CN1401770A

CN1401770A - Human amino acid transporter, its coding sequence, preparing method and use

Info

Publication number: CN1401770A
Application number: CN02137292A
Authority: CN
Inventors: 余龙; 唐丽莎; 郭金虎
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2002-09-29
Filing date: 2002-09-29
Publication date: 2003-03-12

Abstract

A human N system amino acids transporter (hNAT-1), its coding sequence, its preparing process and its use are disclosed. The nucleic acid and antisence strand or fragment of said hNAT-1 can be used to prepare the probe for detecting the abnormality of hNAT-1 gene. The hNAT-1 protein or its suppressor can be used to prepare medicines or reagent kit for regulating the bioactivity of living body, especially the liver cells.

Description

A kind of human amino acid transporter, its encoding sequence, method for making and purposes

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 a kind of people N system's amino acid transporter (hNAT-1), the invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.

Background technology

Amino acid is the fundamental unit that constitutes protein molecule, and protein is the basis of vital movement.Intravital most protein all constantly decomposes and anabolism, ceaselessly utilizes amino acid synthetic protein and decomposing protein to become amino acid in the cell.Amino acid and protein are changed mutually, and are transported to appointed part performance function, instruct the physiological activity of organism.So, pair cell and even whole organism, amino acid whose transhipment all is very important.

Amino acid whose transhipment mainly by systems such as A, ASC, B, GLY, IMINO, L, N, PHE and X finish (physiological reviews Vol.78 No.4 October 1998, pp.969-1054).

Wherein, the N system also has three hypotypes: the N type that acts in liver, the Nm that acts in skeletal muscle and the Nb that acts in neural system.All these hypotypes are all to show as the sodium ion dependency at transhipment L-glutamic acid and arginine.N system transporter albumen in the liver will show better activity under the condition that replaces sodium ion with lithium ion (Issue 31,23707-23717) for J.Biol.Chem., Vol.275.

N system amino acid transport body all plays an important role for new synthetic L-glutamic acid output in ornithine cycle and the liver.These transhipment activities can be blocked by Histidine.In addition, N system amino acid transport studies show that in cerebral tissue, it may participate in the effect of hemato encephalic barrier, and (Issue 7,3230-3235) for Proc.Natl.Acad.Sci.USA, Vol.97.

People such as Gu S separate and have identified the cDNA of an encoding murine film amino acid transport body.The polypeptide that this cDNA supposition obtains is made up of 505 highly hydrophobic amino-acid residues, and formed albumen has 9 to stride membrane structure, and N-terminal is placed tenuigenin and C-terminal is positioned cell surface.This transporter is mainly expressed in liver, and expression is also arranged in kidney, brain, heart.In liver, it mainly is positioned on the plasma membrane of cell, expression amount is higher near important vessel, progressively successively decrease along its direction of leading to less important blood vessel then, it may play very important effect (Proc Natl Acad Sci USA2000 Mar 28 this express spectra demonstration on liver cell physiology; 97 (7): 3230-5).

People such as Nakanishi T have cloned second hypotype of N system amino acid transporter again from the cerebral tissue of rat, and are referred to as SN2 (Am J Physiol Cell Physiol 2001 Dec; 281 (6): C1757-68).

Yet up to the present, hNAT-1 of the present invention someone as yet reports.

Summary of the invention

An object of the present invention is to provide a kind of polynucleotide sequence, a kind of human amino acid transporter of this polynucleotide sequence coding ( hUman NSystem aMino acid tRansporter 1, hNAT-1).

Another object of the present invention provides a kind of albumen, and this albumen is named as human amino acid transporter (hNAT-1).

A further object of the present invention provides a kind of recombinant technology that utilizes and produces the proteic method of described hNAT-1.

The present invention also provides this hNAT-1 nucleotide sequence and proteic application.

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 hNAT-1 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 117-1487 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 117-1487 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 Nucleotide 117-1487 position.

