CN1394959A - Human ring finger proteinase code sequence, its preparation method and application - Google Patents

Abstract

The present invention relates to the field of gene engineering technology, in particular it relates to a new human gene nucleotide sequence, in the more concrete, it relates to cDNA sequence of human ring finger proteinase (hPAI) and said sequence coded polypeptide. Said invention also relates to production method of the described polynucleotide sequence and described polypeptide and their application.

Description

Human protease associated encoding sequence, Preparation Method And The Use

Technical field

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of human protease associated and the polypeptide of this sequence encoding.The invention still further relates to the production method of described polynucleotide sequence and described polypeptide, and the purposes of these polynucleotide and described polypeptide.

Background technology

Zinc refers to that gene family is one of maximum Human genome family, and its protein product is mainly transcribed factor effect by having interacted with DNA, regulates target gene expression to adapt to the needs of organism growth, differentiation, ripening process.Zinc finger protein is not only closely related with hematopoiesis, affect growth, atomizations such as erythron, megakaryocytic series and lymphocyte series (as GATA family and EKLF), granulocyte series (MZF1), monocytic series (Egr-1), but also relevant with some hemopoietic system malignant tumours, play an important role in the acute promyelocytic leukemia morbidity as PLZF.

Fourth finger (RING finger) is a specific zinc fingers that is made of 40-60 amino-acid residue, and it combines with two zinc atoms, may participate in regulating the interaction between the albumen.Two kinds of varients are arranged, and C3HC4 and C3H2C3 type all have certain relation with dissimilar halfcystine/Histidine, and Histidine structure wherein is also called " fourth finger-H2 " structure.Known in the three-dimensional structure of some fourth finger, the connected system of zinc atom is unique for ring finger, and " intersect and support " structure is otherwise known as." intersection supports " structure and two zinc atom bonded modes can be represented with following table:

Wherein, the position of conservative halfcystine/residue when " C " expression combines with zinc atom, the position of conservative Histidine/residue when " H " expression combines with zinc atom, the position of " Zn " expression zinc atom.

Recently result of study shows, ring finger protein has specific action to some albumen, possible all ring finger proteins all relevant with the effect of E3 ubiquitin protein ligase (Freemont PS, 2000, Curr Biol 10 (2): R84-7).The effect of ubiquitin is the target protein of degrading, and is the potential regulation and control person of cell protein function.In time rejecting the albumen that makes the cell malignant proliferation is very important for the normal physiological activity that keeps organism.Yet before this albumen is eliminated, must add that a kind of albumen marker that is called as ubiquitin sticks to them.Discover that recently the fourth finger molecular structure helps sticking of ubiquitin.At present, ring finger (Marcia Barinaga, 1999,286 (5438): 223) in 200 multiple protein, have been found.Yet hPA1 of the present invention is not disclosed as yet or delivered.

Summary of the invention

An object of the present invention is to provide a kind of new proteolytic enzyme family member, this albumen be named as the human protease associated associated protein ( hUman pRotease aSsociated protein 1, hPA1).

Another object of the present invention provides a kind of new polynucleotide, a newcomer of this polynucleotide encoding proteolytic enzyme family, and Nucleotide of the present invention is named as hPA1.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described people hPA1.

The present invention also provides the application of hPA1 gene order and polypeptide.

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 hPA1 protein-active, shows at least 90% homology from the nucleotides sequence of Nucleotide 237-1364 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 237-1364 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.2.More preferably, this sequence has among the SEQ ID NO.1 nucleotide sequence from the 237-1364 position.

In another aspect of this invention, provide a kind of isolating hPA1 protein polypeptide, it comprises: have polypeptide or its active fragments of SEQ ID NO.2 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ IDNO.2 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, the method that provides a kind of generation to have the polypeptide of hPA1 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hPA1 protein-active operationally is connected in expression regulation sequence, form the hPA1 protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 237-1364 position among described nucleotide sequence and the SEQ ID NO.1;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of hPA1;

(c) be fit to express under the condition of hPA1 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hPA1 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 2013 Nucleotide, and its detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 237-1364 position Nucleotide.

