CN1256312A - New human glutathione peroxidase and its code sequence, preparation and use - Google Patents
New human glutathione peroxidase and its code sequence, preparation and use Download PDFInfo
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Abstract
The present invention relates to one new kind of glutathione peroxidase gene pGPxH. The present invention provides the cDNA code sequence thereof, the sequence-encoded polypeptide and the production process of utilizing recombination technology to produce the new human glutathione peroxidase. The present invention also provides the application of the new human glutathione peroxidase.
Description
The present invention relates to a kind of new polynucleotide, by the polypeptide of this polynucleotide encoding, the application of these polynucleotide and polypeptide, and the production of described polynucleotide and described polypeptide.More particularly, polypeptide of the present invention is accredited as a newcomer of glutathione peroxidase family by deduction.
The glutathione peroxidase family member extensively is present in each tissue, organ of organism; with the Triptide is the reduction of reductive agent catalysis organo-peroxide and hydrogen peroxide; the protection cell in normal oxygen metabolism, do not sustain damage (Nutri.Rev., 1980 (38), 265-273).
As far back as nineteen fifty-seven, just detect glutathione peroxidase (glutathioneperoxidase abbreviates " GPx " as, has another name called " GSHPx ") in vivo, and find it have the important physical activity (J.Biol.Chem., 1957 (229), 189-197).
Found simultaneously by Rotruck group and Flohe group that each monomer of glutathione peroxidase all had a selenium (Science in 1973,1973 (179), 588-590), found afterwards that this proteic the 21 amino acid was seleno-cysteine, this is first found proteolytic enzyme that contains selenium.
Afterwards, find phosphatide superoxide glutathione peroxidase (phospholipid hydroperoxideGPx successively, abbreviate " PHGPx " as), blood plasma glutathione peroxidase (glycosylated plasma GPx, abbreviate " pGPx " as), stomach glutathione peroxidase (gastrointestinal GPx, abbreviate " GI-GPx " as) (Biochim Biophys Acta, 1982 (710), 197-211, J.Biol.Chem, 1987 (262), 17398-17403, Arch.Biochem.Biophys., 1987 (254), 9-17) wait the peroxidase that contains selenium.
At present, glutathione peroxidase family mainly is made up of above-mentioned four kinds of seleno-protein enzymes, and four genes of coding GPx, GI-GPx, pGPx and PHGPx are called after GPx1, GPx2, GPx3 and GPx4 (Arch.Biochem.Biophys., 1997 respectively, Vol340 (1), 59-63).Dephosphorization lipoperoxide glutathione peroxidase is that molecular weight is outside the monomer of 19kD, and its excess-three kind enzyme is the tetramer, and each monomer molecule amount is about 22kD.Homology between three kinds of tetramer enzymes is higher, and homology has only 30% between the phosphatide superoxide glutathione peroxidase of they and single aggressiveness.But two two exons with tetramer enzyme in should 7 exons of list aggressiveness enzyme have high homology, suffice to show that they belong to same gene family (Methods in Enzymol, 1995 (252), 38-53).
Among this family member, (1) glutathione peroxidase is present in all cells, (2) phosphatide superoxide glutathione peroxidase reduces the peroxide derivative of phosphatide and cholesterol and the cholesterol ester (J.Biol.Chem. in film and the low-density lipoprotein (LDL) specifically, 1990 (265), 454-461), (3) stomach glutathione peroxidase then mainly is present in (J.Biol.Chem, 1993 (268) in the gi tract, 2571-2576), above-mentioned three kinds of enzymes are desmo enzyme.And the blood plasma glutathione peroxidase is a perienzyme, mainly be present in the blood plasma (Nutri.Rev., 1980 (38), 265-273).As perienzyme unique in this family, blood plasma glutathione peroxidase and other three kinds of enzymes have marked difference: it and other three kinds of enzymes only have 44% identity on protein level, therefore the antibody of anti-blood plasma glutathione peroxidase can not have cross reaction (Blood with other three kinds of enzymes, 1986 (68), 640-645); And the blood plasma glutathione peroxidase has glycosylation modified, other three kinds of enzymes do not have (Blood, 1989 (73), 318-320).Though four kinds of enzymes are variant in primary structure, but their avtive spot is seleno-cysteine, tryptophane, the glutamine of catalytic site especially, be very conservative, this three places residue and enzyme and substrate Triptide (glutathione, GSH) relevant (Biomed.Environ.Sci. of bonded specificity, 1997 (10), 327-332).The constitutional features of this family is, all have by usually as the coded seleno-cysteine of terminator codon TGA (EMBO J., 1986, Vol5 (6), 1221-1227).