In another aspect of this invention, provide a kind of isolating hNAT-1 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 ID NO.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 hNAT-1 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hNAT-1 protein-active operationally is connected in expression regulation sequence, form the hNAT-1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 117-1487 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 hNAT-1;

(c) be fit to express under the condition of hNAT-1 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hNAT-1 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1516 Nucleotide, and its detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 117-1487 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 " hNAT-1 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with HNAT-1 protein-active is as 117-1487 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 117-1487 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 117-1487 position nucleotide sequence homology be low to moderate about 70% 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, more preferably under the height stringent condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 117-1487 position.In addition, this term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 117-1487 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.1 sequence of people HNAT-1 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 " hNAT-1 protein polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of hNAT-1 protein-active.This term also comprises having and variant form people hNAT-1 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 hNAT-1 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 hNAT-1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-hNAT-1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises hNAT-1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of hNAT-1 polypeptide.Usually, this fragment have the hNAT-1 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 hNAT-1 albumen or polypeptide.The difference of these analogues and natural hNAT-1 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 hNAT-1 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of hNAT-1 in the cell.

The present invention also comprises a kind of probe molecule, and this molecule has 8-100 of hNAT-1 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 hNAT-1 that encodes.

The present invention also comprises the method that detects the hNAT-1 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 hNAT-1 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 hNAT-1 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 hNAT-1 gene product or fragment.Preferably, refer to that those can combine with hNAT-1 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 hNAT-1, comprise that also those do not influence the antibody of hNAT-1 protein function.The present invention also comprise those can with modify or without the hNAT-1 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 hNAT-1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing hNAT-1 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.6:511,1976; 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 hNAT-1 function and the antibody that does not influence the hNAT-1 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of hNAT-1 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 hNAT-1 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).

In the present invention, the cDNA nucleotide sequence of people hNAT-1 is so to obtain, and is template with people's testis λ gt10cDNA library (available from Clontech company), is primer with two pairs of oligonucleotide: A1GTACGGAATG CCGGAAGGGC CGG; B1 C TTCCAGATTT CAAATGTTAC AG is a forward primer; A2:CACAGTATAT GGGCAATATT GAG; B2 TTCTTGTAAGAAGTAG GAAAATA 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, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, size are respectively the fragment and the impurity of about 900 and 700 base pairs.Remove impurity, with NcoI respectively enzyme cut above-mentioned two fragments, connect then, obtain the purpose fragment.With the nucleotide sequence carrier of packing into, import host cell.Host cell just can give expression to corresponding protein, just will obtain hNAT-1 albumen of the present invention through separation and purification.

HNAT-1 albumen of the present invention carries out nucleic acid and albumen homology retrieval with BLAST software in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, found that it has the characteristic sequence of amino acid transporter, and certain similarity is arranged with the N system amino acid transport body of other species.

The N system amino acid transport body of hNAT-1 albumen of the present invention and other species all has certain similarity.N system amino acid transport body all plays an important role for new synthetic L-glutamic acid output in ornithine cycle and the liver.These transhipment activities can be blocked by Histidine.In addition, N system amino acid transport studies show that in cerebral tissue, it may participate in the effect of hemato encephalic barrier.Such transporter is mainly expressed in liver, and expression is also arranged in kidney, brain, heart.In liver, it is positioned at around the important blood vessels on the hepatocellular cytolemma, and expression amount branches into little blood vessel gradually along with blood vessel and descends, and this hints that this class transporter has the important physical effect in liver.

In sum, hNAT-1 of the present invention has the characteristic sequence of amino acid transporter, and certain similarity is arranged with the N system amino acid transport body of other species, can play regulating and controlling effect to amino acid whose transmembrane transport, thereby the physiological activity of the nutrition of pair cell and even whole organism especially plays regulating and controlling effect to the liver cell physiological activity.These physiological actions comprise the secretion of hormone, the growth of cell, propagation, differentiation and apoptosis, intercellular interaction or the like.