In the present invention, " isolating ", " purifying " or " pure substantially " DNA are 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 in cell, following it.

In the present invention, term " hPA1 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with hPA1 protein-active is as 237-1364 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 237-1364 position Nucleotide of SEQ ID NO.1 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.1 in 237-1364 position nucleotide sequence homology be low to moderate about 90% the degenerate sequence described sequence of SEQID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 237-1364 position.Also term also comprise with SEQ ID NO.1 in from the nucleotide sequence of the homology of nucleotide sequence at least 90% of Nucleotide 237-1364 position.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.1 with people hPA1 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 " hPA1 protein polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of hPA1 protein-active.This term also comprises having and variant form people hPA1 identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of hPA1 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of hPA1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-hPA1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises hPA1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of hPA1 polypeptide.Usually, this fragment have the hPA1 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 hPA1 albumen or polypeptide.The difference of these analogues and natural hPA1 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

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

The present invention also comprises the antisense sequences of hPA1 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of hPA1 in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 of hPA1 polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the hPA1 that encodes.

The present invention also comprises the method that detects the hPA1 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 is corresponding to the encoding sequence of hPA1 polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.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.

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 hPA1 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 hPA1 gene product or fragment.Preferably, refer to that those can combine with hPA1 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 hPA1, comprise that also those do not influence the antibody of hPA1 protein function.The present invention also comprise those can with modify or without the hPA1 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 hPA1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing hPA1 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 prepare (see people such as Kohler, Nature256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,2013; People such as Kohler, Eur.J.Immunol.6:292,2013; 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 hPA1 function and the antibody that does not influence the hPA1 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of hPA1 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 hPA1 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 hPA1 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 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.Certainly, by first synthetic a plurality of small segments, and then connect and obtain the very long fragment of sequence.

In the present invention, the cDNA nucleotide sequence of hPA1 is so to obtain: with people's testis λ gt10cDNA library (Clontech company) is template, is primer A1:5 '-AGAGATGC ATCTACTCAAGGT-3 ' with two pairs of oligonucleotide; Oligonucleotide A2:5 '-GCCATCAAGGTGAAGGATGAA-3 ' 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, 66 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The electrophoresis detection pcr amplification product finds that the gained fragment length is about 1,100 bases.

Find to have the type sequence of signal peptide, protein kinase (PA) and fourth finger (RING finger) functional domain in the hPA1 protein sequence of the present invention by the homology retrieval.Therefore, hPA1 may have the effect of transcription factor, regulates cell proliferation by cutting off peptide bond.

HPA1 of the present invention is made medicine or test kit, can be used for the disease that treated tissue hyperplasia, tumour generation etc. cause owing to cell hyperproliferation; Clinically, also can organize injured, excision or organ partly damages, after the necrosis, be used to promote the cell growth.

In addition, utilize the nucleotide coding sequence (seeing SEQ ID NO 1 for details) of hPA1 of the present invention can make its antisense sequences, also be used to suppress the hyper-proliferative of cell, thereby be used for the treatment of tumour; Aminoacid sequence (seeing SEQ ID NO 3 for details) by hPA1 of the present invention obtains the proteic antibody of hPA1, and is connected with suitable material, makes medicine, can play certain booster action to some cancer therapy.

Embodiment

Embodiment 1

The clone and the mensuration of the cDNA sequence of hPA1

1. primer amplification

In the present invention, the cDNA nucleotide sequence of STP is so to obtain: with people's testis λ gt10cDNA library (Clontech company) is template, is primer A1:5 '-AGAGATGC ATCTACTCAAGGT-3 ' with two pairs of oligonucleotide; Oligonucleotide A2:5 '-GCCATCAAGGTGAAGGATGAA-3 ' 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, 66 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The electrophoresis detection pcr amplification product finds that the gained fragment length is about 1,100 bases.