The glutathione peroxidase family member all can be with SOD disproportionation superoxide radical (O
2) and the H of generation
2O
2Be transformed into H
2O and remove it, thus hydroxy radical qiao (OH), O prevented
2Generation, and then prevent the generation (sick magazine, 1991 (6), 337 learned of place of china) of lipid peroxide.Studies show that the formation of free radical and lipid peroxide can cause cytolemma infringement, the main diseases that is considered to numerous disease because of, as cardiovascular disordeies such as atherosclerosis, coronary heart disease and diabetes.Because this family member can effectively reduce the toxicity of superoxide, therefore relevant with the mechanism of multiple disease, in pathological study and clinical application, all have great importance.Wherein, the blood plasma glutathione peroxidase is a topmost peroxidase in the blood plasma, be secreted in the blood plasma after synthesizing by kidney, the reduction reaction of all superoxide in the responsible blood plasma (J.Biol.Chem, 1994, Vol269 (43), 27066-27073).
These four kinds of glutathione peroxidases are all found in the human body.Wherein, people's blood plasma glutathione peroxidase gene (pGPx) in 1993 by people such as Shinichi Y clone (Gene, 1994 (145), 293-297).
Yet, before the application, human glutathione peroxidase involved in the present invention or its sequence were not disclosed.
An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding glutathione peroxidase gene family, the new PGPXH gene of the present invention is named as pGPxH.
Another object of the present invention provides a kind of new glutathione peroxidase protein family member, and this enzyme is named as pGPxH.
A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new PGPXH.
The invention still further relates to the application of this PGPXH encoding sequence and polypeptide.
New albumen and people's pGPx have higher homology among the present invention, and they have 91% identity on protein level.In view of existing certain difference between the two, therefore with its called after pGPxH.
In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people pGPxH protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 16-696 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 16-696 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from the 16-696 position.
In another aspect of this invention, provide a kind of isolating pGPxH protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.
In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.
In another aspect of this invention, provide a kind of described carrier transformed host cells.
In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people pGPxH protein-active, this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of people pGPxH protein-active operationally is connected in expression regulation sequence, form people pGPxH protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 16-696 position among described nucleotide sequence and the SEQ ID NO.3;
(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people pGPxH;
(c) under the condition that is fit to expressing human pGPxH protein polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with people pGPxH protein-active.
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 707 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 16-696 position Nucleotide.
In the present invention, " isolating ", purifying " or " pure substantially " DNA be meant; this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state; refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of following it in cell.
In the present invention, term " pGPxH albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people pGPxH protein-active is as 16-696 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 16-696 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 16-696 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, preferably under the height stringent condition, with among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 16-696 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 16-696 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.
This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.3 with people pGPxH identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.
In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.
In the present invention, term " pGPxH protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people pGPxH protein-active.This term also comprises having and the variant form PGPXH identical function, SEQ ID NO.4 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 pGPxH 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 pGPxH DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-pGPxH polypeptide to obtain.The present invention also provides other polypeptide, as comprises pGPxH polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of pGPxH polypeptide.Usually, this fragment have the pGPxH 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 pGPxH albumen or polypeptide.The difference of these analogues and natural pGPxH 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 can also comprise 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 pGPxH conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.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; Nor-leucine | Leu |
| Leu(L) | Nor-leucine; 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; Nor-leucine | Leu |
The present invention also comprises pGPxH polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of pGPxH in the cell.
The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of pGPxH nucleotide sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the pGPxH that encodes.
The present invention also comprises the method that detects the pGPxH nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of pGPxH polypeptide.Primer length is generally 20-50 Nucleotide.
In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
On the other hand, the present invention also comprises pGPxH DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into pGPxH gene product or fragment.Preferably, refer to that those can combine with pGPxH 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 pGPxH, comprise that also those do not influence the antibody of pGPxH protein function.The present invention also comprise those can with modify or without the pGPxH 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 pGPxH gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing pGPxH or its has 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 to prepare that (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 the pGPxH function and the antibody that does not influence the pGPxH function.Each antibody-like of the present invention can utilize the fragment or the functional zone of pGPxH gene product, obtains by immunological technique.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 pGPxH 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).
