding sequence is with capitalizing conventional letter representation.
Fig. 2 is the proteic full length amino acid sequence of people GMPR2 of the present invention.Wherein the sequence of underscore is the catalysis territory of guanosine monophosphate(GMP) reductase enzyme.
Fig. 3 is the shown distribution of people GMPR2 Northern blot hybridization of the present invention.Left figure and middle figure are that the healthy tissues of GMPR2 distributes; Right figure is the distribution of GMPR2 in various kinds of cell system.The 1-16 swimming lane is respectively: spleen, thymus gland, prostate gland, testis, ovary, small intestine, colon, PBL, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas.Swimming lane 17-24 is respectively following cell strain: HL-60, Hela, K562, Raji, SW480, A549 and G361.PBL representative peripheral blood leucocyte.Results suggest GMPR2 is a kind of guanosine 5-monophosphate reductase enzyme molecule of extensive distribution.
Fig. 4 is Western marking result.1:GMPR2 transgenic cell crude extract; 2: the GMPR2 albumen of purifying; 3: the empty carrier negative control; 4: elutriant.
Fig. 5 is the people GMPR2 of the present invention guanosine monophosphate(GMP) reductase activity of its intracellular protein and purified GMPR2 expression product and comparison of control group behind the transient expression in cell.: empty carrier negative control; ■: GMPR2 transgenic cell crude extract; ◆: the GMPR2 albumen of purifying; The ◇ elutriant
GMPR2 provided by the present invention and known GMPR molecule have high homology, have the enzyme activity that the catalysis guanosine monophosphate(GMP) generates inosinic acid in cell, and it are intracellular widely distributed.This shows that GMPR2 is a kind of new people's guanosine monophosphate(GMP) reductase enzyme.Make GMPR2 high expression level in HL-60 leukemia cell by transgenosis, what can promote this cell induces following differentiation to the monocyte direction at Buddhist ripple ester.Therefore, GMPR2 may play a significant role in pathologic processes such as infection, tumour, and may have important development and application in a plurality of fields such as anti-infective, antitumor and be worth.
In the present invention, term " GMPR2 albumen ", " GMPR2 polypeptide ", " people's guanosine 5-monophosphate reductase enzyme GMPR2 " or " people's guanosine monophosphate(GMP) reductase enzyme GMPR2 " are used interchangeably, and all refer to have the albumen or the polypeptide of people's guanosine monophosphate(GMP) reductase enzyme GMPR2 aminoacid sequence (SEQ ID NO:2).They comprise the people's guanosine monophosphate(GMP) reductase enzyme GMPR2 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 GMPR2 albumen or polypeptide " is meant that the GMPR2 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 GMPR2 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
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 GMPR2, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human GMPR2 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 GMPR2 polypeptide " refers to have the SEQ IDNO.2 polypeptide of sequence of people GMPR2 protein-active.This term also comprises having and variant form people GMPR2 albumen identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people GMPR2 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 GMPR2 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people GMPR2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people GMPR2 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people GMPR2 polypeptide.Usually, this fragment have people GMPR2 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 GMPR2 albumen or polypeptide.The difference of these analogues and natural human GMPR2 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 GMPR2 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 SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence 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 GMPR2.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People GMPR2 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 GMPR2 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 GMPR2 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people GMPR2 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 GMPR2 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: the expression vector based on T7 of expressing in bacterium; The pMSXND expression vector of in mammalian cell, expressing 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 GMPR2 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, 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 P
LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The people GMPR2 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 GMPR2 protein function as pharmacological agent GMPR2 protein function.The peptide molecule that can suppress or stimulate people GMPR2 protein function that can be used for seeking therapeutic value with the recombinant human GMPR2 protein screening peptide library of expressing.
