CN1152957C - Human 6-phosphoglucosamine isomerase, its code sequence and application - Google Patents
Human 6-phosphoglucosamine isomerase, its code sequence and application Download PDFInfo
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- CN1152957C CN1152957C CNB011059672A CN01105967A CN1152957C CN 1152957 C CN1152957 C CN 1152957C CN B011059672 A CNB011059672 A CN B011059672A CN 01105967 A CN01105967 A CN 01105967A CN 1152957 C CN1152957 C CN 1152957C
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Abstract
The present invention relates to a novel human 6-phosphoglucosamine (GNPI2). The present invention provides polynucleotide for encoding the protein molecule and a method for generating the protein molecule by the recombination technology. The present invention also discloses applications of the polynucleotide for encoding the novel 6-phosphoglucosamine and also verifies the enzymology activity of GNPI2. The present invention also discloses a strategy of using the protein molecular to diagnose and treat diseases, and especially, the protein molecular can be used for diagnosing and treating tumors, inflammation, immune disorder, reproduction abnormality, etc.
Description
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 (glucosamine-6-phosphate isomerase 2 abbreviates " GNPI2 " 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 GNPI2.Specifically, polypeptide of the present invention is a kind of new and cell proliferation, differentiation, apoptosis, and the relevant intracellular protein of inflammatory reaction.
Background technology
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.
GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 (glucosamine-6-phosphate isomerase, GNPI) (EC5.3.1.10) the mutual conversion between catalysis 6-glucose 1-phosphate1-amine and the fructose-1, 6-diphosphate, mainly be 6-glucose 1-phosphate1-amine through the transformation of aldehyde/ketone structure and take off amine, generate fructose, and then enter tricarboxylic acid cycle.GNPI catalytic reaction be the final step of N-acetyl-6-glucose 1-phosphate1-amine pathways metabolism, to regulating glycosaminoglycan, glycolipid and protein-polysaccharide in intracellular metabolism and utilize again vital role is arranged.Glycosaminoglycan, glycolipid and protein-polysaccharide multidigit are in cytolemma or cell surface, participate in the structure composition of cell, intercellular mutual identification and cell signalling, it is the important biomacromolecule of a class, its metabolic unusual normal imbalance that causes the important cells function is as the generation of infection, immune dysfunction, tumour and transfer etc.The relation that glycosaminoglycan, glycolipid and protein-polysaccharide and disease take place is the focus of life science always.Therefore, regulate its metabolic enzyme and also seem important unusually.
GNPI finds that also purifying comes out at first in intestinal bacteria, have only cerebral tissue and endoerythrocytic GNPI activity once to be studied and to report in human body.Deliver one piece research report of Nature magazine makes GNPI become the focus of concern once more in recent years.This research report is purified into a kind of protein (Oscillin) in the Chinese hamster sperm, and finds that this albumen mass-energy starts the interior stream of mouse ovum calcium ion, and then activates the growth of ovum.And this protein and colibacillary GNPI have the homology of height.After this big quantity research thinks that GNPI and fertilization process are closely related.People's clone and separate from people's sperm goes out GNPI, finds that it mainly is distributed in the acrosome position of sperm, and thinks that it may activate the growth of ovum for fertilization process provides energy by the oxidation energy supply.These studies show that GNPI is also bringing into play important effect in the process of growing.
Studies show that, in view of GNPI may play a significant role in pathologic processes such as infection, tumour, inflammation, and may have important development and application in a plurality of fields such as treatment impotence generandi, anti-infective, anti-inflammatory response, antitumor and immunologic function adjustings and be worth.Therefore, significant for the new GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 of therapeutic purpose research and development.Yet, before the present invention, the GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 of other types was not also disclosed.
Summary of the invention
The purpose of this invention is to provide a kind of new GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2 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.GNPI2 albumen of the present invention is a kind of GNPI homolgous molecule.
Another object of the present invention provides the enzyme activity of GNPI2.
In a first aspect of the present invention, novel isolated GNPI2 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 GNPI 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 158-988 position among the SEQ ID NO:1; (b) has the sequence of 1-1939 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 GNPI2 protein-active, this method comprises: (a) under the proteic condition of suitable expressing human GNPI2, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people GNPI2 protein-active.