HNAT-1 nucleic acid of the present invention, its antisense strand and fragment are made probe, can detect the hNAT-1 gene unconventionality; With hNAT-1 protein sequence generate a reagent box or medicine, adjustable ganglion cell's growing multiplication differentiation promotes the wound healing after cutting tissue and even organ are extractd.With hNAT-1 nucleic acid antisense strand of the present invention hNAT-1 antibody generate a reagent box can be used for alleviating or assisting therapy owing to the too high disease that causes of hNAT-1 expression amount, also can assist the diagnosis of tumour and cancer clinically, prevention and treatment.In addition, hNAT-1 of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen, for example the N end of hNAT-1 of the present invention and the N end of mouse TF can be exchanged, to produce the albumen that new activity is higher or have new features.At the antibody of hNAT-1 of the present invention, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment

Embodiment 1

The clone and the mensuration of the cDNA sequence of hNAT-1

1. primer amplification

With people's testis λ gt10cDNA library (available from Clontech company) is template, is primer with two pairs of oligonucleotide: A1 GTACGGAATG CCGGAAGGGC CGG; B1 C TTCCAGATTTCAAATGTTAC AG is a forward primer; A2:CACAGTATAT GGGCAATATT GAG; B2TTCTTG TAAGAAGTAG GAAAATA 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, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, size are respectively the fragment and the impurity of about 900 and 700 base pairs.Remove impurity, with NcoI respectively enzyme cut above-mentioned two fragments, connect then, obtain the purpose fragment.

2.PCR the order-checking of product

With above-mentioned purpose fragment and pGEM-T ^TMCarrier (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, obtains full length cDNA sequence at last, is total to 1516bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 117-1487 position Nucleotide.

Derive the aminoacid sequence of hNAT-1 according to the full length cDNA sequence that obtains, totally 456 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

The expression of hNAT-1 in intestinal bacteria

In this embodiment,, use PCR Oligonucleolide primers to increase, obtain hNAT-1 cDNA as inserting fragment corresponding to 5 of this dna sequence dna ' and 3 ' end with the cDNA sequence of coding hNAT-1.

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

5 ' GGAG GGATCCATGG AGGCGTCCTG GGGGA, this primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence of the hNAT-1 that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 ' ATTT CTGCAGTTTA TTAATCCAAT CAAAA, this primer contains the part encoding sequence of restriction enzyme site, translation termination and the hNAT-1 of PstI restriction enzyme.

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 PstI 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 (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid cuts with the NcoI enzyme and to identify and insert clip size and direction, and the cDNA fragment of sequence verification hNAT-1 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 into then in the large volume substratum, 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 hNAT-1 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out hNAT-1 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps use the dialysis step to remove Guanidinium hydrochloride, perhaps isolate purifying protein from nickel-chelate column, the albumen behind the purifying can be 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 to containing 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.2 with ordinary method.

Embodiment 3

The expression of hNAT-1 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, the following Oligonucleolide primers of cDNA sequence of coding hNAT-1 is increased, obtain hNAT-1 cDNA as inserting fragment.

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

5’-GGAG?GGATCCATGG?AGGCGTCCTG?GGGGA

This primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence of the hNAT-1 that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-ATTT?CTGCAGTTTA?TTAATCCAAT?CAAAA

This primer contains the part encoding sequence of the restriction enzyme site of PstI restriction enzyme, translation termination and hNAT-1.

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 (fl 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 PstI 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 cuts evaluation with the NcoI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification hNAT-1 has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to use lipofection, carries out with Lipofectin (GiBco Life), and transfection is after 48 hours, and 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.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (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 50mMTrisHCl (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.

Embodiment 4

Homology relatively

HNAT-1 albumen (SEQ ID NO.2) the hNAT-1 albumen of the present invention that obtains with embodiment 2 and embodiment 3 carries out nucleic acid and albumen homology retrieval with BLAST software in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundantGenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, found that it has the characteristic sequence of amino acid transporter, and certain similarity is arranged with the N system amino acid transport body of other species.

