CN1377885A - Human signal conductive protein coding sequence, its preparing method and use - Google Patents

Abstract

The present invention relates to the field of gene engineering, and is especially human signal transducing protein (hGPSTP 1) and its cNDA coding sequence. The present invention also relates to the production process of said polynucleotides sequence and said polypeptide as well as their use. The hGPSTP1 protein, antibody, its coding sequence and antisense sequence are produced into medicine preparation and kit for preventing, diagnosing and treating cancer, diabetes, inflammation and autoimmune disease clinically.

Description

Human signal conductive protein 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 signal conductive 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 signal conduction is an important basic biological phenomena.No matter be unicellular organism, or multicellular organism, their cell is not all taking place to get in touch with surrounding environment all the time, is carrying out exchanging and coordinating, to keep balance and the unification of organism and world around and organism itself.The signal of outside or stimulation all will be crossed over cytolemma and be entered cell, and through different signal transduction pathways signal finally are transmitted into nucleus in tenuigenin, and induction phase is answered genetic expression, cause the cell phenotype variation and produce various biological effects.Many vital processes are regulated and are being controlled in the signal conduction.These processes comprise growth, growth, nerve conduction, hormone and internal secretion effect, learning and Memory, disease, aging and death of organism or the like; The differentiation that also comprises the propagation of cell and cell cycle regulating, cell migration, cellular form and function with keep, immunity, stress, malignant change of cell and cell wither or the like.Therefore, researchdevelopment to transmembrane signal transduction is rapid in the world in recent years, especially find that Tyrosylprotein kinase plays important effect in transmembrane signal transduction, find that again many basic biological phenomenas all have the participation of oncogene expression product, and these product majorities have the effect of transmitting signal, and these results more make the coverage rate of transmembrane signal transduction research field enlarge gradually.

Foremost theory is when counting " second messenger " theory that nineteen sixty-five is proposed by Sutherland in the signal transduction mechanism research, its main argument is: hormone produces cAMP by activated adenyl cyclase again as the receptors bind on " first messenger " and the target cell membrane.CAMP causes a series of intramicellar reaction and produces physiological effect as the second messenger.In the research process to the adenylate cyclase regulation mechanism subsequently, it is found that has a kind of heat-stable protein in the plasma membrane, Here it is G albumen.

The proteic full name of G is that gtp binding protein or GTP are in conjunction with regulating albumen (G protein/GTP bindingprotein).It can combine with guanylic acid, has hydrolysis GTP and generates GDP, promptly has the active albumen of GTP enzyme (GTPase).

The signal transducting system of G albumen coupling by g protein coupled receptor (G protein coupledrecptors, GPRs), G albumen and effector molecular composition.G albumen is a molecular switch, and conduction is dissociated subunit as transmitter with G albumen again by the signal that GPRs receives, and activates corresponding enzyme and ionic channel, produces important second messenger, thereby causes corresponding biological respinse in the born of the same parents.When external stimulus exists, activated receptors activates special G albumen, and catalysis α subunit bonded guanosine diphosphate(GDP) (GDP) changes guanosine triphosphate (GTP) into, thus α subunit conformational change, reduce the avidity of α subunit and β γ subunit, make tripolymer be separated into G α (GTP) and G β γ.Dissociated G α (GTP) and G β γ pass to signal in the cell nuclear by direct regulation and control downstream effector separately; When G α subunit combines with GDP,, suppress the signal conduction again in conjunction with G β γ.

G albumen is a super family (GTP-binding proteins superfamily), except that above-mentioned film is subjected to link coupled heterotrimeric G protein heterotrimeric G protein, also has small G-protein.Small G-protein not only has the GTP binding site, also has the GTPase active zone, can be divided into ras, rho, three families of rab, is commonly referred to as the ras superfamily.

Discover recently, contain rasGEF, rhoGEF, rhoGEF, rhoGAP, RGS, PDZ has a kind of name to be called the functional domain of DEP in the albumen of PH functional domain, cause is as far back as Dishevelled, find in three kinds of albumen of Egl-10and Pleckstrin and gain the name (Ponting C.P., Bork P. 1996, TrendsBiochem.Sci.21:245-246).Studies show that DEP territory and the proteic signal conduction of G relevant (Burchett S.A.2000, J.Neurochem.75:1335-1351; Wong H.C.et al.2000; Nat.Struct.Biol.7:1178-1184).

Summary of the invention

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding signal conductive protein family, signal conductive protein of the present invention be named as the hGPSTP1 (G protein signal transduction related protein 1, hGPSTP1).