In a fifth aspect of the present invention, provide and above-mentioned people GNPI2 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-1939 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people GNPI2 polypeptide active is provided, and the compound that suppresses people GNPI2 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 GNPI2 polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of GNPI2 in the test sample, it comprises: sample is contacted with the proteic specific antibody of GNPI2, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample GNPI2 albumen.
In a eighth aspect of the present invention, a kind of disease relevant with people GNPI2 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 GNPI2 polypeptide active, and perhaps screening suppresses the antagonist of people GNPI2 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people GNPI2 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 GNPI2 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as tumour, inflammation, infection and impotence generandi.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 is the cDNA sequence of people GNPI2 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 GNPI2 of the present invention.Wherein the sequence of underscore is the catalysis territory of 6-glucose 1-phosphate1-amine isomerase.
Fig. 3 is the shown distribution of people GNPI2 Northern blot hybridization of the present invention.Left figure and middle figure are that the healthy tissues of GNPI2 distributes; Right figure is the distribution of GNPI2 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 GNPI2 is a kind of 6-glucose 1-phosphate1-amine isomerase molecule of extensive distribution.
Fig. 4 is Western marking result.1: the empty carrier negative control; 2: elutriant; 3:GNPI2 transgenic cell crude extract; 4: the GNPI2 albumen of purifying.
Fig. 5 is the people GNPI2 of the present invention 6-glucose 1-phosphate1-amine isomerase activity of its intracellular protein and comparison of control group behind the transient expression in cell.1: the empty carrier negative control; 2:GNPI2 transgenic cell crude extract; 3: the GNPI2 albumen of purifying.
Fig. 6 is the enzyme kinetics activity curve of purified GNPI2 expression product of the present invention.
Summary of the invention
In the present invention, term " GNPI2 albumen ", " GNPI2 polypeptide " or " people 6-phosphoric acid grape Osamine isomerase GNPI2 " be used interchangeably, all refer to have GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2 Albumen or the polypeptide of amino acid sequence (SEQ ID NO:2). They comprise contains or does not contain initial methionine GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2.
As used herein, " separation " refers to that material separates (if natural from its primal environment Material, primal environment namely is natural surroundings). Such as the polynucleotide under the native state in the active somatic cell Do not have separation and purification with polypeptide, but same polynucleotide or polypeptide are as existing together from native state Other materials in separately, then for separation and purification.
As used herein, " GNPI2 albumen or the polypeptide of separation " refers to that the GNPI2 polypeptide is substantially free of Natural relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can be with marking Accurate purified technology of protein purifying GNPI2 albumen. Basically pure polypeptide is at non-reduced polyacrylamide Can produce single master tape on the gel. The purity of GNPI2 can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide. Polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis, or uses the restructuring skill Art is from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell) Produce. The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, or can Being nonglycosylated. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analog of people GNPI2 albumen. As used herein, art Language " fragment ", " derivative " and " analog " refer to basically keep natural human GNPI2 of the present invention The biological function that albumen is identical or active polypeptide. Polypeptide fragment of the present invention, derivative or analog can Being that (i) has one or more conservative or non-conservation amino acid residues (preferred conservative amino acid residue) to be got The polypeptide in generation, 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, have the polypeptide of substituted radical, or (iii) mature polypeptide and another Individual compound (such as the compound that prolongs the polypeptide half-life, for example polyethylene glycol) merges formed polypeptide, Or (iv) additional amino acid sequence be fused to this peptide sequence and the polypeptide that forms (such as targeting sequencing or secretion sequence Row or be used for sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion egg of the formation of antigen I gG fragment In vain). According to the instruction of this paper, these fragments, derivative and analog belong to those skilled in the art Known scope.
In the present invention, term " people GNPI2 polypeptide " refers to have the SEQ ID NO. of people GNPI2 protein active The polypeptide of 2 sequences. This term also comprise have with people GNPI2 albumen identical function, SEQ ID NO.2 The variant form of sequence. These variant forms comprise (but being not limited to): several (be generally 1-50, 1-30 preferably, more preferably 1-20,1-10 best) amino acid whose disappearance, insert and/or get Generation, and add one or several and (be generally in 20, preferably be in that C end and/or N are terminal In 10, more preferably be in 5) amino acid. For example, in the art, with the close or phase of performance Like amino acid when replacing, usually can not change the function of protein. Again such as, at C terminal and/ Or one of the terminal interpolation of N or the common function that also can not change protein of several amino acid. This term also comprises Active fragment and the reactive derivative of people GNPI2 albumen.