The characteristic sequence of amino acid transporter is in the protein sequence of hNAT-1 of the present invention: GVSFGLSVFNLMNAIMGSGILGLAYVMANTGVFGFSFLLLTVALLASYSVHLLLSM CIQTAVTSYEDLGLFAFGLPGKLVVAGTIIIQNIGAMSSYLLIIKTELPAAIAEFL TGDYSRYWYLDGQTLLIIICVGIVFPLALLPKIGFLGYTSSLSFFFMMFFALVVII KKWSIPCPLTLNYVEKGFQISNVTDDCKPKLFHFSKESAYALPTMAFSFLCHTSIL PIYCELQSPSKKRMQNVTNTAIALSFLIYFISALFGYLTFYDKVESELLKGYSKYL SHDVVVMTVKLCILFAVLLTVPLIHFPARKAVTMMFFSNFPFSWIRHFLITLALNI IIVLLAIYVPDIRNVFGVVGASTSTCLIFIFPGLFYLKLSREDFLSWKKLGAFVLL IFGILVGNFSLALIIFDWIN (43-455 among the SEQ ID NO 2).

HNAT-1 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, hNAT-1 of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen, for example the N end of hNAT-1 of the present invention and the N end of mouse CKR can be exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of hNAT-1 of the present invention, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment 5

Preparation antibody

Embodiment 2 or 3 recombinant proteins that obtain are 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 hNAT-1 gene translation product with it.

Example 6

Substrate as micromatrix

Micromatrix is called the DNA chip again.(, select by using some famous biosoftwares hNAT-1 full-length cDNA, EST or gene fragment substrate samples as micromatrix as LASERGENESOFTWARE (DNASTAR).By using ink-jet technology and a series of chemical process such as ray, chemistry, thermodynamics, mechanical means etc. that sample is fixed on the carrier,, obtain chip (Schena, M.et al. (1995) Science 270:467-470 then as on glass; And Shalon, D.et al. (1996) Genome Res.6:639-645.), the element of formation has point-like, bar shaped or the like.A typical chip contains the element of some amount usually.Behind the hybridization, the unconjugated probe of flush away detects the element of generation hybridization and the degree of reaction with scanner then.The complementarity of probe and each chip component and binding capacity all can be judged by the analysis to the image that scans.

Results of hybridization can be used for detecting hNAT-1 genovariation, sudden change and polymorphism, understands because hNAT-1 expresses the molecular basis of the undesired disease that causes, and can improve and monitor the activity of related agents.

Embodiment 7:

The preparation of test kit

Test kit 1:

The primer that it contains (1) specific amplification hNAT-1 to and operation instruction.This test kit also can contain or not contain the required reagent that the mRNA reverse transcription is become cDNA.Wherein, the sequence of this Auele Specific Primer is derived from the sequence of SEQ ID NO:1.The cDNA that reverse transcription is become carries out specific amplification, whether to contain the nucleotide sequence of hNAT-1 in the test sample.This test kit also can contain and carries out other required reagent of PCR reaction, for example damping fluid etc.

In addition, preferably, at least one used primer has been crossed over two exons, because will not produce amplified production corresponding to the genome sequence of hNAT-1 this moment.

Test kit 2:

This test kit contains the specific antibody (for example polyclonal antibody of preparation among the embodiment 5, the perhaps monoclonal antibody of the anti-hNAT-1 of the hybridoma technology generation of usefulness standard) at hNAT-1, and operation instruction.Whether this test kit is used for direct test sample and exists or lack hNAT-1 albumen.Form immunocomplex by specific immune response earlier, detect immunocomplex with routine techniques then.

Test kit 3:

This test kit contains can be specifically and the probe of the mRNA hybridization of hNAT-1.It also can contain or not contain hybridization buffer.This test kit comes whether to exist in the test sample nucleic acid molecule of hNAT-1 by the hybridization between nucleic acid molecule and the hNAT-1 specific probe.

Test kit also can be used for detecting genovariation, sudden change and polymorphism, and detects the relevant gene of hNAT-1 a series of and of the present invention, thereby diagnosis, the treatment of relative disease helped out.

