CN1371995A - Human peptide transport protein coding sequence, preparation method and use thereof - Google Patents

Abstract

The present invention relates to a novel human gene nucleotide sequence, in particular it relates to cDNA sequence of human peptide transport protein and polypeptide of said sequence code. Said invention also relates to the described polynucleotide sequence and production method of the described polypeptide and their application.

Description

Human peptide transport protein coding 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 peptide transport protein 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

The peptide transhipment is that a kind of soluble carrier is under the external world provides energy condition, the specificity biological process of peptide molecule transhipment by cytolemma.The small-molecular peptides transhipment plays an important role to the normal physiological function of organism.For example, in mammalian nervous system, each seed amino acid and neuropeptide are being implemented nerve signal conduction, various functions such as cellular metabolism and cell volume adjusting.Neuropeptide just need be removed from born of the same parents' external space after finishing its effect, simultaneously unnecessary glutaminate is transported out synaptic vesicle.In brain, the peptide transport process has and helps eliminate the neuropeptide that is degraded, and also might help to be absorbed with bioactive peptide section (Yamashita T, Shimada S, Guo W, et al, 1997, J Biol Chem272 (15): 10205-11).

Recent study finds that some oral peptide analogs absorb by intestines polypeptide movement system (IPTS), and the drug absorption approach has been expanded in the discovery of IPTS mechanism, absorbs approach safely and efficiently for many polypeptide drugs provide.Medicinal design is become the prodrug of dipeptides, tripeptide analog thing form, pass through intestines line velum through IPTS, prodrug is positioned epithelial cell membrane, discharged parent drug at the epithelium outside surface by enzymic hydrolysis, or before discharging cell, discharged parent drug, the performance drug effect after absorbing fully by intracellular polypeptidase or cruel enzymic hydrolysis.At present, can p-beta-lactam antibiotics, cynnematin, penicillin, ACE inhibitor, renin inhibitor, bestatin, thrombin inhibitors, thyroliberin and analogue thereof be arranged by the medicine that IPTS absorbs.Wherein, bestatin is the dipeptides that contains unique amino acid, has antitumour activity, and the normal clinically form with oral dosage form is used for cancer therapy (" foreign medical science pharmacy fascicle " 2000,27 (5): 301～304).

The peptide transport process extensively is present in bacterium, plant and the mammalian tissues.Studies show that pattern eukaryote Saccharomyces cerevisiae has two kinds of distinct peptide transporting mechanisms, a kind of is the PTR system of transhipment two peptides/tripeptides; Another kind is the OPT system of transhipment tetrapeptide/pentapeptide.The PTR system has three genes, PTR1, PTR2 and PTR3.Be one by the transport vehicle PTR2P of PTR2 genes encoding and contain 12 integrated proteins of striding diaphragm area that mainly transport dipeptides/tripeptides, the PTR family member is identified in multiple biology.(Hauser?M，Narita?V，Donhardt?AM，Naider?F，Becker?JM.(2001)Mol?Membr?Biol，18(1)：105-12)。Yet human peptide transport protein of the present invention is not in the news as yet or delivered.

Summary of the invention

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding translocator family, albumen of the present invention be named as human peptide transport protein (human peptide transport protein, hPTR).

Another object of the present invention provides a kind of new translocator family member, and this albumen is named as people hPTR.

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

The present invention also provides the application of hPTR 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 hPTR protein-active, shows at least 90% homology from the nucleotides sequence of Nucleotide 4-1903 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 4-1903 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 4-1903 position.

