CN1249346A - Human gene sequence, its encoded polypeptide and its preparing process - Google Patents

Abstract

The present invention discloses a new human gene nucleotide sequence, specifically, the cDNA sequence of human WSB1. The protein coded by said sequence is the homolog of mouse WSB1. The present invention also relates to the polypeptide coded by said nucleotide sequence and the application and preparing process of said polynucleotide and said polypeptide.

Description

A kind of new people's gene sequence, its encoded polypeptides and preparation method

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 people WSB1, this albumen is the homologue of mouse WSB1.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.

WSB1 belongs to the member in the subfamily (WD-40) in the cytokine signaling conduction repressor family (SOC).WSB1, WSB2 are two genes finding in the mouse, and they are considered to relevant with the restraining effect of cytokine signaling conduction.The cytokine signaling conduction is a universal phenomenon that all exists in all eucaryons and the prokaryotic organism.Especially in eukaryote, cytokine is that a class has the factor that important biomolecule is learned function, regulates the important proteic secretion of control in vivo, and these albumen play an important role again in the growth of biology and atomization.Cytokine is to bring into play the function that its inducing cell divides and breaks up by a signal pipeline that is called JAK-STAT.Cytokine signaling conduction repressor can be produced by numerous cytokine inductions that activate the JAK-STAT approach, and inhibited to the JAK-STAT approach.

At present, also at the early-stage for WSB subfamily member's research, for the research of SOC (cytokine signaling conduction repressor family) then relatively earlier.Difference according to the contained center function of member district in the SOC family is divided into four subfamilies, CIS, WSB, SRPY and ASB with it.The earliest member is CIS in the SOC gene family, by people such as Yoshimura A. in nineteen ninety-five in the mouse body, find (EMMO J.1995,14,2816-2826).Then, people such as Starr R. separated in 1997 and the clone has obtained gene (Nature1997,387 (6636): 917-921) such as CIS family member SOCS-1, SOCS-2.Recently, people have obtained the clone of member in SPRY and the ASB family again respectively from mouse.WSB1 is a gene of finding in the mouse.It is that (Proc.Natl.Acad.Sci.USA 1998 by people such as the Hilton D.J. gene that the clone obtained from mouse spermatocyte cDNA library in 1998,95:114-119), it has higher homology and has similar function to another gene WSB2 in the mouse.WSB1, WSB2 and genes more noted earlier all belong to SOC family, discover, though their some differences on structural domain, their function is closely similar, play a part to regulate protein transport in cell.

Yet, before the present invention, still do not have to belong among the finder member of WSB subfamily.

An object of the present invention is to provide a kind of new polynucleotide sequence, this polynucleotide sequence coding people's a kind of and mouse WSB1 homologous albumen, of the present invention and mouse WSB1 homologous people's gene is named as people WSB1 (GenBank AccessionNo.AF064257) (annotating: because of applying for secret for some time, so the sequence of logining is open to the public) before the application.

Another object of the present invention provides a kind of new albumen, and this albumen is named as people WSB1 albumen.

A further object of the present invention provides a kind of recombinant technology that utilizes and produces the described new proteic method of people WSB1.

The present invention also provides this people WSB1 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 WSB1 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 31-1296 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 31-1296 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has the nucleotide sequence of 31-1296 position among the SEQ ID NO.3.

In another aspect of this invention, provide a kind of isolating people WSB1 protein polypeptide, it comprises: have polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.

In another aspect of this invention, provide a kind of described carrier transformed host cells.

In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people WSB1 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people WSB1 protein-active operationally is connected in expression regulation sequence, form people WSB1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 31-1296 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people WSB1;

(c) under the condition that is fit to expressing human WSB1 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people WSB1 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1325 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 31-1296 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 " people WSB1 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people WSB1 protein-active is as 31-1296 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.4.This degenerate sequence is meant, is arranged in the encoder block 31-1296 position Nucleotide of SEQ ID NO.4 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.4 in 31-1296 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition with SEQ ID NO.3 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 31-1296 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 31-1296 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.3 with people WSB1 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 " people WSB1 protein polypeptide " refers to have the SEQ IDNO.4 polypeptide of sequence of people WSB1 protein-active.This term also comprises having and variant form people WSB1 identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people WSB1 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 people WSB1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people WSB1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people WSB1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of people WSB1 polypeptide.Usually, this fragment have people WSB1 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 people WSB1 albumen or polypeptide.The difference of these analogues and natural human WSB1 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 people WSB1 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of people WSB1 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 people WSB1 nucleotide sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people WSB1.