Example 8:

Make medicine

HNAT-1 albumen of the present invention and antibody, inhibitor, antagonist or acceptor etc. can be used as medicine, can promote the wound healing after scalds and burns, cutting tissue and even organ are extractd, and prevent wound infection.With hNAT-1 nucleic acid antisense strand of the present invention hNAT-1 antibody generate a reagent box can be used for alleviating or assisting therapy owing to the too high disease that causes of hNAT-1 expression amount, also can assist the diagnosis of tumour and cancer clinically, prevention and treatment.

Albumen of the present invention and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is generally about 5-8, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.

In addition, hNAT-1 nucleic acid of the present invention (encoding sequence or antisense sequences) can directly be introduced cell, with expression level that improves hNAT-1 or the overexpression that suppresses hNAT-1.HNAT-1 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of hNAT-1 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.

When hNAT-1 protein polypeptide of the present invention is used as medicine, the treatment effective dose is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Sequence table＜210〉1＜211〉1516＜212〉Nucleotide＜213〉human＜220〉＜221〉encoding sequence＜222〉(117) .. (1487)＜223〉＜400〉1gtacggaatg ccggaagggc cgggctcaaa gctccgcctc tggcgcgacc gacgactgga 60gcgcagggca ggggtagagg ctcgtagatg gaactggtag tcagctggag agcagc atg 119

Met

1gag?gcg?tcc?tgg?ggg?agc?ttc?aac?gct?gag?cgg?ggc?tgg?tat?gtc?tct 167Glu?Ala?Ser?Trp?Gly?Ser?Phe?Asn?Ala?Glu?Arg?Gly?Trp?Tyr?Val?Ser

5 10 15gtc?cag?cag?cct?gaa?gaa?gcg?gag?gcc?gaa?gag?ttg?agt?ccg?ttg?cta 215Val?Gln?Gln?Pro?Glu?Glu?Ala?Glu?Ala?Glu?Glu?Leu?Ser?Pro?Leu?Leu

20 25 30agc?aac?gaa?ctt?cac?aga?cag?cga?tcc?cca?ggt?gtt?tca?ttt?ggt?tta 263Ser?Asn?Glu?Leu?His?Arg?Gln?Arg?Ser?Pro?Gly?Val?Ser?Phe?Gly?Leu

35 40 45tca?gtg?ttt?aat?ttg?atg?aat?gcc?atc?atg?gga?agt?ggc?atc?ctt?ggc 311Ser?Val?Phe?Asn?Leu?Met?Asn?Ala?Ile?Met?Gly?Ser?Gly?Ile?Leu?Gly50 55 60 65tta?gct?tat?gtt?atg?gct?aat?acc?ggt?gtc?ttt?gga?ttt?agc?ttc?ttg 359Leu?Ala?Tyr?Val?Met?Ala?Asn?Thr?Gly?Val?Phe?Gly?Phe?Ser?Phe?Leu

70 75 80ctg?ctg?aca?gtt?gct?ctc?ctg?gct?tct?tac?tca?gtc?cat?ctt?ctg?ctt 407Leu?Leu?Thr?Val?Ala?Leu?Leu?Ala?Ser?Tyr?Ser?Val?His?Leu?Leu?Leu

85 90 95agt?atg?tgt?att?cag?aca?gct?gta?aca?tct?tat?gaa?gat?ctt?gga?ctc 455Ser?Met?Cys?Ile?Gln?Thr?Ala?Val?Thr?Ser?Tyr?Glu?Asp?Leu?Gly?Leu

100 105 110ttt?gca?ttt?gga?tta?cct?gga?aag?ttg?gtg?gtg?gca?ggc?acc?ata?ata 503Phe?Ala?Phe?Gly?Leu?Pro?Gly?Lys?Leu?Val?Val?Ala?Gly?Thr?Ile?Ile

115 120 125att?cag?aat?att?gga?gct?atg?tca?tct?tat?ctt?tta?att?att?aaa?aca 551Ile?Gln?Asn?Ile?Gly?Ala?Met?Ser?Ser?Tyr?Leu?Leu?Ile?Ile?Lys?Thr130 135 140 145gag?ctt?cct?gct?gct?att?gca?gaa?ttt?ttg?act?gga?gac?tat?agt?aga 599Glu?Leu?Pro?Ala?Ala?Ile?Ala?Glu?Phe?Leu?Thr?Gly?Asp?Tyr?Ser?Arg