On the other hand, the present invention also comprises people GMPR2 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people GMPR2 gene product or fragment.Preferably, refer to that those can combine with people GMPR2 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 GMPR2, comprise that also those do not influence the antibody of people GMPR2 protein function.The present invention also comprise those can with modify or without the people GMPR2 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 GMPR2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human GMPR2 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 be utilized hybridoma technology to prepare (to see people such as Kohler, Nature 256:495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people GMPR2 protein function and the antibody that does not influence people GMPR2 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people GMPR2 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 GMPR2 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 GMPR2 can be used in the immunohistochemistry technology, detects the people GMPR2 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people GMPR2, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.
Antibody among the present invention can be used for treating or prevention and the relevant disease of people GMPR2 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people GMPR2 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 GMPR2 albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell (for example lymph-node cell etc.) of people GMPR2 protein positive.
The production of polyclonal antibody can choose GMPR2 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 GMPR2 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 aspects such as tumour, infection.When using GMPR2 albumen of the present invention, also can use the other treatment agent simultaneously, as IFN-α, IFN-β, TNF-α, TNF-β etc.
The present invention also provides a kind of pharmaceutical composition, and it contains GMPR2 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the GMPR2 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 GMPR2 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 GMPR2 of the proteic nothing expression of GMPR2 or unusual/non-activity.The GMPR2 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic GMPR2 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the GMPR2 transgenosis to cell.The method that structure carries the recombinant viral vector of GMPR2 gene is found in existing document (Sambrook, et al.).Recombinant human GMPR2 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 GMPR2 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 GMPR2 obtains.During screening, must carry out mark to people GMPR2 protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people GMPR2 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people GMPR2 protein level that is detected in the test can be with laying down a definition the importance of people GMPR2 albumen in various diseases and be used to the disease of diagnosing GMPR2 albumen to work.
Whether having the proteic method of GMPR2 in a kind of detection test sample is to utilize the proteic specific antibody of GMPR2 to detect, and it comprises: sample is contacted with the GMPR2 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample GMPR2 albumen.
The proteic polynucleotide of GMPR2 can be used for the diagnosis and the treatment of GMPR2 protein related diseases.Aspect diagnosis, the proteic polynucleotide of GMPR2 can be used for detecting the proteic expression of GMPR2 GMPR2 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of GMPR2 as the GMPR2 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 GMPR2 albumen and also can detect the proteic transcription product of GMPR2.
The sudden change that detects the GMPR2 gene also can be used for the disease of diagnosing GMPR2 albumen relevant.The form of GMPR2 protein mutation comprises that the point mutation compared with normal wild type GMPR2 dna sequence dna, 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 GMPR2 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, MendelianInheritance in Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
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 IDNO:2.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1875 bases, and its open reading frame is positioned at the 348-1394 position, and the coding total length is 348 amino acid whose people GMPR2 albumen (SEQ ID NO:2).This GMPR2 albumen belongs to the enzyme molecule, and with GMPR aminoacid sequence height homology, consistence can be up to 79%, and similarity then can reach 91%.In addition, GMPR2 albumen also comprises the conservative catalysis motif (176-188 residue) of GMPR.The Northern engram analysis shows wide expression in tissue.The research prompting of having carried out, GMPR2 may be new people's guanosine monophosphate(GMP) reductase enzyme, the tool potential is antitumor, anti-infectious effect.Therefore, GMPR2 albumen or its relevant antagonist, agonist etc. can be diseases such as treatment tumour, infection new treatment approach are provided, thereby have great application prospect.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of people GMPR2 cDNA
Extract the total RNA of human dendritic cell with Trizol (Gibco company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is cloned test kit (available from Gibco) with SuperScriptII cDNA fragment orientation is inserted on the multiple clone site of carrier, transform DH5 α bacterium and form the cDNA plasmid library.Measure random choose clone's 5 ' terminal sequence 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 people GMPR2, and its encoding gene name is people GMPR2 gene.
Sequence SEQ ID NO:1 total length is 1875bp, comprises 3 ' end non-coding region of 5 of 347 bp ' end non-coding region and 471bp, and coding contains 348 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 37.8kD.