Another object of the present invention provides a kind of new signal conductive protein family member, and this albumen is named as people hGPSTP1.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described people hGPSTP1.

The present invention also provides the application of hGPSTP1 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 hGPSTP1 protein-active, shows at least 90% homology from the nucleotides sequence of Nucleotide 263-1864 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 263-1864 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 263-1864 position.

In another aspect of this invention, provide a kind of isolating hGPSTP1 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 hGPSTP1 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hGPSTP1 protein-active operationally is connected in expression regulation sequence, form the hGPSTP1 protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 263-1864 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 hGPSTP1;

(c) be fit to express under the condition of hGPSTP1 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hGPSTP1 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 263-1864 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 " hGPSTP1 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with hGPSTP1 protein-active is as 263-1864 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 263-1864 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 263-1864 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 ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 263-1864 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 263-1864 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 hGPSTP1 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 " hGPSTP1 protein polypeptide " refers to have the SEQ IDNO.2 polypeptide of sequence of hGPSTP1 protein-active.This term also comprises having and variant form people hGPSTP1 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 hGPSTP1 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 hGPSTP1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-hGPSTP1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises hGPSTP1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of hGPSTP1 polypeptide.Usually, this fragment have the hGPSTP1 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 hGPSTP1 albumen or polypeptide.The difference of these analogues and natural hGPSTP1 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 hGPSTP1 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of hGPSTP1 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 hGPSTP1 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 hGPSTP1 that encodes.

The present invention also comprises the method that detects the hGPSTP1 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 hGPSTP1 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 hGPSTP1 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 hGPSTP1 gene product or fragment.Preferably, refer to that those can combine with hGPSTP1 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 hGPSTP1, comprise that also those do not influence the antibody of hGPSTP1 protein function.The present invention also comprise those can with modify or without the hGPSTP1 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 hGPSTP1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing hGPSTP1 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 hGPSTP1 function and the antibody that does not influence the hGPSTP1 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of hGPSTP1 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 hGPSTP1 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 hGPSTP1 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 hGPSTP1 is so to obtain: with human brain λ gt11cDNA library (Clontech company) is template, with two pairs of oligonucleotide is primer---A1:5 '-CCTGTCTCAACCACAAACCTT-3 ', B1:5 '-TAAGAGGCAGTCCACCATGGT-3 ' they are forward primer; Oligonucleotide A2:5 '-TAAGTCTGTTCGGTCTGGTTG-3 ', B2:5 '-ATTTAGAGCTTCCACGCTTAC-3 ' are reverse primer, carry out PCR.The PCR condition of A1/A2 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 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 about respectively about 1,000 and 800 bases.With Restriction Enzyme NcoI 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 by the homology retrieval, the type sequence that has signal peptide, DEP functional domain in the hGPSTP1 protein sequence of the present invention, therefore, hGPSTP1 and above-mentioned protein seemingly can influence fetal development and coagulate hemolytic action by the regulating and controlling effect to G protein signal conduction path.HGPSTP1 albumen of the present invention, antibody, its encoding sequence or antisense sequences are made medicament and test kit, be applied to clinical, can play cancer, diabetes, inflammation and the autoimmune disease that causes unusually by G albumen coupling approach prevention, the diagnosis and therapeutic action.These diseases comprise: thrombocytasthenia that cancer of the stomach, multiple endocrine neoplasm, erythroleukemia, sex-linkage pessimum hereditary diabetes insipidus, myotonia atrophica disease, false Parathyroid underactivity syndrome, chronic myelogenous leukemia, the cell adhesion factor that Hirschsprung (Hirschsprung ' s diseases), Duchenne-Arandisease, acute former myelogenous leukemia, receptor abnormality cause causes unusually or the like.

HSTP1 albumen and nucleotide sequence can be used to prevent, diagnose and treat the disease that the polypeptide unconventionality expression causes.For example, the hSTP1 nucleotide sequence can be used for correcting disappearance or other sudden changes of hSTP1 gene in patient's genome, makes patient self cell expressing intact proteins, thereby treatment is because decline of hSTP1 expression amount or the undesired disease that causes; Also can produce polypeptide, this peptide species is applied to patient by insertion hSTP1 nucleotide sequence, the proteic cell of culture expression hSTP1 in other species host cells.HSTP1 nucleotide sequence and complementary sequence thereof can be used as dna probe and survey similar nucleotide sequence in the also quantitative sample, and patient is diagnosed and observation of curative effect.