People pGPxH nucleotide sequence 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 relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about 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 with its human cloning carrier, changes cell over to again, 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.
In one embodiment of the invention, polynucleotide total length of the present invention is 707 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at the upright Nucleotide of 16-696.These polynucleotide are so to obtain: with people's kidney λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer A:5 '-GCGCCCCAGCCCGCCATGGC-3 ' and reverse primer B:5 '-ACGGCAATCAGTTACTTCCTC TTC-3 ' carry out PCR, obtain the purpose fragment of 707bp, obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.
According to homology result relatively, the glutathione peroxidase of nucleotide sequence of the present invention and encoded protein matter sequence and different sources has shown significant homology, therefore, this shows that it is a newcomer of glutathione peroxidase family, and has some critical functions of glutathione peroxidase family protein.
The glutathione peroxidase family member is reductive agent with the Triptide, the reduction of catalysis organo-peroxide and hydrogen peroxide, the protection cell in normal oxygen metabolism, do not sustain damage (Nutri.Rev., 1980 (38), 265-273).Concrete mechanism is: this family member all can be with SOD disproportionation superoxide radical (O
2) and the H of generation
2O
2Be transformed into H
2O and remove it, thus hydroxy radical qiao (OH), O prevented
2Produce, and then prevent the generation (sick magazine, 1991 (6), 337 learned of place of china) of lipid peroxide.PGPxH of the present invention can reduce the toxicity of superoxide, thereby may be relevant with the mechanism of multiple disease, all has great importance in pathological study and clinical application.
In the accompanying drawings, Fig. 1 is the homology comparison diagram of the nucleotide sequence of people pGPxH of the present invention and mouse pGPx (MpGPx).Wherein, identical Nucleotide marks with " | ".
Fig. 2 is the homology comparison diagram of the aminoacid sequence of people pGPxH of the present invention and mouse pGPx (MpGPx).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.
Fig. 3 is the homology comparison diagram of the aminoacid sequence of people pGPxH of the present invention and people pGPx (pGPx).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.
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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The clone and the mensuration of the cDNA sequence of pGPxH
1. primer amplification
With people's kidney λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A:5 '-GCGCCCCAGCCCGCCATGGC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B:5 '-ACGGCAATCAGTTACTTCCTCTTC-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains is the purpose fragment of about 700bp.
2.PCR the order-checking of product
With pcr amplification product and the pGEM-T that as above obtains
Carrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL
TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 707bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 16-696 position Nucleotide.
Derive the aminoacid sequence of pGPxH according to the full length cDNA sequence that obtains, totally 226 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.
Homology relatively
Full length cDNA sequence and proteins encoded thereof with pGPxH of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with BLAST software.Found that the member of it and glutathione peroxidase family has very high homology.Wherein the blood plasma glutathione peroxidase gene pGPx with the people reaches 91% identity on protein level, 95% similarity (Fig. 3), on nucleic acid level, reach 98.4% identity (Fig. 1) with the blood plasma glutathione peroxidase gene pGPx of mouse, on protein level, reach 97.8% identity, 98.2% similarity (Fig. 2).Therefore, this shows that new albumen of the present invention is a kind of new blood plasma glutathione peroxidase.
Particularly in the aminoacid sequence of pGPxH of the present invention, exist by the coded seleno-cysteine of the terminator codon TGA on the ordinary meaning (abbreviating " SeCys " as) (the 73rd amino acids among the SEQ ID NO.4).This TGA codon connects when translation to be read, thereby makes seleno-cysteine just enter the protein polypeptide chain when protein translation, but not the formation of modification afterwards.