They are different with known for the BLAST analysis revealed, and with people GMPR aminoacid sequence height homology, consistence reaches 79%, and similarity reaches 91%, belongs to the enzyme molecule.In addition, also comprise a proteic conservative catalysis motif of GMPR (176-188 residue) (Fig. 2) in GMPR2 albumen, this also further points out it may be new people's guanosine monophosphate(GMP) reductase enzyme.
Embodiment 2: with the encoding sequence of RT-PCR method human cloning GMPR2
Be in logarithmic phase B lymphoma cell strain Raji cell total rna with Trizol (Gibco company) extraction, get 6mg cell total rna and 0.5 μ g Oligo-dT
12-18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ l ddH after finishing
2O dilutes.The used primer of pcr amplification is as follows: adopted primer 5 '-G is arranged
GAATTCCGCCATCATGCCTCATATTGACAACGATG-3 ' (SEQID NO:3), antisense primer 5 '-G
GAATTCGTCGACGCACGCCTCACTGAAGATTG-3 ' (SEQ ID NO:4).The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4mM primer, 0.2mM dNTP and 1U ExTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 25 circulations.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the coding region shown in the SEQ ID NO:1 are in full accord.
The Northern engram analysis of embodiment 3 GMPR2
Carry out the Northern trace by following ordinary method: filter membrane to be checked places the hybridization solution of 10ml through 68 ℃ of preheatings, in hybrid heater (Bellco) in 68 ℃ of prehybridizations 30 minutes; The cDNA probe that mark is good was in 95～100 ℃ of sex change 2～5 minutes, and (cDNA probe final concentration is 2～10ng/ml or 1～2 * 10 to put rapid cooling back adding hybridization solution on ice
6Cpm/ml), fully mixing was hybridized 2 hours in 68 ℃.After hybridization finishes, filter membrane with 2 * SSC, the drip washing of 0.05%SDS room temperature for several times, the washing lotion several is changed in continuing vibration flushing 30～40 minutes therebetween.Use 0.1 * SSC, 0.1%SDS in 50 ℃ of vibration flushings 20～40 minutes subsequently.Last filter membrane wrapped up with plastic fresh-keeping membrane, in-70 ℃ of exposure X line films 24～48 hours.
Northern blot hybridization result shows: in many healthy tissuess such as liver, heart, skeletal muscle, placenta and in the various kinds of cell systems such as Molt-4, Raji expression (4.5kb) is arranged all, this shows that GMPR2 albumen is a kind of albumen (Fig. 3) of wide expression.Embodiment 4 people GMPR2 are recombinant expressed
In this embodiment, be template with the pcr amplification product among the embodiment 2, increase with the PCR Oligonucleolide primers of 5 ' and 3 following ' end of sequence, obtain people GMPR2 DNA as inserting fragment.
5 ' end Oligonucleolide primers sequence of using in the PCR reaction is:
5’-G
GAATTCCGCCATCATGCCTCATATTGACAACGATG-3′(SEQ?IDNO：3)
This primer contains the restriction enzyme site of EcoRI restriction enzyme, is the part encoding sequence that is begun by initiator codon after this restriction enzyme site;
5’-G
GAATTCGTCGACGCACGCCTCACTGAAGATTG-3′(SEQ?ID?NO：4)
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people GMPR2 of EcoRI restriction enzyme.
With the PCR product purification that obtains after the EcoRI enzyme cut and recombinate according to a conventional method with plasmid pGEM-3ZF again and be converted into the competence bacillus coli DH 5 alpha, 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 GMPR2 cDNA EcoRI endonuclease bamhi of correct sequence is cloned into expression vector pcDNA3.1 (Pharmacia company), forms carrier pcDNA3.1-GMPR2, transform people DH5 α then.Positive colony is cut evaluation with the EcoRI enzyme, and enzyme is cut the capable 2% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted complete GMPR2 encoding sequence.