The hSTP1 polypeptide can be used as antigen to produce antibody or to be used to identify expression of polypeptides regulation and control person and activity, and the hSTP1 protein antibodies can be with surveying this proteic existence and downward modulation expression amount and activity in the sample.

HSTP1 polypeptide and Nucleotide also can be used as nutritive substance, realize a series of physiological functions, as: regulate cytokine and cell-proliferation activity, excite or suppress immune response (as treatment infected by microbes or autoimmunization system), regulate hematopoiesis, regulate tissue growth activity, regulate activator and statin activity, regulate chemotactic activity, regulate hemostasis and is connected active, inhibition tumor growth etc. with hemolytic activity, adjusting acceptor.

Embodiment

Embodiment 1

The clone and the mensuration of the cDNA sequence of hGPSTP1

1. primer amplification

In the present invention, the cDNA nucleotide sequence of STP is so to obtain: with human brain λ gt11cDNA library (Clontech company) is template, with two pairs of oligonucleotide is primer A1:5 '-CCTGTCTCAACCACAAACCT T-3 ', and B1:5 '-TAAGAGGCAGTCCACCATGGt-3 ' is a forward primer; Oligonucleotide A2:5 '-TAAGTCTGTTCGGTCTGGTTG-3 ', B2:5 '-ATTTAGAGCTTCCACGCTTAC-3 ' are reverse primer, carry out PCR.The PCR condition of A1/A2 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 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 about respectively about 1,000 and 800 bases.With Restriction Enzyme NcoI 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

To be connected with pGEM-T carrier (Promega) with above-mentioned amplified production, 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 EXCEL dna 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 SEQ ID NO.1, and wherein open reading frame is positioned at 263-1864 position Nucleotide.Derive the aminoacid sequence of hGPSTP1 according to the full length cDNA sequence that obtains, totally 534 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

Structural analysis

In Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with the full length cDNA sequence of hGPSTP1 and proteins encoded thereof, found that the type sequence that has signal peptide, DEP functional domain in the protein sequence of hGPSTP1 with BLAST.

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 hGPSTP1 of the present invention is LHPETSPGRGHLLAVLLALLGTAWAE (among the SEQ ID NO 2 from the 2-20 position).

DEP (Domain found in Dishevelled, Egl-10 and Pleckstrin) generally is a globular domain that constitutes by about 80 amino acid, cause is as far back as Dishevelled, find and (the Ponting C.P. that gains the name in three kinds of albumen of Egl-10 and Pleckstrin, Bork P. 1996, Trends Biochem.Sci.21:245-246).One of function of Di shevelled is that polar cell among the embryo and fragment are carried out the consistent arrangement of polarity; Egl-10 regulates G protein signal conduction in the central nervous system; Vertebrate Pleckstrin albumen then is the main substrate of blood platelet albumen kinase c.Except that above-mentioned three kinds of albumen, contain PH, rasGEF, rhoGEF, rhoGAP, RGS, also contain the DEP functional domain in the albumen of PDZ structural domain, these albumen are the modulin of G protein signal conduction in Mammals, thereby conduct by the GTP enzymic activity conditioning signal of DEP territory increase G protein alpha subunit, make G albumen become GDP bonded inactive state.Other has research to think, the DEP functional domain still is (Burchett S.A.2000, the J.Neurochem.75:1335-1351 of playing a decisive role in the specific G protein coupling signal path selecting the albumen that contains the DEP functional domain navigated to ubcellular film site; Wong H.C., Mao J., Nguyen J.T., Srinivas S., Zhang W., Liu B., Li L., Wu D., Zheng are J.2000; Nat.Struct.Biol.7:1178-1184).

In albumen of the present invention, also has the aminoacid sequence that meets the DEP functional domain: QTQVEVKKRRHRLKRHNDCLVGSEAVDVIFSHLIQNKYFGDVDIPRAKVVRVCQAL MDYKVFEAVPTKVFGKDKKPTFEDSSCSLYRFTT (49-139 position among the SEQ ID NO.3).This shows, hGPSTP1 of the present invention may with above-mentioned protein seemingly, in G protein signal conduction path, play regulating and controlling effect, and influence fetal development and coagulate hemolytic action by this regulating and controlling effect.

HGPSTP1 albumen of the present invention, its encoding sequence or antisense sequences and antibody are made medicament and test kit, be applied to clinical, cancer, diabetes and autoimmunity that can cause unusually and inflammatory diseases for approach by the G albumen coupling play prevention, the diagnosis and therapeutic action.These diseases comprise: thrombocytasthenia that cancer of the stomach, multiple endocrine neoplasm, erythroleukemia, sex-linkage pessimum hereditary diabetes insipidus, myotonia atrophica disease, false Parathyroid underactivity syndrome, chronic myelogenous leukemia, the cell adhesion factor that Hirschsprung (Hirschsprung ' s diseases), Duchenne-Arandisease, acute former myelogenous leukemia, receptor abnormality cause causes unusually or the like.