The present invention also comprises the method that detects people WSB1 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 people WSB1 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 people WSB1 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 people WSB1 gene product or fragment.Preferably, refer to that those can combine with people WSB1 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 people WSB1, comprise that also those do not influence the antibody of people WSB1 protein function.The present invention also comprise those can with modify or without the people WSB1 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 people WSB1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human WSB1 or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people WSB1 function and the antibody that does not influence people WSB1 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people WSB1 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 people WSB1 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 WSB1 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.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

In one embodiment of the invention, the cDNA nucleotide sequence of people WSB1 is so to obtain, with people's testis λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer A1:5 ＇-GTATTCCCGGAATCAGACGGTGC-3 ＇ and reverse primer A2:5 ＇-GTCCCTACTACTAGGGAAGGCAG-3 ＇, carry out PCR, obtain the purpose fragment of 1325bp (A1/A2).Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.

WSB1, WSB2 belong to the member in cytokine signaling conduction repressor family (SOC) subfamily (WSB).It is relevant with the restraining effect of the signal conduction of cytokine that WSB1 and WSB2 are considered to.Cytokine is that a class has the factor that important biomolecule is learned function, regulates important proteic secretion in vivo, and these albumen play an important role again in the growth of biology and atomization.Cytokine signaling conduction repressor can be produced by numerous cytokine inductions that activate the JAK-STAT approach, and inhibited to the JAK-STAT approach.The WSB1 height homology of people WSB1 gene of the present invention and mouse, simultaneously also higher with the homology of WSB2, this shows that it is the homologous gene of mouse WSB1 in the people, and to WSB1 (and WSB2) similar biology is arranged.

In the accompanying drawings,

Fig. 1 is the homology comparison diagram of the nucleotide sequence of people WSB1 of the present invention (hWSB1) and mouse WSB1 (mWSB1).Wherein, identical Nucleotide marks with " | ".

Fig. 2 is the homology comparison diagram of the aminoacid sequence of people WSB1 albumen of the present invention (hWSB1) and mouse WSB1 albumen (mWSB1).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone and the mensuration of the cDNA sequence of people WSB1

1. primer amplification

With people's testis λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 ＇-GTATTCCCGGAATCAGACGGTGC-3 ＇ (SEQ ID NO:1) is a forward primer, oligonucleotide A2:5 ＇-GTCCCTACTACTAGGGAAGGCAG-3 ＇ (SEQ ID NO:2) is a reverse primer, carries out PCR.A1, the right PCR condition of A2 primer be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 70 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes; The PCR segment that electrophoresis detection obtains is that primer is about more than 1300 base pair to the purpose fragment that amplifies with A1, A2.

2.PCR the order-checking of product

The pcr amplification product that as above obtains is connected with pGEM-T carrier (Promega) respectively, 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.Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 1325bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 31-1296 position Nucleotide.

Derive the aminoacid sequence of people WSB1 according to the full length cDNA sequence that obtains, totally 421 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.

Embodiment 2

Homology relatively

The full length cDNA sequence of personnel selection WSB1 and proteins encoded thereof carry out nucleic acid and albumen homology retrieval with blast program in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database.Found that they and mouse WSB1 gene and proteins encoded thereof have high homology, about the identity on the nucleic acid level has reached 87% (Fig. 1), identity on the protein level and similarity are respectively 92% and 95% (Fig. 2), so, people WSB1 of the present invention is the homologue of mouse WSB1 in the people, and has the same or analogous function with WSB family member (especially mouse WSB1).