The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural prominent Variant, induced mutation body, under high or low stringency condition can with the DNA of people GNPI2 DNA hybridization Coded albumen and the polypeptide or the albumen that utilize the antiserum acquisition of anti-people GNPI2 polypeptide. This Brightly also provide other polypeptide, as comprised the fusion of people GNPI2 polypeptide or its fragment. Except almost Outside the polypeptide of total length, the present invention has also comprised the soluble fragments of people GNPI2 polypeptide. Usually, this fragment Have people GNPI2 peptide sequence at least about 10 continuous amino acids, usually at least about 30 Continuance ammines Base acid is preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, the best Ground is at least about 100 continuous amino acids.
Invention also provides the analog of people GNPI2 albumen or polypeptide. These analogs and natural human GNPI2 The difference of polypeptide can be the difference on the amino acid sequence, also can be not affect on the modified forms of sequence Difference perhaps haves both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can Obtaining by various technology, as by radiation or be exposed to mutagens and produce random mutagenesis, also can pass through Direct mutagenesis method or the biological technology of other known moleculars. Analog also comprises having and is different from natural L-ammonia The analog of the residue (such as D-amino acid) of base acid, and have that non-natural exists or synthetic amino acid (as β, gamma-amino acid) analog. Should be understood that polypeptide of the present invention is not limited to the above-mentioned representativeness that exemplifies Polypeptide.
(usually the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external Such as acetylation or carboxylated. Modify and also to comprise glycosylation, such as those in the synthetic and processing of polypeptide or advance one Carry out glycosylation modified and polypeptide that produce in the step procedure of processing. This modification can be by being exposed to polypeptide Carry out glycosylated enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finish. Modified forms also wraps Draw together have the phosphorylated amino acid residue sequence of (such as phosphotyrosine, 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 GNPI2 albumen conservative variation polypeptide " refers to the amino with SEQ ID NO:2 Acid sequence is compared, and has 10 at the most, and preferably at the most 8, more preferably at the most 5, at the most 3 best Amino acid is replaced by similar performance or close amino acid and is formed polypeptide. These conservatives variation polypeptide Carrying out amino acid substitution according to table 1 well produces.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotides of the present invention can be dna form or rna form. Dna form comprise cDNA, Genomic DNA or artificial synthetic DNA. DNA can be strand or double-stranded. DNA can Coding strand or noncoding strand. The coding region sequence of encoding mature polypeptide can be with shown in the SEQ ID NO:1 The variant of the identical or degeneracy of coding region sequence. As used herein, " variant of degeneracy " is at this Referring in bright encodes has the protein of SEQ ID NO:2, but with the code area shown in the SEQ ID NO:1 The differentiated nucleotide sequence of sequence.
The polynucleotides of the mature polypeptide of coding SEQ ID NO:2 comprise: the coding of an encoding mature polypeptide Sequence; The coded sequence of mature polypeptide and various additional code sequence; The coded sequence of mature polypeptide (with appoint The additional code sequence of choosing) and non-coding sequence.
Term " polynucleotides of coded polypeptide " can be the polynucleotides that comprise this polypeptide of encoding, and also can The polynucleotides that also comprise additional code and/or non-coding sequence.
The invention still further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid order with the present invention The polypeptide of row or fragment, analog and the derivative of polypeptide. The variant of these polynucleotides can be natural The variant that the allelic variant of giving birth to or non-natural take place. These nucleotide diversity bodies comprise replace variant, Deletion mutation body and insertion variant. As known in the art, allelic variant is replacing of polynucleotides Change form, it may be replacement, disappearance or the insertion of one or more nucleotides, but can be from not changing in fact Become the function of the polypeptide of its coding.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the 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 GNPI2.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People GNPI2 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 GNPI2 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 GNPI2 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people GNPI2 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 GNPI2 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 GNPI2 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 GNPI2 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 GNPI2 protein function as pharmacological agent GNPI2 protein function.The peptide molecule that can suppress or stimulate people GNPI2 protein function that can be used for seeking therapeutic value with the recombinant human GNPI2 protein screening peptide library of expressing.