People hGPSTP1 of the present invention is used for further functional study except can be used as signal protein 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 hGPSTP1 can also merge with other members of this family or exchange fragment, to produce new albumen.

At the antibody of inventor hGPSTP1, be used to screen other albumen with similar structures territory, perhaps be used for the affinity purification associated protein.

For example, inventor hGPSTP1 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people hGPSTP1 or the overexpression that suppresses people hGPSTP1.People hGPSTP1 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people hGPSTP1 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 hGPSTP1 in intestinal bacteria

In this embodiment, use the PCR Oligonucleolide primers corresponding to 5 ' and 3 ' end of the cDNA sequence of coding hGPSTP1 to increase, the cDNA that obtains hGPSTP1 is as the insertion fragment.

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

5’-ACGAGGATCCATGGCCAGGGGCCTGAGCT-3’

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

3 ' end primer sequence is:

5’-TAAAGTCGACGTCTCCAAAATGCTCAATA-3’

This primer contains the restriction enzyme site of SalI restriction enzyme, the partial nucleotide sequence of the encoding sequence of translation termination and hGPSTP1.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (QiagenInc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp ^r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With BamHI and SalI digestion pQE-9 carrier 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 hGPSTP1 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 hGPSTP1 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out hGPSTP1 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 1-20 position.

Embodiment 4

The expression of hGPSTP1 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, use PCR Oligonucleolide primers to increase, obtain hGPSTP1 cDNA as the insertion fragment corresponding to 5 ' and 3 ' end of the cDNA sequence of coding hGPSTP1.

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

5’-ACGAGTCGACATGGCCAGGGGCCTGAGCT-3’

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

3 ' end primer sequence is:

5’-TAAAGGATCCGTCTCCAAAATGCTCAATA-3’

This primer contains the partial nucleotide sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and hGPSTP1.

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 (SV40ori), 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 SalI 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 hGPSTP1 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 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.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 50mMTrisHCl (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 1-20 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 hGPSTP1 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: 2013bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： b.：cDNA c.：SEQ ID NO.1 aacgcccccg ccgagcgggc ggatcgagtt cctctctggc tggtgggctg caagcggagg gcccgcccgc acagcccagc ttttttcttt ctacccagac tctcaacttg accgcgcgtt ggtctgtgcg cccgctaacg tgccaggcac gcgccgggga gctccgggat atgctccgcg gtgctaccgg tgacaagtgt ggtatctgga atttgtgtag ttctttgccc cctgtctcaa ccacaaacct tgacgacaca gtatggccag gggcctgagc tcctcccttg cccactttgt tctccagact gctaggagtc aagagtcagc ttgtctcccc agactcttcc tgggagggat gagaggttta tgtgagttct actggcagga atttggcata aaaggtttca gtgtagctca gaagccattt ggagccacgt atgtatggag cagcatcata aacactcttc aaacacaagt ggaagtgaaa aaacgaaggc accgtttaaa acgacataat gactgcttgg ttggttcaga agctgtggat gtcatttttt ctcacctaat tcagaataag tattttggtg atgtagatat tcctcgagcc aaagtggtga gagtgtgtca agcgcttatg gactacaaag tatttgaagc agttccaacc aaagtctttg gaaaagacaa aaaacctaca tttgaagata gtagttgcag cctttataga ttcaccacaa tacctaacca agacagtcag ttaggcaaag agaacaaact atattcacct gccaggtatg cagatgcatt atttaagtca tccgatatca gatcagccag tttagaggac ctgtgggaaa atctgagttt aaagcctgcc aactcccctc atgtaaatat ctctgcaacc ttgtctccac aagttattaa tgaagtgtgg caagaagaaa caattgggcg tctactacaa cttgtagacc ttccacttct tgactcctta ctgaaacagc aagaggctgt acctaaaatt cctcaaccta agaggcagtc caccatggtc aacagcagta actatctgga tcgagggatt ctcaaggctt atagtgactc tcaggaagat gagtggctct cggcagcaat tgactgttca gaataccttc cagaccaaat ggtggtggaa ataagcagaa gctttcctga gcaaccagac cgaacagact tagtgaaaga acttctgttt gatgccattg gcagatatta cagtagtagg gaacctctgt taaatcactt atctgacgtt cataatggaa ttgcagaact cttagtgaat gggaagacgg aaatagcttt agaagctacc cagctccttc taaagctttt agatttccaa aatagagaag aatttagaag actactgtat ttcatggctg ttgcagcaaa tccttctgag tttaaattac agaaagaaag tgacaaccga atggttgtga aaaggatatt ctcaaaagct attgttgaca ataaaaattt atccaaaggc aaaacagatc ttctggtact ctttttaatg gatcatcaga aagatgtttt taagattcct ggaactctac ataaaattgt aagtgttaag cttatggcca tacagaacgg aagagatcca aatagagatg caggatatat ttattgccag agaattgatc aacgtgacta ttccaacaat acagagaaga caaccaaaga tgagctgttg aatttactaa aaactcttga tgaggattca aaactttctg ccaaagagaa gaaaaaattg ctaggtcaat tctataagtg tcacccagac atctttattg agcattttgg agactgagtt tttaatatct gtatataagt tgtgtatttt aagaataaat tatgtatcct aaatatccaa tcacatttgt aagcgtggaa gctctaaatt tgaaactgta cttaataaaa atttttttgt ataaaaaaaa aaaaaaaaaa aaa ( 2 ) SEQ ID NO.2： a.：