Order-checking is turned out to be correct carrier for expression of eukaryon pcDNA3.1-GMPR2 import in the people COS7 cell, collect cell after 72 hours, (contain 20mM Tris, PH7.5 with ice-cold lysate with liposome; 150mMNacl; 1mM EDTA; 1% Triton; 2.5mM trisodium phosphate; 1mM β phospho-glycerol; 1mM NaVO
3With 1mM PMSF) lysing cell, use ultrasonication then 30 times, 13, centrifugal 10 minutes of 000rpm gets supernatant and is used for subsequent experimental.The part supernatant obtains people GMPR2 albumen with HiTrap Chelating columns (PharmaciaBiotech) affinity chromatography purifying.
Carry out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 10 amino-acid residues of N-end shown in 10 amino acid of N-end and the SEQID NO:2 are identical as a result.
Embodiment 5: anti-people GMPR2 production of antibodies
The recombinant protein people GMPR2 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 GMPR2 gene translation product with it.Found that antibody can combine with GMPR2 albumen of the present invention specifically.
The enzyme activity of embodiment 6:GMPR2
The people GMPR2 cDNA EcoRI endonuclease bamhi of correct sequence in the reorganization pGEM-3ZF carrier that contains the GMPR2 complete encoding sequence that is obtained among the embodiment 4 is cloned into the pcDNA3.1 carrier for expression of eukaryon, identify direction with the XbalI/SalI double digestion, the clone who gets forward imports in the people COS7 cell with liposome, collect cell after 72 hours, (contain 20mM Tris, PH7.5 with ice-cold lysate; 150mM Nacl; 1mM EDTA; 1% Triton; 2.5mM trisodium phosphate; 1mM β phospho-glycerol; 1mM NaVO
3With 1mM PMSF) lysing cell, use ultrasonication then 30 times, 13, centrifugal 10 minutes of 000rpm gets supernatant and is used for subsequent experimental.The part supernatant all uses BCA protein quantification reagent (Pierce) quantitative with HiTrap Chelating columns (Pharmacia Biotech) affinity chromatography purifying, purifying protein and cell crude extract.Purifying protein and cell crude extract confirm that through the Western electrophoresis and with fluorescently-labeled anti-myc antibodies there be (Fig. 4) in the GMPR2 albumen that wherein has molecular weight to coincide.
The detection of the enzyme activity is carried out under 37 ℃ of conditions, represents the activity of enzyme with the minimizing of NADPH content in the product.The 50mM Tris-HCl (pH7.8) that contains 2ml in the reaction system, 2mM 2-mercaprol, 1mM EDTA, 10 μ l guanosine monophosphate(GMP)s (substrate), 4 μ l 0.01mM NADPH and 30 μ l zymoproteins comprise the negative control that purifying protein, elutriant, transgenic cell crude extract and pcDNA3.1 empty carrier are done.After reaction is carried out 15,30,45,60,120 minutes respectively, detect the light absorption ratio at 340nm place with the spectrophotometric note.NADPH in the enzyme reaction has special absorption peak at the 340nm place.Found that the product of GMPR2 instantaneous overexpression in the COS7 cell has the activity (Fig. 5) that the catalysis guanosine monophosphate(GMP) is converted into inosinic acid.
The relation of embodiment 7:GMPR2 and tumour cell differentiation
Change the GMPR2 expression carrier over to HL-60 leukemia cell with liposome, transfection adds the Buddhist ripple ester of 50ng/ml concentration and induced 72 hours under 37 ℃ of conditions after 48 hours.The differentiation of HL-60 cell detects with the blue method of tetrazole (NBT), and the intracellular peroxidase of the HL-60 of differentiation and maturation can be oxidized to xanchromatic tetrazole indigo plant the first hairpin of blue look.Add 0.1% NBT, 3 μ M Buddhist ripple ester continuation effects 25 minutes in the HL-60 cell after inducing, get cell and make smear, formaldehyde fixed is used in dry back, and the quantity of blue look positive cell in per 200 cells of numeration is carried out check analysis with empty carrier cells transfected group under opticmicroscope.As a result, the HL-60 cell of overexpression GMPR2 is induced down at Buddhist ripple ester, and the noble cells number is more than a times of control group (P＜0.01).