150 155 160tat?tgg?tat?ctt?gat?gga?caa?aca?cta?cta?ata?atc?ata?tgt?gtt?ggc 647Tyr?Trp?Tyr?Leu?Asp?Gly?Gln?Thr?Leu?Leu?Ile?Ile?Ile?Cys?Val?Gly

165 170 175att?gtg?ttc?cct?ctt?gca?ctt?ctt?ccc?aaa?ata?ggc?ttt?ctt?ggc?tac 695Ile?Val?Phe?Pro?Leu?Ala?Leu?Leu?Pro?Lys?Ile?Gly?Phe?Leu?Gly?Tyr

180 185 190aca?agt?agt?tta?tca?ttt?ttc?ttt?atg?atg?ttc?ttt?gct?ctt?gtg?gta 743Thr?Ser?Ser?Leu?Ser?Phe?Phe?Phe?Met?Met?Phe?Phe?Ala?Leu?Val?Val

195 200 205ata?att?aaa?aaa?tgg?tcc?atc?cct?tgt?cct?ctg?aca?tta?aat?tat?gta 791Ile?Ile?Lys?Lys?Trp?Ser?Ile?Pro?Cys?Pro?Leu?Thr?Leu?Asn?Tyr?Val210 215 220 225gag?aaa?ggc?ttc?cag?att?tca?aat?gtt?aca?gat?gat?tgt?aag?cca?aag 839Glu?Lys?Gly?Phe?Gln?Ile?Ser?Asn?Val?Thr?Asp?Asp?Cys?Lys?Pro?Lys

230 235 240ctc?ttt?cat?ttc?tcc?aaa?gag?agt?gct?tat?gcc?tta?cca?acc?atg?gct 887Leu?Phe?His?Phe?Ser?Lys?Glu?Ser?Ala?Tyr?Ala?Leu?Pro?Thr?Met?Ala

245 250 255ttt?tca?ttt?ctc?tgc?cat?acc?tca?ata?ttg?ccc?ata?tac?tgt?gaa?ctt 935Phe?Ser?Phe?Leu?Cys?His?Thr?Ser?Ile?Leu?Pro?Ile?Tyr?Cys?Glu?Leu

260 265 270caa?agt?cct?tca?aag?aaa?aga?atg?cag?aat?gtt?acc?aat?aca?gca?att 983Gln?Ser?Pro?Ser?Lys?Lys?Arg?Met?Gln?Asn?Val?Thr?Asn?Thr?Ala?Ile

275 280 285gct?tta?agt?ttt?ctc?att?tat?ttt?ata?tct?gca?ctc?ttt?ggg?tac?ctc 1031Ala?Leu?Ser?Phe?Leu?Ile?Tyr?Phe?Ile?Ser?Ala?Leu?Phe?Gly?Tyr?Leu290 295 300 305act?ttt?tat?gac?aaa?gtg?gag?tca?gaa?tta?cta?aaa?ggt?tat?agt?aaa 1079Thr?Phe?Tyr?Asp?Lys?Val?Glu?Ser?Glu?Leu?Leu?Lys?Gly?Tyr?Ser?Lys

310 315 320tac?tta?tca?cat?gat?gtt?gtt?gtc?atg?act?gtg?aag?tta?tgc?ata?cta 1127Tyr?Leu?Ser?His?Asp?Val?Val?Val?Met?Thr?Val?Lys?Leu?Cys?Ile?Leu

325 330 335ttt?gct?gtg?ctt?ttg?aca?gtc?cct?cta?atc?cac?ttc?cct?gcc?aga?aaa 1175Phe?Ala?Val?Leu?Leu?Thr?Val?Pro?Leu?Ile?His?Phe?Pro?Ala?Arg?Lys