2.PCR the order-checking of product

To be connected with pGEM-T  carrier (Promega) with the product that A1, A2 amplification obtains, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque with double-stranded nested type disappearance test kit (Pharmacia) to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then.Check order with disappearance of SequiTherm EXCELTMDNA sequencing kit (Epicentre Technologies) to brachymemma successively, at last with computer software splicing order, obtain full length cDNA sequence, be total to 2013bp, detailed sequence is seen SEQID NO.1, and wherein open reading frame is positioned at 237-1364 position Nucleotide.Derive the aminoacid sequence of hPA1 according to the full length cDNA sequence that obtains, totally 376 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

Structural analysis

Full length cDNA sequence and proteins encoded thereof with hPA1 carry out nucleic acid and albumen homology retrieval with BLAST in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database.Found that the type sequence that has signal peptide, protein kinase (PA) and fourth finger (RING finger) functional domain in the protein sequence of hPA1.

Signal peptide is meant that the segment length after the N end initiator codon is the peptide fragment of 15-40 amino-acid residue, and its effect is by combining with signal recognition particle (SRPs), guiding follow-up synthetic peptide chain to enter endoplasmic reticulum.Subsequently SRPs again with the SRPs receptors bind.In case after protein changed endoplasmic reticulum over to, signal peptide was just cut.Protein is encapsulated in the film vesicle, and the film vesicle is through golgi body then, and the mode to merge with the cell cytosol film is discharged into specific position with its content and has an effect.The signal peptide sequence of hPA1 of the present invention is HLLKVGTWRNNTASSWLMKFSVLWLVSQNCCRAS (among the SEQ ID NO 2 from the 2-35 position).

Proteolytic enzyme is relevant, and (Protease associated, PA) functional domain is the insert type structural domain of finding in various proteolytic enzyme.The proteolytic enzyme rough segmentation; Can be divided into solation enzyme and proteolytic enzyme, they can both resolve into peptide bond acid and amine two portions, but often show different and adaptability tensio-active agent, latter's (proteolytic ferment) only with anionic in the straight chain benzene sulfonate compatible, its activity is unaffected; The former (solation enzyme), to negatively charged ion and non-ionic LAS, the alkyl polyoxyethylene ether AE of LES, AOS and non-ionic type is all compatible.The part that hZF1 of the present invention meets PA functional domain conserved sequence is STLKRVAGVIVPPEGKIQNACNPNTIFSRSKYSETWLALIERGGCTFTQKIKVATE KGASGVIIYNVPGTGNQVFPMFHQAFEDVVVVMIGNLKGTEIFHLI (among the SEQ ID NO 3 from the 30-132 position).Therefore, hZF1 of the present invention may have by cutting off peptide bond and regulates the effect of cell physiological active.

HZF1 albumen of the present invention also has the sequence fragment that meets fourth finger (RING finger) structural domain.Fourth finger is a specific zinc fingers that is made of 40-60 amino-acid residue, and it combines with two zinc atoms, may participate in regulating the interaction between the albumen.The structural pattern of halfcystine is as follows in the ring finger: the part that meets the RING conserved sequence among C-x (2)-C-x (9to 39)-C-x (1 to 3)-H-x (2 to 3)-C-x (2)-C-x (4 to 48)-C-x (2)-C hZF1 of the present invention is VICFERYKPNDIVRILTCKHFFHKNCIDPWILPHGTCPIC (among the SEQ IDNO 2 from the 221-261 position).So hZF1 may have the function of transcription factor, participate in the adjusting of on cell proliferation.

By to further investigation of the present invention, one finds it and many clinical cases surely, as the relation of apoptosis, hamartoplasia and tumour, and final for addressing these problems developing new thinking and method.

People hZF1 of the present invention except can be used as protein kinase, transcription factor family a member is used for the further function, also can be used for producing fusion rotein, such as producing fusion rotein with immunoglobulin (Ig) with other albumen.In addition, inventor hPA1 can also merge with other members of this family or exchange fragment, to produce new albumen.