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.
＜110〉＜120〉、＜130〉011998＜160〉4＜170〉PatentIn version 3.0＜210〉1＜211〉1875＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222〉 ( 348 ) .. ( 1394 )＜400〉1ggcatccagt ttagcaacag cagagatgac gactctgcga ttctgagagt ccctggcgag 60cccgggctag cgaaaagtgg gggcagaacg aactacatct cccatcgtgc caggaggcgg 120tcccgcccgt ttccccctgg gagttgtagt ctaaccccct cggatccaac agcaacctca 180gtgcgtgaac tctgttatcc agaaggcctc gccctgccgc cgccgaagct ggaattcgtc 240ggctagtagt tctcgccggc aactagagga acctgttggc gtggcccaga aggcttagcg 300ggattgcacg agccctcaga ttcatcgcta ccccgaggct aagcgcc atg cct cat 356
Met?Pro?His
1att?gac?aac?gat?gtg?aaa?ctg?gac?ttc?aag?gat?gtc?ctt?ttg?agg?ccc??????404Ile?Asp?Asn?Asp?Val?Lys?Leu?Asp?Phe?Lys?Asp?Val?Leu?Leu?Arg?Pro
5???????????????????10??????????????????15aaa?cgc?agt?acc?ctt?aag?tct?cga?agt?gag?gtg?gat?ctc?aca?aga?tcc??????452Lys?Arg?Ser?Thr?Leu?Lys?Ser?Arg?Ser?Glu?Val?Asp?Leu?Thr?Arg?Ser20??????????????????25??????????????????30??????????????????35ttt?tca?ttt?cgg?aac?tca?aag?cag?aca?tac?tct?ggg?gtt?ccc?atc?att??????500Phe?Ser?Phe?Arg?Asn?Ser?Lys?Gln?Thr?Tyr?Ser?Gly?Val?Pro?Ile?Ile
40??????????????????45??????????????????50gct?gcc?aat?atg?gat?act?gtg?ggc?acc?ttt?gag?atg?gcc?aag?gtt?ctc??????548Ala?Ala?Asn?Met?Asp?Thr?Val?Gly?Thr?Phe?Glu?Met?Ala?Lys?Val?Leu
55??????????????????60??????????????????65tgt?aag?ttc?tct?ctc?ttc?act?gct?gtc?cat?aag?cac?tat?agc?ctc?gtt??????596Cys?Lys?Phe?Ser?Leu?Phe?Thr?Ala?Val?His?Lys?His?Tyr?Ser?Leu?Val
70??????????????????75??????????????????80cag?tgg?caa?gag?ttt?gct?ggc?cag?aat?cct?gac?tgt?ctt?gag?cat?ctg??????644Gln?Trp?Gln?Glu?Phe?Ala?Gly?Gln?Asn?Pro?Asp?Cys?Leu?Glu?His?Leu
85??????????????????90??????????????????95gct?gcc?agc?tca?ggc?aca?ggc?tct?tct?gac?ttt?gag?cag?ctg?gaa?cag??????692Ala?Ala?Ser?Ser?Gly?Thr?Gly?Ser?Ser?Asp?Phe?Glu?Gln?Leu?Glu?Gln100?????????????????105?????????????????110?????????????????115atc?ctg?gaa?gct?att?ccc?cag?gtg?aag?tat?ata?tgc?ctg?gat?gtg?gca??????740Ile?Leu?Glu?Ala?Ile?Pro?Gln?Val?Lys?Tyr?Ile?Cys?Leu?Asp?Val?Ala
120?????????????????125?????????????????130aat?ggc?tac?tct?gaa?cac?ttt?gtt?gaa?ttt?gta?aaa?gat?gta?cgg?aag??????788Asn?Gly?Tyr?Ser?Glu?His?Phe?Val?Glu?Phe?Val?Lys?Asp?Val?Arg?Lys
135?????????????????140?????????????????145cgc?ttc?ccc?cag?cac?acc?atc?atg?gca?ggg?aat?gtg?gta?aca?gga?gag??????836Arg?Phe?Pro?Gln?His?Thr?Ile?Met?Ala?Gly?Asn?Val?Val?Thr?Gly?Glu
150?????????????????155?????????????????160atg?gta?gaa?gag?ctc?atc?ctt?tct?ggg?gct?gac?atc?atc?aaa?gtg?gga??????884Met?Val?Glu?Glu?Leu?Ile?Leu?Ser?Gly?Ala?Asp?Ile?Ile?Lys?Val?Gly