In another aspect of this invention, provide a kind of isolating hPTR 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 hPTR protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hPTR protein-active operationally is connected in expression regulation sequence, form the hPTR protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 4-1903 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 hPTR;

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

(d) isolate polypeptide with hPTR 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 4-1903 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 " hPTR albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with hPTR protein-active is as 4-1903 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 4-1903 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 SEQID NO.1 in 4-1903 position nucleotide sequence homology be low to moderate about 90% the degenerate sequence described sequence of SEQ ID 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 IDNO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 4-1903 position.Also term also comprise with SEQ IDNO.1 in from the nucleotide sequence of the homology of nucleotide sequence at least 90% of Nucleotide 4-1903 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 hPTR 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 " hPTR protein polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of hPTR protein-active.This term also comprises having and variant form people hPTR 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 hPTR 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 hPTR DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-hPTR polypeptide to obtain.The present invention also provides other polypeptide, as comprises hPTR polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of hPTR polypeptide.Usually, this fragment have the hPTR 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 hPTR albumen or polypeptide.The difference of these analogues and natural hPTR 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 hPTR polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of hPTR 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 hPTR 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 hPTR that encodes.

The present invention also comprises the method that detects the hPTR 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 hPTR 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 hPTR 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 hPTR gene product or fragment.Preferably, refer to that those can combine with hPTR 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 hPTR, comprise that also those do not influence the antibody of hPTR protein function.The present invention also comprise those can with modify or without the hPTR 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 hPTR gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing hPTR 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.6:511,2013; People such as Kohler, Eur.J.Immunol.6:292,2013; People such as Hammerling, In Monoclonal Antibodies and T CellHybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the hPTR function and the antibody that does not influence the hPTR function.Each antibody-like of the present invention can utilize the fragment or the functional zone of hPTR 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 hPTR 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 hPTR 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 hPTR is so to obtain: with people's testis λ gt10cDNA library (Clontech company) is template, with two pairs of oligonucleotide is primer A1:5 '-CGCATGGAGG GCTCTGGGGGC-3 ', B1:5 '-CATGTTTG ATGCTGTGCT CAT-3 '; Oligonucleotide A2:5 '-ATGCCCACGGCGATCCTCT TC-3 ', B2:5 '-CACTTACC AAGCTCCTTT GGA-3 ' is a reverse primer, carries out PCR.The PCR condition of A1/A2 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 condition of B1/B2 be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The electrophoresis detection pcr amplification product finds that the gained fragment length is respectively about 1,200 and 800 bases.With Restriction Enzyme DrdI both are carried out enzyme then and cut, connect, obtain purpose cDNA sequence and assorted band.The methods such as being with available electrophoresis of mixing is removed, thereby obtains the higher purpose cDNA sequence of concentration.

Find to have the type sequence of signal peptide and peptide transport protein (PTR2) functional domain in the hPTR protein sequence of the present invention by the homology retrieval.The albumen of PTR2 genes encoding is one and contains 12 integrated proteins of striding diaphragm area, mainly transports dipeptides/tripeptides.The present invention has PTR translocator family member's conserved domain, and therefore, hPTR albumen of the present invention may be the newcomer of translocator family, the transhipment of peptide molecule is absorbed have regulating and controlling effect.In fact the present invention provides a kind of method of improving the bad patient's symptom of dietetic alimentation from gene level like this.In addition, often medicinal design is become dipeptides, tripeptide analog thing form clinically, utilize hPTR of the present invention to absorb approach safely and efficiently for more polypeptide drugs provide by the oral way administration.

Embodiment

Embodiment 1

The clone and the mensuration of the cDNA sequence of hPTR

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, with two pairs of oligonucleotide is primer A1:5 '-CGCATGGAGG GCTCTGGGGGC-3 ', B1:5 '-CATGTTTG ATGCTGTGCT CAT-3 '; Oligonucleotide A2:5 '-ATGCCCACGGCGATCCTCT TC-3 ', B2:5 '-CACTTACC AAGCTCCTTT GGA-3 ' is a reverse primer, carries out PCR.The PCR condition of A1/A2 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 condition of B1/B2 be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The electrophoresis detection pcr amplification product finds that the gained fragment length is respectively about 1,200 and 800 bases.With Restriction Enzyme DrdI both are carried out enzyme then and cut, connect, obtain purpose cDNA sequence and assorted band.The methods such as being with available electrophoresis of mixing is removed, thereby obtains the higher purpose cDNA sequence of concentration.