340 345 350gct?gta?aca?atg?atg?ttt?ttc?tcc?aat?ttt?cca?ttc?tca?tgg?att?cgc 1223Ala?Val?Thr?Met?Met?Phe?Phe?Ser?Asn?Phe?Pro?Phe?Ser?Trp?Ile?Arg

355 360 365cat?ttt?ttg?atc?act?cta?gca?ctc?aat?att?atc?atc?gtt?tta?ctt?gca 1271His?Phe?Leu?Ile?Thr?Leu?Ala?Leu?Asn?Ile?Ile?Ile?Val?Leu?Leu?Ala370 375 380 385ata?tat?gtt?cct?gac?att?aga?aat?gta?ttt?ggt?gta?gtt?ggt?gcc?agt 1319Ile?Tyr?Val?Pro?Asp?Ile?Arg?Asn?Val?Phe?Gly?Val?Val?Gly?Ala?Ser

390 395 400aca?tca?aca?tgt?ttg?att?ttt?ata?ttc?cca?gga?cta?ttt?tat?ctt?aaa 1367Thr?Ser?Thr?Cys?Leu?Ile?Phe?Ile?Phe?Pro?Gly?Leu?Phe?Tyr?Leu?Lys

405 410 415ctt?agc?aga?gag?gat?ttt?ctg?tca?tgg?aaa?aag?ctt?ggg?gca?ttc?gtt 1415Leu?Ser?Arg?Glu?Asp?Phe?Leu?Ser?Trp?Lys?Lys?Leu?Gly?Ala?Phe?Val

420 425 430ttg?ctc?atc?ttt?gga?att?ttg?gtt?ggg?aat?ttt?agt?tta?gca?ctc?atc 1463Leu?Leu?Ile?Phe?Gly?Ile?Leu?Val?Gly?Asn?Phe?Ser?Leu?Ala?Leu?Ile

435 440 445att ttt gat tgg att aat aaa taa aagaaatatt ttcctacttc ttacaagaa 1516Ile Phe Asp Trp Ile Asn Lys450 455＜210〉2＜211〉456＜212〉amino acid＜213〉mankind＜400〉2Met Glu Ala Ser Trp Gly Ser Phe Asn Ala Glu Arg Gly Trp Tyr Val1 5 10 15Ser Val Gln Gln Pro Glu Glu Ala Glu Ala Glu Glu Leu Ser Pro Leu

20 25 30Leu?Ser?Asn?Glu?Leu?His?Arg?Gln?Arg?Ser?Pro?Gly?Val?Ser?Phe?Gly

35 40 45Leu?Ser?Val?Phe?Asn?Leu?Met?Asn?Ala?Ile?Met?Gly?Ser?Gly?Ile?Leu

50 55 60Gly?Leu?Ala?Tyr?Val?Met?Ala?Asn?Thr?Gly?Val?Phe?Gly?Phe?Ser?Phe65 70 75 80Leu?Leu?Leu?Thr?Val?Ala?Leu?Leu?Ala?Ser?Tyr?Ser?Val?His?Leu?Leu

85 90 95Leu?Ser?Met?Cys?Ile?Gln?Thr?Ala?Val?Thr?Ser?Tyr?Glu?Asp?Leu?Gly

100 105 110Leu?Phe?Ala?Phe?Gly?Leu?Pro?Gly?Lys?Leu?Val?Val?Ala?Gly?Thr?Ile

115 120 125Ile?Ile?Gln?Asn?Ile?Gly?Ala?Met?Ser?Ser?Tyr?Leu?Leu?Ile?Ile?Lys

130 135 140Thr?Glu?Leu?Pro?Ala?Ala?Ile?Ala?Glu?Phe?Leu?Thr?Gly?Asp?Tyr?Ser145 150 155 160Arg?Tyr?Trp?Tyr?Leu?Asp?Gly?Gln?Thr?Leu?Leu?Ile?Ile?Ile?Cys?Val