At the antibody of inventor hPA1, be used to screen other albumen with similar structures territory, perhaps be used for the affinity purification associated protein.

For example, inventor hPA1 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people hPA1 or the overexpression that suppresses people hPA1.People hPA1 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people hPA1 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 hPA1 in intestinal bacteria

In this embodiment, use the PCR Oligonucleolide primers corresponding to 5 ' and 3 ' end of the cDNA sequence of coding hPA1 to increase, the cDNA that obtains hPA1 is as the insertion fragment.

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

5’-GAAGGGATTCATGCATCTACTCAAGGTTG-3’

This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is the N-terminal partial nucleotide sequence of hPA1 encoding sequence;

3 ' end primer sequence is:

5’-CATGAAGCTTAGGTGAAGGATGAACATCT-3’

This primer contains the restriction enzyme site of HindIII restriction enzyme, the partial nucleotide sequence of the encoding sequence of translation termination and hPA1.

The restriction enzyme site of the restriction enzyme on the primer 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 HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep 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 (Kanr).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, and the cDNA fragment of sequence verification hPA1 has correctly been inserted carrier.

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).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when reaching 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 hPA1 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out hPA1 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps from nickel-chelate column, isolate purifying protein.Protein behind the purifying is incorporated in second post, 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.

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.3 with ordinary method.Because this albumen has signal peptide sequence, signal peptide is cut when expressing, so this sequence does not contain among the SEQ ID NO.2 aminoacid sequence from the 1-35 position.

Embodiment 4

The expression of hPA1 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, use PCR Oligonucleolide primers to increase, obtain hPA1 cDNA as inserting fragment corresponding to 5 ' and 3 ' end of the cDNA sequence of coding hPA1.

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

5’-GAAGAAGCTTATGCATCTACTCAAGGTTG-3’

This primer contains the restriction enzyme site of HindIII restriction enzyme, is the aminoterminal partial nucleotide sequence of hPA1 encoding sequence after this restriction enzyme site;

3 ' end primer sequence is:

5’-CATGGGATCCAGGTGAAGGATGAACATCT-3’

This primer contains the partial nucleotide sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and hPA1.

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 (Invitrogen company), 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 BamHI and HindIII digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be 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, and the cDNA fragment of sequence verification hPA1 has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI 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 and are expressed the ring finger 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.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant.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 50mM TrisHCl (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.

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.3 with ordinary method.Because this albumen has signal peptide sequence, signal peptide is cut when expressing, so this sequence does not contain among the SEQ ID NO.2 aminoacid sequence from the 1-35 position.

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, specific as follows.Recombinant protein 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 hPA1 gene translation product with it.

Correlated series of the present invention is as follows: the information of (1) SEQ ID NO.1: a. sequence signature:

(A) length: 1446bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： b.：cDNA c.：SEQ ID NO.1gcagtatcta gagttaacag taggggattt atttttcaat ctaaagtttt tcacctctctctagtaagac atctgaaact cataagccat ctttaagaat ttcttacaaa aacatgctggaagacagtgg atcttgtttg tatcctgaag atttttttct cttcgttatt ttaaattaattgtcaacaga tgtgcaactg ttaggttggt tcttaagtca ctcggagaag agagagatgcatctactcaa ggttggcact tggagaaaca acactgcctc ttcctggctt atgaagttcagtgttctttg gcttgttagt cagaactgtt gcagagcaag tgttgtttgg atggcttatatgaacatatc atttcatgtt gggaatcatg tgttgtcaga gttgggagag actggagtctttggaagaag ctccactttg aagagagtgg caggagttat agtgccacca gagggaaaaatccaaaatgc atgtaatccc aataccattt tcagccgatc aaagtactca gagacctggcttgcacttat tgaacgggga ggttgtacct tcacacagaa aattaaagtg gcaactgagaagggagccag tggagtgatc atctataacg ttccaggtac tggcaaccag gtgttccccatgtttcatca ggcatttgaa gatgtcgttg tggttatgat tggtaactta aaaggcacggaaattttcca tttaattaag aagggagttc tcattacagc cgtggttgag gtggggagaaagcacatcat ctggatgaat cactatttgg tctcttttgt gattgtcaca actgctaccttagcatattt catcttttat cacattcata gactttgttt agcaaggatt cagaaccggagatggcagcg attaacaaca gatcttcaga acacatttgg acaactccaa cttcgagtagtaaaagaggg ggatgaagaa ataaatccaa atggggatag ctgcgtaatt tgctttgaacgctataagcc taatgacata gttcgtattc tgacttgtaa acattttttc cacaagaattgcattgaccc ctggatttta ccccatggga catgccccat ttgcaaatgt gatattcttaaagttttggg gattcaagtg gttgttgaaa atggaacaga acctttgcaa gttctaatgtcaaatgaact gcctgaaacc ttatcaccta gtgaagagga gacaaataat gaagtttctcctgcaggaac ctcagataaa gtaatccatg tggaggagaa ccctacttct cagaataatgacatccagcc tcattcagta gtggaagatg ttcatccttc accttgatgg catgacttttgaggaagtgt attaaacctg tatgtgaaat caggtcctaa tactgacaag cagtttgtctgttaaa ( 2 ) SEQ ID NO.2： a.：