In order to realize correct deciphering TGA (in mRNA, being UGA) terminator codon, seleno-cysteine is inserted seleno-protein, need the complicated translating mechanism of a cover.This mechanism in prokaryotic organism, illustrate substantially (TIBS, 1991 (16), 463-467), but the mechanism in the eukaryote is still waiting to continue research.Known, in prokaryotic organism, relate to four genes, be respectively SelA, B, C and D (TIBS, 1991 (16), 463-467).By SelC a kind of special tRNA that encodes
Ser, this tRNA
SerMore common tRNA is big, and anticodon is and terminator codon UGA complementary UCA, the amino acid that carries be Serine (Nature, 1988 (331), 723-725).At tRNA by the SelA coding
SeCysUnder the catalysis of (carrying the tRNA of seleno-cysteine) synthetic enzyme and selenium Phosphoric acid esterase, tRNA
SerBe converted into tRNA
SeCys(Proc.Natl.Acad.Sci.USA, 1990 (87), 543-547).Cofactor by SelB coding both can be discerned specific UGA, again can with tRNA
SeCysCombination specifically (Nature, 1989 (342), 453-456).Under the help of cofactor, tRNA
SeCysOn the position of UGA correspondence, inserted seleno-cysteine.In the said process, the selenium Phosphoric acid esterase be the enzyme by SelD coding utilize ATP and selenium synthetic (J.Bacteriol, 1988 (170), 540-546).SelD be considered to express seleno-protein factor of determination (Biomed.Environ.Sci., 1997 (10), 163-176).Above-mentioned four kinds of genes all find in E.coli at first, and at present, except that SelB, its excess-three kind gene has all found homologous gene in eukaryote, and are similar in function and the prokaryotic organism.
It is common constitutional features (the EMBO J of glutathione peroxidase family that terminator codon TGA is translated as seleno-cysteine, 1986, Vol5 (6), 1221-1227), therefore, this has determined that more blood plasma glutathione peroxidase of the present invention belongs to this family, and has the correlation function of this glutathione peroxidase family protein.
Discover that the 5 ' end of the pGPxH of the present invention segment signal peptide (between SEQ ID NO.4 the 24th, 25 amino acids for cleavage site) of having encoded is secreted in cell with the blood plasma glutathione peroxidase that these characteristics meet in the blood plasma after synthetic.
Studies show that lipid peroxide is the primary product of unsaturated fatty acids enzyme digestion reaction and non-enzyme digestion reaction.They are the intermediates in the bioactive molecules building-up processes such as prostaglandin(PG), leukotriene.Lipid peroxide can with transition metal composite and the very high free radical (Cytochrome, 1986,29-76, Plenum Press) of metalloprotein qualitative response toxigenicity.Except that the toxicity of free radical, the toxicity of superoxide also show they in the arachidonic acid cascade reaction, have the active effect of regulatory enzyme (N.Engl.J.Med., 1979 (300), 1142-1147).The formation of free radical and lipid peroxide causes cytolemma infringement, the main diseases that is considered to numerous disease because of, as cardiovascular disordeies such as atherosclerosis, coronary heart disease and diabetes.Recent study finds, artery congee sample pathology be attended by the lipid peroxide level rising (trace element and health research, 1996,13 1 phases of volume, 61-63).In normal metabolism, mainly lipid peroxide is reduced to corresponding alcohol by peroxidase.The glutathione peroxidase family member all can be with SOD disproportionation superoxide radical (O
2) and the H of generation
2O
2Be transformed into H
2O and remove it, thus hydroxy radical qiao (OH), O prevented
2Generation, and then prevent the generation (sick magazine, 1991 (6), 337 learned of place of china) of lipid peroxide.The generation of following multiple disease is relevant with this family member.As:
(1) in heart, this family member eliminates H
2O
2Important enzyme.When enzymic activity reduces, the active oxygen of in time not eliminated makes and unsaturated fatty acids active oxidation on the cytolemma generates lipid peroxide, makes that the concentration of lipid peroxide increases in the heart, infringement membrane structure and function, make the flowability and the permeability of film unusual, cardiac muscle obviously reduces the detoxification ability of superoxide, finally causes the irreversible damage of myocardial cell, and chief cell necrosis (the sick magazine of learning of place of china, 1992,11 (2), 81).
(2) atherosclerosis is a feature with the lipid metabolism disorders, is the pathologic basis of cardiovascular and cerebrovascular diseases such as coronary heart disease.Atherosclerotic's body lipid peroxide concentrations raises, and easily forms thrombus, and vascular damaged district cholesterol deposits and vascular smooth muscle hyperplasia.The activity that body includes the selenium glutathione peroxidase increases the cholesterol that the energy considerable damage gathers at the vascular damaged place, and blood lipid metabolism in the control agent prevents or reverse the generation (gerontology magazine, 1991,11 (1), 49) of Atherosclerosis.Glutathione peroxidase is active in the patients with coronary heart disease body increases, and can alleviate lipid peroxidation, quickens the repair process of myocardial infarction cell, increases the volume of blood flow of coronary vasodilator, reduces myocardial consumption of oxygen, helps the improvement of myocardial cell's function.