165 170 175Gly?Ile?Val?Phe?Pro?Leu?Ala?Leu?Leu?Pro?Lys?Ile?Gly?Phe?Leu?Gly

180 185 190Tyr?Thr?Ser?Ser?Leu?Ser?Phe?Phe?Phe?Met?Met?Phe?Phe?Ala?Leu?Val

195 200 205Val?Ile?Ile?Lys?Lys?Trp?Ser?Ile?Pro?Cys?Pro?Leu?Thr?Leu?Asn?Tyr

210 215 220Val?Glu?Lys?Gly?Phe?Gln?Ile?Ser?Asn?Val?Thr?Asp?Asp?Cys?Lys?Pro225 230 235 240Lys?Leu?Phe?His?Phe?Ser?Lys?Glu?Ser?Ala?Tyr?Ala?Leu?Pro?Thr?Met

245 250 255Ala?Phe?Ser?Phe?Leu?Cys?His?Thr?Ser?Ile?Leu?Pro?Ile?Tyr?Cys?Glu

260 265 270Leu?Gln?Ser?Pro?Ser?Lys?Lys?Arg?Met?Gln?Asn?Val?Thr?Asn?Thr?Ala

275 280 285Ile?Ala?Leu?Ser?Phe?Leu?Ile?Tyr?Phe?Ile?Ser?Ala?Leu?Phe?Gly?Tyr

290 295 300Leu?Thr?Phe?Tyr?Asp?Lys?Val?Glu?Ser?Glu?Leu?Leu?Lys?Gly?Tyr?Ser305 310 315 320Lys?Tyr?Leu?Ser?His?Asp?Val?Val?Val?Met?Thr?Val?Lys?Leu?Cys?Ile

325 330 335Leu?Phe?Ala?Val?Leu?Leu?Thr?Val?Pro?Leu?Ile?His?Phe?Pro?Ala?Arg

340 345 350Lys?Ala?Val?Thr?Met?Met?Phe?Phe?Ser?Asn?Phe?Pro?Phe?Ser?Trp?Ile

355 360 365Arg?His?Phe?Leu?Ile?Thr?Leu?Ala?Leu?Ash?Ile?Ile?Ile?Val?Leu?Leu

370 375 380Ala?Ile?Tyr?Val?Pro?Asp?Ile?Arg?Asn?Val?Phe?Gly?Val?Val?Gly?Ala385 390 395 400Ser?Thr?Ser?Thr?Cys?Leu?Ile?Phe?Ile?Phe?Pro?Gly?Leu?Phe?Tyr?Leu

405 410 415Lys?Leu?Ser?Arg?Glu?Asp?Phe?Leu?Ser?Trp?Lys?Lys?Leu?Gly?Ala?Phe

420 425 430Val?Leu?Leu?Ile?Phe?Gly?Ile?Leu?Val?Gly?Asn?Phe?Ser?Leu?Ala?Leu

435 440 445Ile?Ile?Phe?Asp?Trp?Ile?Asn?Lys

450 455

Claims

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people hNAT-1 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 117-1487 position among described nucleotide sequence and the SEQ ID NO.1.

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 is the sequence of Nucleotide 117-1487 position among the SEQ ID NO.1.

4. isolating hNAT-1 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 hNAT-1 protein-active, it is characterized in that this method comprises:

(d) isolate polypeptide with hNAT-1 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 117-1487 position among the SEQ ID NO.1.

12. energy and the described hNAT-1 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 8-100 continuous nucleotide in the described dna molecular of claim 1

15. a test kit is characterized in that, it contains the described dna molecular of claim 1.

16. a test kit is characterized in that, it contains the described antibody of claim 12.

17. the application of the described dna molecular of claim 1 is characterized in that, it is used for nucleic acid amplification reaction as primer or with making gene chip; Perhaps be used to prepare the disease that medicine causes owing to the hNAT-1 encoding gene unusually with prevention, diagnosis or treatment.

18. the application of the described protein polypeptide of claim 4 is characterized in that, it uses agonist or inhibitor with the screening human amino acid transporter; Perhaps be used to prepare the disease that medicine causes owing to the hNAT-1 abnormal expression with prevention, diagnosis or treatment.