WSB1 belongs to a member in cytokine signaling repressor family (SOC) subfamily (WSB).In vivo, cytokine mainly plays the important proteic effect of control emiocytosis of regulating, and these albumen also play an important role in the growth of biology and atomization.The cytokine protein product is to bring into play the function that its inducing cell divides and breaks up by a signal pipeline that is called JAK-STAT.Cytokine signaling conduction repressor can be produced by numerous cytokine inductions that activate the JAK-STAT approach, and inhibited (Nature 1995,377 (19), 591-594) to the JAK-STAT approach.The member of all SOC families all has similar constructional feature, and their N-terminal contains the amino acid composition of different lengths, and C-terminal then all has the fancy structure of a non-identity, i.e. SOCS frame.SOC family is by the composition difference in division center territory, be divided into four subfamilies: contain the SH2 structural domain the CIS subfamily, contain the WD-40 structural domain the WSB subfamily, contain the SSB subfamily of SPRY structural domain and contain ASB subfamily (J Leukoe Biol1998,63 (6): 665-668) of ankyrin repeat structural domain.According to the structures shape function, the intimate principle of structural similitude can be inferred the function of people WSB1 from these genes or proteic function.

As everyone knows, conduction of the signal of cytokine and inhibition thereof play an important role for important protein excretion and transportation in the adjusting organism, so WSB1 plays an important role in vivo.

The WSB family member had both been contained the WD-40 tumor-necrosis factor glycoproteins, contained the SOCS functional zone again.Its function in vivo also has this two functional zone decisions.The characteristic sequence that particularly in the aminoacid sequence of people WSB1, has 2 WD-40 tumor-necrosis factor glycoproteinss of forming by 15 amino acid:

——(L/I/V/M/S/T/A/C)-(L/I/V/M/F/Y/W/S/T/A/G/C)-(L/I/M/S/T/A/G)-(L/I/V/M/S/T/A/G/C)-X2-(D/N)-X2-(L/I/V/M/W/S/T/A/C)-X-(L/I/V/M/F/S/T/A/G)-W-(D/E/N/)-(L/I/V/M/F/S/T/A/G/C/N)

[annotate: X is an arbitrary amino acid in this sequence, and numerals such as " 2 " is an amino acid number, and an amino acid is selected in " (L/I/V/M/S/T/A/C) " expression arbitrarily from these 8 amino acid].

The sequence fragment that meets above-mentioned pattern in albumen of the present invention is: LATGLNNGRIKIWDV (140-155 position among the SEQID NO.4), LVSASRDKTLRVWDL (183-198 position among the SEQ ID NO.4).This has proved that further people WSB1 of the present invention is a member in the WD-40 family.

In addition, also find also to exist among the people WSB1 of the present invention 1 GH-WD tumor-necrosis factor glycoproteins

-X _6-94-(GH-X _23-41-WD)-

About the description of this GH-WD tumor-necrosis factor glycoproteins, can be referring to people such as Eva J.Neer, Nature1994,371 (22): 297-300.Wherein, X _6-946-94 amino acid whose variable-length of expression, X _23-41Expression 23-41 amino acid whose variable-length preferably is spaced apart 27 ± 2 amino acid between GH and WI.

The sequence fragment that albumen of the present invention meets above-mentioned pattern is: GHHHDVVACDFSPDGALLATASYDTRVVIWD (253-284 position among the SEQ ID NO.4), the GH-WD tumor-necrosis factor glycoproteins is the WD tumor-necrosis factor glycoproteins, 27 amino acid at interval between GH and the WD wherein, this has proved that further people WSB1 of the present invention is a member in the WD-40 family.

The distribution of member in the WD-40 family is very extensive, all can find in from the nucleus to the cytoplasmic membrane, and they also are considered to the moiety of cytoskeleton.Yeast is considered to study the optimal mode of WD-40 tumor-necrosis factor glycoproteins function, and this tumor-necrosis factor glycoproteins is relevant with proteic interaction.WD-40 tumor-necrosis factor glycoproteins tumor-necrosis factor glycoproteins common and the TPR family member interacts, thereby regulates proteic interaction and transhipment.Discover that the interaction of WD-40 and TPR tumor-necrosis factor glycoproteins is (FEBS Lett.1992307 (2) 131-134) that finishes by the transcriptional expression that suppresses nucleic acid.In sum, people's WSB belongs to a member in the WD-40 family, and has WD-40 family member's above-mentioned correlation function.