2.PCR the order-checking of product

Above-mentioned amplified production is connected with pGEM-T  carrier (Promega), 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 SequiThermEXCELTMDNA sequencing kit (Epicentre Technologies) to brachymemma successively, at last with computer software splicing order, obtain full length cDNA sequence, be total to 2736bp, detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 4-1903 position Nucleotide.Derive the aminoacid sequence of hPTR according to the full length cDNA sequence that obtains, totally 633 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 hPTR 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 and peptide transport protein (PTR2) functional domain in the protein sequence of hPTR.

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 part that meets signal peptide sequence among the hPTR of the present invention is EGSGGGAGERAPLLGARRAAAAAAAAGAFAG (among the SEQ ID NO 2 from the 2-32 position).

The part that meets the PTR2 conserved sequence among the hPTR of the present invention is RAILLSLALYLLGMLAFPLLAAPATRAALCGSARLLNCTAPGPDAAARCCSPATFA GLVLVGLGVATVKANITPFGADQVKDRGPEATRRFFNWFYWSINLGAILSLGGIAY IQQNVSFVTGYAIPTVCVGLAFVVFLCGQSVFITKPPDGSAFTDMFKILTYSCCSQ KRSGERQSNGEGIGVFQQSSKQSLFDSCKMSHGGPFTEEKVEDVKALVKIVPVFLA LIPYWTVYFQMQTTYVLQSLHLRIPEISNITTTPHTLPAAWLTMFDAVLILLLIPL KDKLVDPILRRHGLLPSSLKRIAVGMFFVMCSAFAAGILESKRLNLVKEKTINQTI GNVVYHAADLSLWWQVPQYLLIGI (among the SEQ ID NO 3 from the 71-464 position). The albumen of PTR2 genes encoding is one and contains 12 integrated proteins of striding diaphragm area, mainly transports dipeptides/tripeptides.The present invention has PTR translocator family member's conserved domain, and therefore, hPTR albumen of the present invention may be the newcomer of translocator family, the transhipment of peptide molecule is absorbed have regulating and controlling effect.In fact the present invention provides a kind of method of improving the bad patient's symptom of dietetic alimentation from gene level like this.In addition, often medicinal design is become dipeptides, tripeptide analog thing form clinically, utilize hPTR of the present invention to absorb approach safely and efficiently for more polypeptide drugs provide by the oral way administration.By to further investigation of the present invention, one finds the relation of it and many clinical cases surely, and final for addressing these problems developing new thinking and method.

People hPTR of the present invention is used for further functional study except can be used as translocator family a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor hPTR can also merge with other members of this family or exchange fragment, to produce new albumen.

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

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

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

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

5′-ATCGGGATCCATGGAGG?GCTCTGGGGG?CG-3′

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

3 ' end primer sequence is:

5’-AA?GGAAGCTTCC?AAGCTCCTTT?GGATAGA-3’

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

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Ampr), 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 hPTR 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) (OD600) 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 hPTR from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out hPTR 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 1-32 position.

Embodiment 4

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

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

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

5′-ATCGAAGCTTATGGAGG?GCTCTGGGGG?CG-3′

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

3 ' end primer sequence is:

5’-AA?GGGGATCCCC?AAGCTCCTTT?GGATAGA-3’