Glutathione peroxidase family member's activity is relevant with trace element-selenium, because in the active centre of all glutathione peroxidases, selenium is connected on the peptide chain of enzyme with the form of seleno-cysteine.In clinical application, can in sufferer (as chronic kidney hypofunction, the patients with coronary heart disease) body of being badly in need of the raising resistance of oxidation, increase the content of selenium, regulate peroxidase activity.But in some tissue, glutathione peroxidase reduction lipid peroxide generates the precursor of leukotriene, and this precursor participates in as inflammatory reactions such as asthma and rheumatoid arthritiss.Therefore in the arthritic medicine of design, can consider with the antagonist of glutathione peroxidase as the medicine that reduces inflammation (J.Biol.Chem., 1984 (259), 1043-1050).
The blood plasma glutathione peroxidase is a topmost peroxidase in the blood plasma, in the blood plasma reduction reaction of nearly all superoxide by its be responsible for (J.Biol.Chem., 1987, Vol262 (36), 17398-17403).Because the specific position of blood plasma glutathione peroxidase in blood plasma, therefore except that above-mentioned this family member's common function, the blood plasma glutathione peroxidase is also relevant with the mechanism of multiple disease specifically.As:
Because the main generation of blood plasma glutathione peroxidase and secretion are therefore relevant with the pathology of uremia in renal prostration disease with arteriosclerosis all at kidney.There are a large amount of reactive oxygen specieses in chronic renal failure patients's the kidney, resistance of oxidation but descends, thereby can't in time remove reactive oxygen species, make reactive oxygen species more outstanding to the effect that renal function worsens, concrete toxicity shows as: lipid peroxidation, membrane permeability increase, the protein biological activity is impaired, cell is subjected to damaging widely, hypofunction or forfeiture lose and recover and multiplication capacity.Improve peroxidase activity, can improve the development of removing the ability delaying chronic kidney hypofunction of reactive oxygen species in chronic kidney hypofunction or the body.(J.Lab?Clin?Med.，1994(124)，468-469)
The new blood plasma glutathione peroxidase of the present invention is understood in depth for the 26S Proteasome Structure and Function of blood plasma glutathione peroxidase, and pathological study and clinical application all have great importance.
People pGPxH of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor pGPxH can also merge with other members of this family or exchange fragment, to produce new albumen.For example the C end of the C of inventor pGPxH end with the pGPx of mouse exchanged, to produce the albumen that new activity is higher or have new features.
At the antibody of inventor pGPxH, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family, such as mouse pGPx albumen).
In addition, inventor pGPxH nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people pGPxH or the overexpression that suppresses people pGPxH.People pGPxH albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people pGPxH disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.
Embodiment 3
The expression of pGPxH in intestinal bacteria
The PCR Oligonucleolide primers of the cDNA sequence of coding pGPxH corresponding to 5 of this dna sequence dna ' and 3 ' end is that template increases with the pcr amplification product among the embodiment 1, with synthetic insertion fragment.
5 ' Oligonucleolide primers sequence is
5′-TTGCGGATCCATGCAAGAGAAGTCTAAGA-3′(SEQ?ID?NO.5)，
This primer contains the restriction enzyme site of BamHI restriction enzyme, connect be initiator codon and the pGPxH encoding sequence that the 88th Nucleotide begins from SEQ IDNO.3 16 Nucleotide (in SEQ ID NO.3 from the 16th Nucleotide (A) to the 87th Nucleotide (A) coding be a segment signal peptide sequence, when expression in escherichia coli, this section sequence is removed);
3 ' Oligonucleolide primers sequence is
5’-GTTCGTCGACTTACTTCCTCTTGACCCCC-3’(SEQ?ID?NO.6)，
This primer contains the restriction enzyme site of SalI restriction enzyme, the encoding sequence of translation termination and pGPxH.
The restriction enzyme site of restriction enzyme is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp
r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.
With BamHI and SalI digestion pQE-9 carrier, the insertion fragment that will digest equally subsequently is connected to the pQE-9 carrier and keeps open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E-coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan
r).Screen transformant containing on the LB culture dish of Amp and Kan, incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).The extracting plasmid is cut with the ApaI enzyme and identify to be inserted people's clip size and direction, and sequence verification result shows that the cDNA of pGPxH inserts the fragment carrier of correctly packing into.
Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum and (in substratum, be added with the Sodium Selenite of 10% foetal calf serum and 5ng/ml), culturing cell grows to 600 optical density(OD) (OD
600) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved pGPxH from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out pGPxH from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.At first, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein can be incorporated in second post from nickel-chelate column, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.
SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 23kDa.
In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.
Embodiment 4
The expression of pGPxH in eukaryotic cell (Chinese hamster ovary celI strain)
In this embodiment, the cDNA sequence of coding pGPxH being used PCR Oligonucleolide primers corresponding to 5 of this dna sequence dna ' and 3 ' end, is that template increases with the pcr amplification product among the embodiment 1, with synthetic insertion fragment.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5′-TTGCAAGCTTATGGCCCGGATCCTCCGGG-3′(SEQ?ID?NO.7)，
This primer contains the restriction enzyme site of HindIII restriction enzyme, and what connect is 19 Nucleotide of the pGPxH encoding sequence that begun by initiator codon;
3 ' end primer sequence is:
5’GTTCGGATCCTTACTTCCTCTTGACCCCC-3’(SEQ?ID?NO.8)
This primer contains the encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and pGPxH.
The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp
rAnd Neo
r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.
With HindIII, BamHI digestion pcDNA3 carrier, the insertion fragment that will digest equally subsequently is connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, sequence verification result shows that the cDNA of pGPxH inserts the fragment carrier of correctly packing into.
Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.Cultivate above-mentioned positive subclone and (and in substratum, add the Sodium Selenite of 10% foetal calf serum and 7.5ng/ml in a large number according to a conventional method.Though seleno-cysteine mixes proteic expression mechanism and does not illustrate fully as yet in eukaryote, but studies show that, in substratum, improve the content of selenium, just can promote the expression (Biomed.Environ.Sci. of seleno-protein significantly, 1997 (10), 163-176)).After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mM TrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.
SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 25kDa.
In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.
Embodiment 5
Preparation antibody
The recombinant protein that as above obtains is used for immune animal to produce antibody, specific 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 pGPxH gene translation product with it.Found that antibody can precipitate with albumen of the present invention specifically.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table (1) general information: (i) applicant: Fudan University is denomination of invention (ii): new PGPXH, its encoding sequence and method for making and purposes be the sequence number (iii): information (i) sequence signature of 8 (2) SEQ ID NO.1
(A) length: 20 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.1:GCGCCCCAGC CCGCCATGGC 20 (2) SEQ ID NO.2
(A) length: 24 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:2ACGGCAATCA GTTACTTCCT CTTC 24 (2) SEQ ID NO.3:
(i) sequence signature:
(A) length: 707bp
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: cDNA
( xi ) ：SEQ ID NO.3GCGCCCCAGC CCGCCATGGC CCGGATCCTC CGGGATTCCT GCCTTCTGTC CCTGCTCCTG 60GCCGGGTTTG TTCCGCCGGG CCGGGGACAA GAGAAGTCTA AGACAGACTG CCATGGCGGT 120ATGAGTGGTA CCATCTACGA GTATGGAGCC CTCACCATCG ATGGGGAGGA ATACATTCCT 180TTTAAGCAGT ATGCAGGCAA ATATATCCTC TTTGTCAACG TAGCCAGCTA CTGAGGTCTG 240ACAGACCAAT ACCTTGAACT GAATGCACTA CAAGAAGAAC TTGGGCCATT TGGCTTGGTC 300ATTCTGGGCT TCCCTTCCAA CCAATTTGGC AAACAGGAGC CAGGCGAGAA CTCGGAGATA 360CTCCCCAGTC TCAAGTATGT TCGACCAGGT GGGGGCTTTG TGCCTAATTT CCAGCTCTTT 420GAGAAAGGAG ATGTGAACGG GGAGAAAGAG CAGAAATTCT ACACTTTCCT GAAGAACTCC 480TGTCCTCCCA CTGCAGAGCT CCTGGGCTCA CCTGGCCGCC TCTTTTGGGA ACCCATGAAG 540ATCCATGACA TCCGCTGGAA CTTTGAGAAG TTCCTGGTGG GGCCAGATGG TATACCGGTT 600ATGCGCTGGT ACCACCGGAC CACAGTCAGC AACGTCAAGA TGGACATCCT GTCCTACATG 660AGGCGGCAGG CAGCCCTGGG GGTCAAGAGG AAGTAACTGA TTGCCGT 707 ( 2 ) SEQ ID NO.4：
(i) sequence signature:
(A) length: 226 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: polypeptide
(A) describe: the Xaa that/desc=＂ is the 73rd is the seleno-cysteine ＂ by the TGA coding