(A) length: 534 amino acid

(B) type: amino acid

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

MARGLSSSLAHFVLQTARSQESACLPRLFLGGMRGLCEFYWQEFGIKGFS

VAQKPFGATYVWSSIINTLQTQVEVKKRRHRLKRHNDCLVGSEAVDVIFS

HLIQNKYFGDVDIPRAKVVRVCQALMDYKVFEAVPTKVFGKDKKPTFEDS

SCSLYRFTTIPNQDSQLGKENKLYSPARYADALFKSSDIRSASLEDLWEN

LSLKPANSPHVNISATLSPQVINEVWQEETIGRLLQLVDLPLLDSLLKQQ

EAVPKIPQPKRQSTMVNSSNYLDRGILKAYSDSQEDEWLSAAIDCSEYLP

DQMVVEISRSFPEQPDRTDLVKELLFDAIGRYYSSREPLLNHLSDVHNGI

AELLVNGKTEIALEATQLLLKLLDFQNREEFRRLLYFMAVAANPSEFKLQ

KESDNRMVVKRIFSKAIVDNKNLSKGKTDLLVLFLMDHQKDVFKIPGTLH

KIVSVKLMAIQNGRDPNRDAGYIYCQRIDQRDYSNNTEKTTKDELLNLLK

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

(A) length: 514 amino acid

(B) type: amino acid

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

ESACLPRLFLGGMRGLCEFYWQEFGIKGFSVAQKPFGATYVWSSIINTLQ

TQVEVKKRRHRLKRHNDCLVGSEAVDVIFSHLIQNKYFGDVDIPRAKVVRVCQALMDYKVFEAVPTKVFGKDKKPTFEDSSCSLYRFTTIPNQDSQLGKENKLYSPARYADALFKSSDIRSASLEDLWENLSLKPANSPHVNISATLSPQVINEVWQEETIGRLLQLVDLPLLDSLLKQQEAVPKIPQPKRQSTMVNSSNYLDRGILKAYSDSQEDEWLSAAIDCSEYLPDQMVVEISRSFPEQPDRTDLVKELLFDAIGRYYSSREPLLNHLSDVHNGIAELLVNGKTEIALEATQLLLKLLDFQNREEFRRLLYFMAVAANPSEFKLQKESDNRMVVKRIFSKAIVDNKNLSKGKTDLLVLFLMDHQKDVFKIPGTLHKIVSVKLMAIQNGRDPNRDAGYIYCQRIDQRDYSNNTEKTTKDELLNLLKTLDEDSKLSAKEKKKLLGQFYKCHPDIFIEHFGD

Claims (14)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people hGPSTP1 protein-active, shows at least 90% homology from the nucleotides sequence of Nucleotide 263-1864 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 864 of Nucleotide 263-1.

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 has the sequence of Nucleotide 263-1864 position among the SEQ ID NO.1.

4. isolating hGPSTP1 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 hGPSTP1 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hGPSTP1 protein-active operationally is connected in expression regulation sequence, form the hGPSTP1 protein expression vector, show at least 90% homology from the nucleotides sequence of Nucleotide 263-1864 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 hGPSTP1;

(c) be fit to express under the condition of hGPSTP1 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hGPSTP1 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 263-1864 position among the SEQ ID NO.1.

12. energy and the described hGPSTP1 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.