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

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), and this plasmid vector coding antibiotics resistance (Ampr and Neor), 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 are in proper 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 hPTR 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 expression of peptides translocator vigor.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 1-32 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 hPTR 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: 2736bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： b.：cDNA c.：SEQ ID NO.1cgcatggagg gctctggggg cggtgcgggc gagcgggcgc cgctgctggg cgcgcggcgggcggcggcgg ccgcggcggc ggctggggcg ttcgcgggcc ggctcttggc gtgcggggccgtgctgctga cggagctgct ggagcgcgcc gctttctacg gcatcacgtc caacctggtgctattcctga acggggcgcc gttctgctgg gagggcgcgc aggccagcga ggcgctgctgctcttcatgg gcctcaccta cctgggctcg ccgttcggag gctggctggc cgacgcgcggctgggccggg cgcgcgccat cctgctgagc ctggcgctct acctgctggg catgctggccttcccgctgc tggccgcgcc cgccacgcga gccgcgctct gcggttccgc gcgcctgctcaactgcacgg cgcctggtcc cgacgccgcc gcccgctgct gctcaccggc caccttcgcggggctggtgc tggtgggcct gggcgtggcc accgtcaagg ccaacatcac gcccttcggcgccgaccagg ttaaagatcg aggtccggaa gccactagga gattttttaa ttggttttattggagcatta acctgggagc gatcctgtcg ttaggtggca ttgcctatat tcagcagaacgtcagctttg tcactggtta tgcgatcccc actgtctgcg tcggccttgc ttttgtggtcttcctctgtg gccagagcgt tttcatcacc aagcctcctg atggcagtgc cttcaccgacatgttcaaga tactgacgta ttcctgctgt tcccagaagc gaagtggaga gcgccagagtaatggtgaag gcattggagt ctttcagcaa tcttctaaac aaagtctgtt tgattcatgtaagatgtctc atggtgggcc atttacagaa gagaaagtgg aagatgtgaa agctctggtcaagattgtcc ctgttttctt ggctttgata ccttactgga cagtgtattt ccaaatgcagacaacatatg ttttacagag tcttcatttg aggattccag aaatttcaaa tattacaaccactcctcaca cgctccctgc agcctggctg accatgtttg atgctgtgct catcctcctgctcatccctc tgaaggacaa actggtcgat cccattttga gaagacatgg cctgctcccatcctccctga agaggatcgc cgtgggcatg ttctttgtca tgtgctcagc ctttgctgcaggaattttgg agagtaaaag gctgaacctt gttaaagaga aaaccattaa tcagaccatcggcaacgtcg tctaccatgc tgccgatctg tcgctgtggt ggcaggtgcc gcagtacttgctgattggga tcagcgagat ctttgcaagt atcgcaggcc tggaatttgc atactcagctgcccccaagt ccatgcagag tgccataatg ggcttgttct ttttcttctc tggcgtcgggtcgttcgtgg gttctggact gctggcactg gtgtctatca aagccatcgg atggatgagcagtcacacag actttggtaa tattaacggc tgctatttga actattactt ttttcttctggctgctattc aaggagctac cctcctgctt ttcctcatta tttctgtgaa atatgaccatcatcgagacc atcagcgatc aagagccaat ggcgtgcccc accagcagga gggcctgaccttcctgaggc catgtgcggt ttctgaggct gacatgtcag taactgactg gggtgcactgagaacaggca agactttaaa ttcccataaa atgtctgact tcactgaaac ttgcatgttgcctggattga tttcttcttt ccctctatcc aaaggagctt ggtaagtgcc ttactgcagcgtgtctcctg gcacgctggg ccctccggga ggagagctgc agatttcgag tatgtcgcttgtcattcaag gtctctgtga atcctctagc tgggttccct tttttacaga aactcacaaatggagattgc aaagtcttgg ggaactccac gtgttagttg gcatcccagt ttcttaaacaaatagtatca cctgcttccc atagccatat ctcactgtaa aaaaaaaaat taataaactgttacttatat ttaaagataa aagcatggtc agatgctgca aggattttac ataaatgccatatttatggt ttccttcctg agaacaatct tgctcttgcc atgttctttg atttaggctggtagtaaaca catttcatct gctgcttcaa aaagtactta ctttttaaac catcaacattacttttcttt cttaaggcaa ggcatgcata agagtcattt gagaccatgt gtcccatctcaagccacaga gcaactcacg gggtacttca caccttacct agtcagagtg cttatatatagctttatttt ggtacgattg agactaaaga ctgatcatgg ttgtatgtaa ggaaaacattcttttgaaca gaaatagtgt aattaaaaat aattgaaagt gttaaatgtg aacttgagctgtttgaccag tcacattttt gtattgttac tgtacgtgta tctggggctt ctccgtttgttaatactttt tctgtatttg ttgctgtatt tttggcataa ctttattata aaaagcatctcaaatgcgaa atccaaaaaa aaaaaaaaaa aaaaaa ( 2 ) SEQ ID NO.2： a.：