165?????????????????170?????????????????175att?ggg?cca?ggc?tct?gtg?tgt?act?act?cgg?aag?aaa?act?gga?gtg?ggg??????932Ile?Gly?Pro?Gly?Ser?Val?Cys?Thr?Thr?Arg?Lys?Lys?Thr?Gly?Val?Gly180?????????????????185?????????????????190?????????????????195tat?cca?cag?ctc?agc?gca?gtg?atg?gag?tgt?gca?gat?gct?gct?cat?ggc??????980Tyr?Pro?Gln?Leu?Ser?Ala?Val?Met?Glu?Cys?Ala?Asp?Ala?Ala?His?Gly
200?????????????????205?????????????????210ctc?aaa?ggc?cac?atc?att?tca?gat?gga?ggt?tgc?agc?tgt?cct?ggg?gat?????1028Leu?Lys?Gly?His?Ile?Ile?Ser?Asp?Gly?Gly?Cys?Ser?Cys?Pro?Gly?Asp
215?????????????????220?????????????????225gtg?gcc?aag?gct?ttt?ggg?gca?gga?gct?gac?ttc?gtg?atg?ctg?ggt?ggc?????1076Val?Ala?Lys?Ala?Phe?Gly?Ala?Gly?Ala?Asp?Phe?Val?Met?Leu?Gly?Gly
230?????????????????235?????????????????240atg?ctg?gct?ggg?cac?agt?gag?tca?ggt?ggt?gag?ctc?atc?gag?agg?gat?????1124Met?Leu?Ala?Gly?His?Ser?Glu?Ser?Gly?Gly?Glu?Leu?Ile?Glu?Arg?Asp
245?????????????????250?????????????????255ggc?aag?aag?tac?aag?ctc?ttc?tat?gga?atg?agt?tct?gaa?atg?gcc?atg?????1172Gly?Lys?Lys?Tyr?Lys?Leu?Phe?Tyr?Gly?Met?Ser?Ser?Glu?Met?Ala?Met260?????????????????265?????????????????270?????????????????275aag?aag?tat?gct?ggg?ggc?gtg?gct?gag?tac?aga?gcc?tca?gag?gga?aag?????1220Lys?Lys?Tyr?Ala?Gly?Gly?Val?Ala?Glu?Tyr?Arg?Ala?Ser?Glu?Gly?Lys
280?????????????????285?????????????????290aca?gtg?gaa?gtt?cct?ttt?aaa?gga?gat?gtg?gaa?cat?acc?atc?cga?gac?????1268Thr?Val?Glu?Val?Pro?Phe?Lys?Gly?Asp?Val?Glu?His?Thr?Ile?Arg?Asp
295?????????????????300?????????????????305atc?cta?gga?ggg?atc?cgc?tct?acg?tgt?acc?tat?gtg?gga?gca?gct?aag?????1316Ile?Leu?Gly?Gly?Ile?Arg?Ser?Thr?Cys?Thr?Tyr?Val?Gly?Ala?Ala?Lys
310?????????????????315?????????????????320ctc?aaa?gag?ttg?agc?agg?aga?act?acc?ttc?atc?cga?gtc?acc?cag?cag?????1364Leu?Lys?Glu?Leu?Ser?Arg?Arg?Thr?Thr?Phe?Ile?Arg?Val?Thr?Gln?Gln
325?????????????330?????????????????????335gtg?aat?cca?atc?ttc?agt?gag?gcg?tgc?tag?acctgagcag?ttctaccctc???????1414Val?Asn?Pro?Ile?Phe?Ser?Glu?Ala?Cys340?????????????????345ccaaggcacc?agtactctac?catggggcat?cccaagtggg?tcctcaccca?tcccagctac???1474tgcagcttct?gtattacttt?gtcatttcct?gttgtctcac?tcctgagggc?tcctgcagta???1534actctgtact?tctctatctg?cacacacaaa?atgcccaagg?cactcactgg?ggaggaagca???1594aggaagcaaa?cagtctgaga?aaatgatgca?agaaaatcaa?atgggaatct?ggggacccaa???1654cacaacatcc?tgaagattat?taaaaggaaa?agatgctgat?tggtacataa?atcttttaca???1714tggccttggt?ctagaggagg?caggctttta?gaatcatgtt?ttgttaatcc?gcttcactaa???1774attggacctt?cacatatcta?aaaagctctg?aagtgtttgt?atatttgaaa?tacctcaata???1834aagagagagc?tcattgactg?taaaaaaaaa?aaaaaaaaaa?a???????????????????????1875<210>2<211>348<212>PRT<213>Homo?sapiens<400>2Met?Pro?His?Ile?Asp?Asn?Asp?Val?Lys?Leu?Asp?Phe?Lys?Asp?Val?Leu1???????????????5???????????????????10??????????????????15Leu?Arg?Pro?Lys?Arg?Ser?Thr?Leu?Lys?Ser?Arg?Ser?Glu?Val?Asp?Leu