( xi ) ：SEQ ID NO.4Met Ala Arg Ile Leu Arg Asp Ser Cys Leu Leu Ser Leu Leu Leu 15Ala Gly Phe Val Pro Pro Gly Arg Gly Gln Glu Lys Ser Lys Thr 30Asp Cys His Gly Gly Met Ser Gly Thr Ile Tyr Glu Tyr Gly Ala 45Leu Thr Ile Asp Gly Glu Glu Tyr Ile Pro Phe Lys Gln Tyr Ala 60Gly Lys Tyr Ile Leu Phe Val Asn Val Ala Ser Tyr Xaa Gly Leu 75Thr Asp Gln Tyr Leu Glu Leu Asn Ala Leu Gln Glu Glu Leu Gly 90Pro Phe Gly Leu Val Ile Leu Gly Phe Pro Ser Asn Gln Phe Gly 105Lys Gln Glu Pro Gly Glu Asn Ser Glu Ile Leu Pro Ser Leu Lys 120Tyr Val Arg Pro Gly Gly Gly Phe Val Pro Asn Phe Gln Leu Phe 135Glu Lys Gly Asp Val Asn Gly Glu Lys Glu Gln Lys Phe Tyr Thr 150Phe Leu Lys Asn Ser Cys Pro Pro Thr Ala Glu Leu Leu Gly Ser 165Pro Gly Arg Leu Phe Trp Glu Pro Met Lys Ile His Asp Ile Arg 180Trp Asn Phe Glu Lys Phe Leu Val Gly Pro Asp Gly Ile Pro Val 195Met Arg Trp Tyr His Arg Thr Thr Val Ser Asn Val Lys Met Asp 210Ile Leu Ser Tyr Met Arg Arg Gln Ala Ala Leu Gly Val Lys Arg 225Lys 226
(2) information of SEQ ID NO.5
(i) sequence signature
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: the information of SEQ ID NO.5:TTGCGGATCC ATGCAAGAGA AGTCTAAGA 29 (2) SEQ ID NO.6
(i) sequence signature
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: information (i) sequence signature of SEQ ID NO.6:GTTCGTCGAC TTACTTCCTC TTGACCCCC 29 (2) SEQ ID NO.7
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.7:TTGCAAGCTT ATGGCCCGGA TCCTCCGGG 29 (2) SEQ ID NO.8
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.8:GTTCGGATCC TTACTTCCTC TTGACCCCC 29
Claims (14)
1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people pGPxH protein-active,
Show at least 70% homology from the nucleotides sequence of Nucleotide 16-696 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps
Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 16-696 position.
2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.4.
3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 16-696 position.
4. isolating pGPxH protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.
5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.
6. a carrier is characterized in that, it contains the described DNA of claim 1.
7. one kind with the described carrier transformed host cells of claim 6.
8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.
9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.
10. a generation has the method for the polypeptide of pGPxH protein-active, it is characterized in that this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of pGPxH protein-active operationally is connected in expression regulation sequence, form the pGPxH protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 16-696 position among described nucleotide sequence and the SEQ ID NO.3;
(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of pGPxH;
(c) be fit to express under the condition of pGPxH protein polypeptide the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with pGPxH protein-active.
11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 16-696 position among the SEQ ID NO.3.
12. energy and the described pGPxH protein polypeptide of claim 4 specificity bonded antibody.
13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.
14. a probe molecule is characterized in that, it contains about 8-100 continuous nucleotide in the described dna molecular of claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001083509A1 (en) * | 2000-04-29 | 2001-11-08 | Shanghai Biowindow Gene Development Inc. | A novel polypeptide, a human peroxydase 25 and the polynucleotide encoding the polypeptide |
| WO2018086126A1 (en) * | 2016-11-14 | 2018-05-17 | 高雄医学大学 | Method for detecting whether glucose metabolism is abnormal, and prevention and treatment therefor |
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1998
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001083509A1 (en) * | 2000-04-29 | 2001-11-08 | Shanghai Biowindow Gene Development Inc. | A novel polypeptide, a human peroxydase 25 and the polynucleotide encoding the polypeptide |
| WO2018086126A1 (en) * | 2016-11-14 | 2018-05-17 | 高雄医学大学 | Method for detecting whether glucose metabolism is abnormal, and prevention and treatment therefor |
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