On the other hand, the present invention also comprises people GNPI2 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 GNPI2 gene product or fragment.Preferably, refer to that those can combine with people GNPI2 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 GNPI2, comprise that also those do not influence the antibody of people GNPI2 protein function.The present invention also comprise those can with modify or without the people GNPI2 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 GNPI2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human GNPI2 albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler,
Nature256; 495,1975; People such as Kohler,
Eur.J.Immunol.6:511,1976; People such as Kohler,
Eur.J.Immunol.6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people GNPI2 protein function and the antibody that does not influence people GNPI2 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people GNPI2 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 GNPI2 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 GNPI2 can be used in the immunohistochemistry technology, detects the people GNPI2 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people GNPI2, 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 GNPI2 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people GNPI2 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 GNPI2 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 GNPI2 protein positive.
The production of polyclonal antibody can choose GNPI2 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 GNPI2 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 tumour, inflammation, infection, the sick aspect of impotence generandi.When using GNPI2 albumen of the present invention, also can use the other treatment agent simultaneously, as IFN-α, IFN-β, TNF-o, TNF-β etc.
The present invention also provides a kind of pharmaceutical composition, and it contains GNPI2 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 GNPI2 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 GNPI2 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 GNPI2 of the proteic nothing expression of GNPI2 or unusual/non-activity.The GNPI2 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic GNPI2 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the GNPI2 transgenosis to cell.The method that structure carries the recombinant viral vector of GNPI2 gene is found in existing document (Sambrook, et al.).Recombinant human GNPI2 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 GNPI2 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 GNPI2 obtains.During screening, must carry out mark to people GNPI2 protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people GNPI2 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people GNPI2 protein level that is detected in the test can be with laying down a definition the importance of people GNPI2 albumen in various diseases and be used to the disease of diagnosing GNPI2 albumen to work.
Whether having the proteic method of GNPI2 in a kind of detection test sample is to utilize the proteic specific antibody of GNPI2 to detect, and it comprises: sample is contacted with the GNPI2 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample GNPI2 albumen.
The proteic polynucleotide of GNPI2 can be used for the diagnosis and the treatment of GNPI2 protein related diseases.Aspect diagnosis, the proteic polynucleotide of GNPI2 can be used for detecting the proteic expression of GNPI2 GNPI2 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of GNPI2 as the GNPI2 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 GNPI2 albumen and also can detect the proteic transcription product of GNPI2.
The sudden change that detects the GNPI2 gene also can be used for the disease of diagnosing GNPI2 albumen relevant.The form of GNPI2 protein mutation comprises that the point mutation compared with normal wild type GNPI2 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 GNPI2 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 MedicalLibrary).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 1939 bases, and its open reading frame is positioned at the 158-988 position, and the coding total length is 276 amino acid whose people GNPI2 albumen (SEQ ID NO:2).This GNPI2 albumen belongs to the enzyme molecule, and with GNPI aminoacid sequence height homology, consistence can be up to 87%, and similarity then can reach 94%.
In addition, GNPI2 albumen also comprises the conservative catalysis motif (125-143 residue) of GNPI.The Northern engram analysis shows wide expression in tissue.The research prompting of having carried out, GNPI2 may be new people 6-phosphoric acid isomerase, the tool potential is antitumor, anti-inflammatory, the effect of diseases such as treatment impotence generandi.Therefore, GNPI2 albumen or its relevant antagonist, agonist etc. can be diseases such as treatment tumour, inflammation, immune dysfunction and impotence generandi new treatment approach are provided, thereby have great application prospect.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of people GNPI2 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 the sequence of random choose clone's 5 ' end with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a cDNA clone's dna sequence dna is new full-length cDNA.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (as SEQ ID NO:1 and shown in Figure 1), the new protein (as SEQ ID NO:2 and shown in Figure 2) of encoding.This protein is named as human dendritic cell deutero-6-glucose 1-phosphate1-amine isomerase or people GNPI2, and its encoding gene name is people GNPI2 gene.
Sequence SEQ ID NO:1 total length is 1939bp, comprises 5 ' the end non-coding region of 157bp and 3 ' the end non-coding region of 951bp, and coding contains 276 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 31kD.