20??????????????????25??????????????????30Thr?Arg?Ser?Phe?Ser?Phe?Arg?Asn?Ser?Lys?Gln?Thr?Tyr?Ser?Gly?Val
35??????????????????40??????????????????45Pro?Ile?Ile?Ala?Ala?Asn?Met?Asp?Thr?Val?Gly?Thr?Phe?Glu?Met?Ala
50??????????????????55??????????????????60Lys?Val?Leu?Cys?Lys?Phe?Ser?Leu?Phe?Thr?Ala?Val?His?Lys?His?Tyr65??????????????????70??????????????????75??????????????????80Ser?Leu?Val?Gln?Trp?Gln?Glu?Phe?Ala?Gly?Gln?Asn?Pro?Asp?Cys?Leu
85??????????????????90??????????????????95Glu?His?Leu?Ala?Ala?Ser?Ser?Gly?Thr?Gly?Ser?Ser?Asp?Phe?Glu?Gln
100?????????????????105?????????????????110Leu?Glu?Gln?Ile?Leu?Glu?Ala?Ile?Pro?Gln?Val?Lys?Tyr?Ile?Cys?Leu
115?????????????????120?????????????????125Asp?Val?Ala?Asn?Gly?Tyr?Ser?Glu?His?Phe?Val?Glu?Phe?Val?Lys?Asp
130?????????????????135?????????????????140Val?Arg?Lys?Arg?Phe?Pro?Gln?His?Thr?Ile?Met?Ala?Gly?Asn?Val?Val145?????????????????150?????????????????155?????????????????160Thr?Gly?Glu?Met?Val?Glu?Glu?Leu?Ile?Leu?Ser?Gly?Ala?Asp?Ile?Ile
165?????????????????170?????????????????175Lys?Val?Gly?Ile?Gly?Pro?Gly?Ser?Val?Cys?Thr?Thr?Arg?Lys?Lys?Thr
180?????????????????185?????????????????190Gly?Val?Gly?Tyr?Pro?Gln?Leu?Ser?Ala?Val?Met?Glu?Cys?Ala?Asp?Ala
195?????????????????200?????????????????205Ala?His?Gly?Leu?Lys?Gly?His?Ile?Ile?Ser?Asp?Gly?Gly?Cys?Ser?Cys
210?????????????????215?????????????????220Pro?Gly?Asp?Val?Ala?Lys?Ala?Phe?Gly?Ala?Gly?Ala?Asp?Phe?Val?Met225?????????????????230?????????????????235?????????????????240Leu?Gly?Gly?Met?Leu?Ala?Gly?His?Ser?Glu?Ser?Gly?Gly?Glu?Leu?Ile
245?????????????????250?????????????????255Glu?Arg?Asp?Gly?Lys?Lys?Tyr?Lys?Leu?Phe?Tyr?Gly?Met?Ser?Ser?Glu
260?????????????????265?????????????????270Met?Ala?Met?Lys?Lys?Tyr?Ala?Gly?Gly?Val?Ala?Glu?Tyr?Arg?Ala?Ser
275?????????????????280?????????????????285Glu?Gly?Lys?Thr?Val?Glu?Val?Pro?Phe?Lys?Gly?Asp?Val?Glu?His?Thr
290?????????????????295?????????????????300Ile?Arg?Asp?Ile?Leu?Gly?Gly?Ile?Arg?Ser?Thr?Cys?Thr?Tyr?Val?Gly305?????????????????310?????????????????315?????????????????320Ala?Ala?Lys?Leu?Lys?Glu?Leu?Ser?Arg?Arg?Thr?Thr?Phe?Ile?Arg?Val
325?????????????????330?????????????????335Thr?Gln?Gln?Val?Asn?Pro?Ile?Phe?Ser?Glu?Ala?Cys
340 345＜210〉3＜211〉36＜212〉DNA＜213〉synthetic primer＜400〉3ggaattccgc catcatgcct catattgaca acgatg 36＜210〉4＜211〉33＜212〉DNA＜213〉synthetic primer＜400〉4ggaattcgtc gacgcacgcc tcactgaaga ttg 33
Claims (12)