In addition, the member in the SOC family is all contained the SOCS frame again, and cytokine signaling conduction inhibition mainly plays a part to suppress the cytokine signaling conduction in vivo.Studies show that its effect may be by combining with the special sequence of recipient cell, thus stop albumen and acceptor combine the normal transhipment of arrestin.When secretory protein has surpassed its normal contents in cell, just produce reverse feedback with these albumen of control cytokine secretion by the cytokine aporepressor.The SOCS frame may be the functional zone that these albumen play a role, target protein is with after the SOCS frame combines, make right judgement by soc protein, the speed that produces with decision albumen, and be stabilized in the tram of cell with position certain in the target protein transporte to cells and with it by signal conductive protein.Signal conduction aporepressor also may combine with kinases by the SOCS frame, suppresses kinase whose catalytic activity and comes the transhipment of aporepressor (Proc.Natl.Acad.Sci.USA 1998,95:114-119).The C-terminal of people WSB1 polypeptide of the present invention also contain one with SOCS frame height homologous zone, this has shown that it also may have the same or analogous function with the SOC family member.

As from the foregoing, the member in the WSB subfamily also may play dual function in this process.It plays a part to regulate and the correct transhipment of pilot protein in vivo.Mainly in numerous physiological functions (as: cell development, differentiation, propagation and the immunity) process that relies on the JAK-STAT approach, play an important role.This also provides foundation for we further study the effect in vivo of signal factor repressor.

People WSB1 of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor WSB1 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of the N of inventor WSB1 end with the WSB1 of mouse exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor WSB1, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

In addition, inventor WSB1 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people WSB1 or the overexpression that suppresses people WSB1.People WSB1 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people WSB1 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 people WSB1 in intestinal bacteria

In this embodiment, the cDNA sequence of coding people WSB1 used corresponding to 5 ＇ of this dna sequence dna and the PCR Oligonucleolide primers of 3 ＇ end increase, obtain people WSB1 cDNA as inserting fragment.

The 5 ＇ Oligonucleolide primers sequences of using in the PCR reaction are:

5＇-CAGAGTCGACATGGCCAGCTTTCCCCCGAG-3＇(SEQ?ID?NO.5)，

This primer contains the restriction enzyme site of SalI restriction enzyme, is 20 Nucleotide of the people WSB1 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ＇ end primer sequence is:

5’-CCAAGTCGACCTAAATACGATACGAGAGAAAC-3’(SEQ?ID?NO.6)，

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people WSB1 of SalI restriction enzyme.

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

With 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 (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people WSB1 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, when culturing cell to 600 optical density(OD) (OD600) is 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 people WSB1 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people WSB1 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.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 48KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 4

The expression of people WSB1 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, the cDNA sequence of coding people WSB1 used corresponding to 5 ＇ of this dna sequence dna and the PCR Oligonucleolide primers of 3 ＇ end increase, obtain people WSB1 cDNA as inserting fragment.

The 5 ＇ Oligonucleolide primers sequences of using in the PCR reaction are:

5 ＇-CAGAGAATTCATGGCCAGCTTTCCCCCGAG-3 ＇ (SEQ ID NO.7), this primer contains the restriction enzyme site of EcoRI restriction enzyme, is 20 Nucleotide of the people WSB1 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ＇ end primer sequence is:

This primer of 5 '-CCAATCTAGACTAAATACGATACGAGAGAAAC-3 ' (SEQ ID NO.8) contains the part encoding sequence of the restriction enzyme site of XbaI restriction enzyme, translation termination and people WSB1.