(A) length: 376 amino acid

(B) type: amino acid

(C) topological framework: linear b. molecule type: polypeptide c. sequence description: SEQ ID NO.2

MHLLKVGTWRNNTASSWLMKFSVLWLVSQNCCRASVVWMAYMNISFHVGNHVLSELGE

TGVFGRSSTLKRVAGVIVPPEGKIQNACNPNTIFSRSKYSETWLALIERGGCTFTQKI

KVATEKGASGVIIYNVPGTGNQVFPMFHQAFEDVVVVMIGNLKGTEIFHLIKKGVLIT

AVVEVGRKHIIWMNHYLVSFVIVTTATLAYFIFYHIHRLCLARIQNRRWQRLTTDLQN

TFGQLQLRVVKEGDEEINPNGDSCVICFERYKPNDIVRILTCKHFFHKNCIDPWILPH

GTCPICKCDILKVLGIQVVVENGTEPLQVLMSNELPETLSPSEEETNNEVSPAGTSDK

The information of VIHVEENPTSQNNDIQPHSVVEDVHPSP (3) SEQ ID NO.3: a. sequence signature:

(A) length: 341 amino acid

(B) type: amino acid

(C) topological framework: linear b. molecule type: polypeptide c. sequence description: SEQ ID NO.3

VVWMAYMNISFHVGNHVLSELGETGVFGRSSTLKRVAGVIVPPEGKIQNACNPNTIFS

RSKYSETWLALIERGGCTFTQKIKVATEKGASGVIIYNVPGTGNQVFPMFHQAFEDVV

VVMIGNLKGTEIFHLIKKGVLITAVVEVGRKHIIWMNHYLVSFVIVTTATLAYFIFYH

IHRLCLARIQNRRWQRLTTDLQNTFGQLQLRVVKEGDEEINPNGDSCVICFERYKPND

IVRILTCKHFFHKNCIDPWILPHGTCPICKCDILKVLGIQVVVENGTEPLQVLMSNEL

PETLSPSEEETNNEVSPAGTSDKVIHVEENPTSQNNDIQPHSVVEDVHPSP

Claims (14)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people hPA1 protein-active, shows at least 90% homology from the nucleotides sequence of Nucleotide 237-1364 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 237-1364 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.2.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has the sequence of Nucleotide 237-1364 position among the SEQ ID NO.1.

4. isolating hPA1 protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.2 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.2 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 hPA1 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hPA1 protein-active operationally is connected in expression regulation sequence, form the hPA1 protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 237-1364 position among described nucleotide sequence and the SEQ ID NO.1;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of hPA1;

(c) be fit to express under the condition of hPA1 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hPA1 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 237-1364 position among the SEQ ID NO.1.

12. energy and the described hPA1 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.