1. isolating people's guanosine 5-monophosphate reductase enzyme GMPR2 polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:
(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ IDNO: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 348-1394 position among the SEQ ID NO:1;
(b) has the sequence of 1-1875 position among the SEQ ID NO:1.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. preparation method with the active polypeptide of people's guanosine 5-monophosphate reductase enzyme GMPR2 is characterized in that this method comprises:
(a) under the condition that is fit to expressing human guanosine 5-monophosphate reductase enzyme GMPR2 polypeptide, cultivate the described host cell of claim 7;
(b) from culture, isolate and have the active polypeptide of people's guanosine 5-monophosphate reductase enzyme GMPR2.
9. energy and the described people's guanosine 5-monophosphate of claim 1 reductase enzyme GMPR2 polypeptid specificity bonded antibody.
10. whether there is the method for guanosine 5-monophosphate reductase enzyme GMPR2 polypeptide in the test sample, it is characterized in that, comprising:
The described antibody of sample and claim 9 is contacted,
Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample guanosine 5-monophosphate reductase enzyme GMPR2 polypeptide.
11. the purposes of polypeptide as claimed in claim 1, it is characterized in that, it is used to screening and promotes the active agonist of people guanosine 5-monophosphate reductase enzyme GMPR2, and perhaps screening suppresses people's guanosine 5-monophosphate active antagonist of reductase enzyme GMPR2 or is used to the peptide finger print identification.
12. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011059664A CN1151251C (en) | 2001-04-12 | 2001-04-12 | New human-phosphoguanosine reductase, its coding sequence and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011059664A CN1151251C (en) | 2001-04-12 | 2001-04-12 | New human-phosphoguanosine reductase, its coding sequence and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1380407A true CN1380407A (en) | 2002-11-20 |
| CN1151251C CN1151251C (en) | 2004-05-26 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011059664A Expired - Lifetime CN1151251C (en) | 2001-04-12 | 2001-04-12 | New human-phosphoguanosine reductase, its coding sequence and application |
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| Country | Link |
|---|---|
| CN (1) | CN1151251C (en) |
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2001
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| CN1151251C (en) | 2004-05-26 |
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