They are different with known for the BLAST analysis revealed, and with people GNPI aminoacid sequence height homology, consistence reaches 87%, and similarity reaches 94% and belongs to the enzyme molecule.In addition, also comprise a proteic conservative catalysis motif of GNPI (125-143 residue) (Fig. 2) in GNPI2 albumen, this also further points out it may be new GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2.
Embodiment 2: with the encoding sequence of RT-PCR method human cloning GNPI2
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
GAATTCCGCCATCATGAGGCTTGTAATTCTTGATAAC-3 ' (SEQ ID NO:3), antisense primer 5 '-GG
GGTACCGTTTCCATCTTTCATACTGAATAG-3 ' (SEQ IDNO: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 158-986 shown in the SEQ ID NO:1 are identical.
The Northern engram analysis of embodiment 3 GNPI2
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 GNPI2 albumen is a kind of albumen (Fig. 3) of wide expression.
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 GNPI2 DNA as inserting fragment.
5 ' end Oligonucleolide primers sequence of using in the PCR reaction is:
5’-G
GAATTCCGCCATCATGAGGCTTGTAATTCTTGATAAC-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;
3 ' end primer sequence is:
5’-GG
GGTACCGTTTCCATCTTTCATACTGAATAG-3′(SEQ?ID?NO：4)
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people GNPI2 of Kpn I restriction enzyme.
With the PCR product purification that obtains after EcoRI/Kpn I 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 GNPI2 cDNA EcoRI/Kpn I endonuclease bamhi of correct sequence is cloned into expression vector pcDNA3.1 (Pharmacia company), forms carrier pcDNA3.1-GNPI2, transform people DH5 α then.Positive colony is cut evaluation with EcoRI/Kpn I enzyme, and enzyme is cut the capable 2% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted complete GNPI2 encoding sequence.
Carrier for expression of eukaryon pcDNA3.1-GNPI2 is imported 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 obtains people GNPI2 albumen with HiTrap Chelating columns (Pharmacia Biotech) 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 SEQ IDNO:2 are identical as a result.
Embodiment 5: anti-people GNPI2 production of antibodies
The recombinant protein people GNPI2 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 GNPI2 gene translation product with it.Found that antibody can combine with GNPI2 albumen of the present invention specifically.
The enzyme activity of embodiment 6:GNPI2
The people GNPI2 cDNA EcoRI/KpnI endonuclease bamhi of correct sequence in the reorganization pGEM-3ZF carrier that contains the GNPI2 complete encoding sequence that is obtained among the embodiment 4 is cloned into the pcDNA3.1 carrier for expression of eukaryon, import in the people COS7 cell with liposome then, collect cell after 72 hours, (contain 20mMTris, 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 GNPI2 albumen that wherein has molecular weight to coincide.
The detection of the enzyme activity is carried out under 30 ℃ of conditions, represents the activity of enzyme with the increase of fructose content in the product.The 75mM Tris-HCl (pH 7.7) that contains 200 μ l in the reaction system, 0.85mM N-acetyl 6-glucose 1-phosphate1-amine (activator), 20mM 6-glucose 1-phosphate1-amine (substrate) and 20 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 minutes respectively, add the hydrochloric acid of 1.8ml 11.6N and 0.05% resorcin, 77 ℃ were heated 8 minutes, and product detects the light absorption ratio at 420nm place with the spectrophotometric note.Fructose in the enzyme reaction product has special absorption peak at the 420nm place after hydrochloric acid and resorcin effect.The result shows that the product of GNPI2 instantaneous overexpression in the COS7 cell has the activity (Fig. 5 and 6) that catalysis 6-glucose 1-phosphate1-amine is converted into fructose.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Immunology Inst., No.2 Military Medical Univ.