(A) length: 633 amino acid

(B) type: amino acid

( C ) ： b.： c.：SEQ ID NO.2MEGSGGGAGERAPLLGARRAAAAAAAAGAFAGRLLACGAVLLTELLERAAFYGITSNLVLFLNGAPFCWEGAQASEALLLFMGLTYLGSPFGGWLADARLGRARAILLSLALYLLGMLAFPLLAAPATRAALCGSARLLNCTAPGPDAAARCCSPATFAGLVLVGLGVATVKANITPFGADQVKDRGPEATRRFFNWFYWSINLGAILSLGGIAYIQQNVSFVTGYAIPTVCVGLAFVVFLCGQSVFITKPPDGSAFTDMFKILTYSCCSQKRSGERQSNGEGIGVFQQSSKQSLFDSCKMSHGGPFTEEKVEDVKALVKIVPVFLALIPYWTVYFQMQTTYVLQSLHLRIPEISNITTTPHTLPAAWLTMFDAVLILLLIPLKDKLVDPILRRHGLLPSSLKRIAVGMFFVMCSAFAAGILESKRLNLVKEKTINQTIGNVVYHAADLSLWWQVPQYLLIGISEIFASIAGLEFAYSAAPKSMQSAIMGLFFFFSGVGSFVGSGLLALVSIKAIGWMSSHTDFGNINGCYLNYYFFLLAAIQGATLLLFLIISVKYDHHRDHQRSRANGVPHQQEGLTFLRPCAVSEADMSVTDWGALRTGKTLNSHKMSDFTETCMLPGLISSFPLSKGAW ( 3 ) SEQ ID NO.3： a.：

(A) length: 601 amino acid

(B) type: amino acid

( C ) ： b.： c.：SEQ ID NO.3RLLACGAVLLTELLERAAFYGITSNLVLFLNGAPFCWEGAQASEALLLFMGLTYLGSPFGGWLADARLGRARAILLSLALYLLGMLAFPLLAAPATRAALCGSARLLNCTAPGPDAAARCCSPATFAGLVLVGLGVATVKANITPFGADQVKDRGPEATRRFFNWFYWSINLGAILSLGGIAYIQQNVSFVTGYAIPTVCVGLAFVVFLCGQSVFITKPPDGSAFTDMFKILTYSCCSQKRSGERQSNGEGIGVFQQSSKQSLFDSCKMSHGGPFTEEKVEDVKALVKIVPVFLALIPYWTVYFQMQTTYVLQSLHLRIPEISNITTTPHTLPAAWLTMFDAVLILLLIPLKDKLVDPILRRHGLLPSSLKRIAVGMFFVMCSAFAAGILESKRLNLVKEKTINQTIGNVVYHAADLSLWWQVPQYLLIGISEIFASIAGLEFAYSAAPKSMQSAIMGLFFFFSGVGSFVGSGLLALVSIKAIGWMSSHTDFGNINGCYLNYYFFLLAAIQGATLLLFLIISVKYDHHRDHQRSRANGVPHQQEGLTFLRPCAVSEADMSVTDWGALRTGKTLNSHKMSDFTETCMLPGLISSFPLSKGAW

Claims (14)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people hPTR protein-active, shows at least 90% homology from the nucleotides sequence of Nucleotide 4-1903 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 4-1903 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 4-1903 position among the SEQ ID NO.1.

4. isolating hPTR 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 hPTR protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hPTR protein-active operationally is connected in expression regulation sequence, form the hPTR protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 4-1903 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 hPTR;

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

(d) isolate polypeptide with hPTR protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 4-1903 position among the SEQ ID NO.1.

12. energy and the described hPTR 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.