＜120〉GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2, its encoding sequence and purposes
<130>011997
<160>4
<170>PatentIn?version?3.0
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<211>1939
<212>DNA
<213>Homo?sapiens
<220>
<221>CDS
<222>(158)..(988)
<400>1
cgcctttatc?tgcatccggg?tccgtgggat?tcgcgctcca?ctggtcagct?ggggtcgctc 60
tcgggtggtt?gggtgttgct?tgttcccgct?gttccagcgt?cgaagaacca?ttgggtctgc 120
cggtttgaac?ttgttctgga?agctgtgcgt?caccgta?atg?agg?ctt?gta?att?ctt 175
Met?Arg?Leu?Val?Ile?Leu
1 5
gat?aac?tat?gac?ttg?gct?agt?gaa?tgg?gca?gcc?aaa?tac?atc?tgt?aat 223
Asp?Asn?Tyr?Asp?Leu?Ala?Ser?Glu?Trp?Ala?Ala?Lys?Tyr?Ile?Cys?Asn
10 15 20
cgc?atc?att?cag?ttc?aaa?cct?gga?cag?gac?aga?tat?ttt?aca?ctg?ggt 271
Arg?Ile?Ile?Gln?Phe?Lys?Pro?Gly?Gln?Asp?Arg?Tyr?Phe?Thr?Leu?Gly
25 30 35
tta?cca?aca?ggg?agt?aca?cct?tta?gga?tgc?tat?aaa?aaa?cta?ata?gaa 319
Leu?Pro?Thr?Gly?Ser?Thr?Pro?Leu?Gly?Cys?Tyr?Lys?Lys?Leu?Ile?Glu
40 45 50
tat?cat?aag?aat?gga?cac?ctt?tct?ttt?aaa?tat?gtg?aag?acc?ttt?aat 367
Tyr?His?Lys?Asn?Gly?His?Leu?Ser?Phe?Lys?Tyr?Val?Lys?Thr?Phe?Asn
55 60 65 70
atg?gat?gaa?tat?gta?gga?ctt?cca?aga?aat?cat?cct?gaa?agc?tac?cat 415
Met?Asp?Glu?Tyr?Val?Gly?Leu?Pro?Arg?Asn?His?Pro?Glu?Ser?Tyr?His
75 80 85
tct?tat?atg?tgg?aat?aat?ttt?ttt?aag?cat?atc?gat?ata?gat?cct?aat 463
Ser?Tyr?Met?Trp?Asn?Asn?Phe?Phe?Lys?His?Ile?Asp?Ile?Asp?Pro?Asn
90 95 100
aat?gca?cat?atc?ctt?gac?ggg?aat?gct?gca?gat?tta?caa?gca?gaa?tgt 511
Asn?Ala?His?Ile?Leu?Asp?Gly?Asn?Ala?Ala?Asp?Leu?Gln?Ala?Glu?Cys
105 110 115
gat?gct?ttt?gaa?aac?aaa?ata?aaa?gaa?gct?gga?gga?ata?gat?ctt?ttt 559
Asp?Ala?Phe?Glu?Asn?Lys?Ile?Lys?Glu?Ala?Gly?Gly?Ile?Asp?Leu?Phe
120 125 130
gtt?gga?gga?att?ggt?cca?gat?ggt?cat?atc?gct?ttc?aat?gag?cct?gga 607
Val?Gly?Gly?Ile?Gly?Pro?Asp?Gly?His?Ile?Ala?Phe?Asn?Glu?Pro?Gly
135 140 145 150
tcc?agt?tta?gtg?tca?agg?aca?aga?tta?aag?act?cta?gca?atg?gat?acc 655
Ser?Ser?Leu?Val?Ser?Arg?Thr?Arg?Leu?Lys?Thr?Leu?Ala?Met?Asp?Thr
155 160 165
atc?ttg?gca?aat?gcc?aaa?tat?ttt?gat?gga?gat?tta?tca?aaa?gtg?cca 703
Ile?Leu?Ala?Asn?Ala?Lys?Tyr?Phe?Asp?Gly?Asp?Leu?Ser?Lys?Val?Pro
170 175 180
act?atg?gct?cta?act?gtt?ggt?gtg?ggg?aca?gtg?atg?gat?gct?aga?gaa 751
Thr?Met?Ala?Leu?Thr?Val?Gly?Val?Gly?Thr?Val?Met?Asp?Ala?Arg?Glu
185 190 195
gta?atg?atc?ctt?ata?aca?ggg?gca?cac?aag?gca?ttt?gcc?ctg?tac?aaa 799
Val?Met?Ile?Leu?Ile?Thr?Gly?Ala?His?Lys?Ala?Phe?Ala?Leu?Tyr?Lys
200 205 210
gca?ata?gaa?gaa?gga?gtc?aat?cac?atg?tgg?act?gtt?tcc?gct?ttc?cag 847
Ala?Ile?Glu?Glu?Gly?Val?Asn?His?Met?Trp?Thr?Val?Ser?Ala?Phe?Gln
215 220 225 230
cag?cat?ccc?cgg?act?att?ttt?gta?tgc?gat?gaa?gat?gct?act?tta?gaa 895
Gln?His?Pro?Arg?Thr?Ile?Phe?Val?Cys?Asp?Glu?Asp?Ala?Thr?Leu?Glu
235 240 245
tta?aga?gtt?aaa?act?gtg?aaa?tac?ttt?aaa?ggt?cta?atg?cat?gtg?cac 943
Leu?Arg?Val?Lys?Thr?Val?Lys?Tyr?Phe?Lys?Gly?Leu?Met?His?Val?His
250 255 260
aat?aaa?ctt?gtg?gat?cca?cta?ttc?agt?atg?aaa?gat?gga?aac?tga 988
Asn?Lys?Leu?Val?Asp?Pro?Leu?Phe?Ser?Met?Lys?Asp?Gly?Asn
265 270 275
aggagactgg?agcaaaattc?agcttgaatg?aacagagcac?tttttactaa?gtagtagatg 1048
aattttcagc?tatgcaatat?gacaaaacat?ggggaatttt?gaagattgtc?attttttcat 1108
tcgagtctct?atgttaaaca?ttccatattt?tgaatattta?tatcttgtac?ttgggtttaa 1168
gagaagtagc?tggctctcaa?gattgactgg?ctatttatta?taaagtactg?aagtcacata 1228
gccacctata?aaacagcata?gaaatgtctg?cctgtttaaa?aagtcatttt?aaaggtagag 1288
tgtccacatc?aggcaccatt?tgtgatatga?ctccagtggc?atatatttca?ttttttaatg 1348
acaagacact?ccaaaccttt?cagataacaa?actatcattg?cagaccttca?cttttggaat 1408
gcaatcttta?tattttctgt?gcatcacaca?catgcttttc?tgcacgtggt?tgccttagtc 1468
atcttcctac?agcaccatct?agacatcaaa?aattgtgcta?tatatcattg?gtaaaggaaa 1528
tttgaagaga?tgacagtgcc?taaaagtaca?gtttacatcc?ttttggaaag?tatgtgtaag 1588
tgcatgtttt?ttgtgcacct?tcttctatag?cactttttta?caaatatctt?atttttattt 1648
aacgacttgg?gttcatgtcc?ctaatataag?tatcttgaca?attatgagct?ttatacctag 1708
caagccactt?caggaaattc?ttttggagaa?tattttctga?ttattgttaa?acttaatata 1768
caattagctt?tattccttat?aaaatgtcta?aaagaataat?acgaagtata?tataaaagga 1828
attactgtaa?actacattgc?catagcaatt?tacataaaag?tatattgttt?tctatcttta 1888
actcaaataa?agcgtgtaat?aaataagtta?aaaaaaaaaa?aaaaaaaaaa?a 1939
<210>2
<211>276
<212>PRT
<213>Homo?sapiens
<400>2
Met?Arg?Leu?Val?Ile?Leu?Asp?Asn?Tyr?Asp?Leu?Ala?Ser?Glu?Trp?Ala
1 5 10 15
Ala?Lys?Tyr?Ile?Cys?Asn?Arg?Ile?Ile?Gln?Phe?Lys?Pro?Gly?Gln?Asp
20 25 30
Arg?Tyr?Phe?Thr?Leu?Gly?Leu?Pro?Thr?Gly?Ser?Thr?Pro?Leu?Gly?Cys
35 40 45
Tyr?Lys?Lys?Leu?Ile?Glu?Tyr?His?Lys?Asn?Gly?His?Leu?Ser?Phe?Lys
50 55 60
Tyr?Val?Lys?Thr?Phe?Asn?Met?Asp?Glu?Tyr?Val?Gly?Leu?Pro?Arg?Asn
65 70 75 80
His?Pro?Glu?Ser?Tyr?His?Ser?Tyr?Met?Trp?Asn?Asn?Phe?Phe?Lys?His
85 90 95
Ile?Asp?Ile?Asp?Pro?Asn?Asn?Ala?His?Ile?Leu?Asp?Gly?Asn?Ala?Ala
100 105 110
Asp?Leu?Gln?Ala?Glu?Lys?Asp?Ala?Phe?Glu?Asn?Lys?Ile?Lys?Glu?Ala
115 120 125
Gly?Gly?Ile?Asp?Leu?Phe?Val?Gly?Gly?Ile?Gly?Pro?Asp?Gly?His?Ile
130 135 140
Ala?Phe?Asn?Glu?Pro?Gly?Ser?Ser?Leu?Val?Ser?Arg?Thr?Arg?Leu?Lys
145 150 155 160
Thr?Leu?Ala?Met?Asp?Thr?Ile?Leu?Ala?Asn?Ala?Lys?Tyr?Phe?Asp?Gly
165 170 175
Asp?Leu?Ser?Lys?Val?Pro?Thr?Met?Ala?Leu?Thr?Val?Gly?Val?Gly?Thr
180 185 190
Val?Met?Asp?Ala?Arg?Glu?Val?Met?Ile?Leu?Ile?Thr?Gly?Ala?His?Lys
195 200 205
Ala?Phe?Ala?Leu?Tyr?Lys?Ala?Ile?Glu?Glu?Gly?Val?Asn?His?Met?Trp
210 215 220
Thr?Val?Ser?Ala?Phe?Gln?Gln?His?Pro?Arg?Thr?Ile?Phe?Val?Cys?Asp
225 230 235 240
Glu?Asp?Ala?Thr?Leu?Glu?Leu?Arg?Val?Lys?Thr?Val?Lys?Tyr?Phe?Lys
245 250 255
Gly?Leu?Met?His?Val?His?Asn?Lys?Leu?Val?Asp?Pro?Leu?Phe?Ser?Met
260 265 270
Lys?Asp?Gly?Asn
275
<210>3
<211>38
<212>DNA
＜213〉synthetic primer
<400>3
ggaattccgc?catcatgagg?cttgtaattc?ttgataac 38
<210>4
<211>32
<212>DNA
＜213〉synthetic primer
<400>4
ggggtaccgt?ttccatcttt?catactgaat?ag 32
Claims (12)
1. isolating GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2 polypeptide is characterized in that this polypeptide is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of 1-10 amino-acid residue, and have function that catalysis 6-glucose 1-phosphate1-amine is converted into fructose by (a) polypeptides derived.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
3. isolating polynucleotide is characterized in that, it contains a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ 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 158-988 position among the SEQ ID NO:1;
(b) has the sequence of 1-1939 position among the SEQ ID NO:1.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. preparation method with polypeptide of GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2 polypeptide active is characterized in that this method contains following steps:
(a) being fit to express under the condition of GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2 polypeptide, cultivate the described host cell of claim 7;
(b) from culture, isolate polypeptide with GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2 polypeptide active.
9. energy and the described GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2 of claim 1 polypeptid specificity bonded antibody.
10. whether there is the method for 6-glucose 1-phosphate1-amine isomerase GNPI2 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 6-glucose 1-phosphate1-amine isomerase GNPI2 polypeptide.
11. the purposes of polypeptide as claimed in claim 1, it is characterized in that, it is used to screen the agonist that promotes GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2 polypeptide active, and perhaps screening suppresses the antagonist of GLUCOSAMINE-6-PHOSPHATE ISOMERASE 2 GNPI2 polypeptide active 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 |
|---|---|---|---|
| CNB011059672A CN1152957C (en) | 2001-04-12 | 2001-04-12 | Human 6-phosphoglucosamine isomerase, its code sequence and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011059672A CN1152957C (en) | 2001-04-12 | 2001-04-12 | Human 6-phosphoglucosamine isomerase, its code sequence and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1380413A CN1380413A (en) | 2002-11-20 |
| CN1152957C true CN1152957C (en) | 2004-06-09 |
Family
ID=4655027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011059672A Expired - Lifetime CN1152957C (en) | 2001-04-12 | 2001-04-12 | Human 6-phosphoglucosamine isomerase, its code sequence and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1152957C (en) |
-
2001
- 2001-04-12 CN CNB011059672A patent/CN1152957C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CN1380413A (en) | 2002-11-20 |
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