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 EcoRI, XbaI 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 HindIII enzyme and insert clip size and direction, and the cDNA fragment of sequence verification people WSB1 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 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 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 48KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 5

Preparation antibody

The recombinant protein that 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 WSB1 gene translation product with it.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table (1) general information: (i) applicant: Fudan University is denomination of invention (ii): a kind of new people's gene sequence, its encoded polypeptides and preparation method be the sequence number (iii): information (i) sequence signature of 8 (2) SEQ ID NO:1

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:1:GTATTCCCGG AATCAGACGG TGC 23 (2) SEQ ID NO:2

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:2GTCCCTACTA CTAGGGAAGG CAG 23 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 1325bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3 1 GTATTCCCGG AATCAGACGG TGCCCCATAG ATGGCCAGCT TTCCCCCGAG GGTCAACGAG 61 AAAGAGATCG TGAGATCACG TACTATAGGT GAACTTTTAG CTCCTGCAGC TCCTTTTGAC 121 AAGAAATGTG GTCGTGAAAA TTGGACTGTT GCTTTTGCTC CAGATGGTTC ATACTTTGCT 181 TGGTCACAAG GACATCGCAC AGTAAAGCTT GTTCCGTGGT CCCAGTGCCT TCAGAACTTT 241 CTCTTGCATG GCACCAAGAA TGTTACCAAT TCAAGCAGTT TAAGATTGCC AAGACAAAAT 301 AGTGATGGTG GTCAGAAAAA TAAGCCTCGT GAACATATTA TAGACTGTGG AGATATAGTC 361 TGGAGTCTTG GTTTTGGGTC ATCAGTTCCA GAAAAACAGA GTCGCTGTGT AAATATAGAA 421 TGGCATCGCT TCAGATTTGG ACAAGATCAG CTACTTCTTG CTACAGGGTT GAACAATGGG 481 CGTATCAAAA TATGGGATGT ATATACAGGA AAACTCCTCC TTAACTTGGT AGATCATACT 541 GAAGTGGTCA GAGATTTAAC TTTTGCTCCA GATGGAAGCT TGATCCTGGT GTCAGCTTCA 601 AGAGACAAAA CTCTCAGAGT ATGGGACCTG AAAGATGATG GAAACATGAT GAAAGTATTG 661 AGGGGGCATC AGAATTGGGT GTACAGCTGT GCATTCTCTC CTGACTCTTC TATGCTGTGT 721 CCAGTCGGAG CCAGTAAAGC AGTTTTCCTT TGGAATATGG ATAAATACAC CATGATACGG 781 AAACTAGAAG GACATCACCA TGATGTGGTA GCTTGTGACT TTTCTCCTGA TGGAGCATTA 841 CTGGCTACTG CATCTTATGA TACTCGAGTA TATATCTGGG ATCCACATAA TGGAGACATT 901 CTGATGGAAT TTGGGCACCT GTTTCCCCCA CCTACTCCAA TATTTGCTGG AGGAGCAAAT 961 GACCGGTGGG TACGATCTGT ATCTTTTAGC CATGATGGAC TGCATGTTGC AAGCCTTGCT1021 GATGATAAAA TGGTGAGGTT CTGGAGAATT GATGAGGATT ATCCAGTGCA AGTTGCACCT1081 TTGAGCAATG GTCTTTGCTG TGCCTTCTCT ACTGATGGCA GTGTTTTAGC TGCTGGGACA1141 CATGACGGAA GTGTGTATTT TTGGGCCACT CCACGGCAGG TCCCTAGCCT GCAACATTTA1201 TGTCGCATGT CAATCCGAAG AGTGATGCCC ACCCAAGAAG TTCAGGAGCT GCCGATTCCT1261 TCCAAGCTTT TGGAGTTTCT CTCGTATCGT ATTTAGAAGA TTCTGCCTTC CCTAGTAGTA1321 GGGAC ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 421 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4 1 Met Ala Ser Phe Pro Pro Arg Val Asn Glu Lys Glu Ile Val Arg 16 Ser Arg Thr Ile Gly Glu Leu Leu Ala Pro Ala Ala Pro Phe Asp 31 Lys Lys Cys Gly Arg Glu Asn Trp Thr Val Ala Phe Ala Pro Asp 46 Gly Ser Tyr Phe Ala Trp Ser Gln Gly His Arg Thr Val Lys Leu 61 Val Pro Trp Ser Gln Cys Leu Gln Asn Phe Leu Leu His Gly Thr 76 Lys Asn Val Thr Asn Ser Ser Ser Leu Arg Leu Pro Arg Gln Asn 91 Ser Asp Gly Gly Gln Lys Asn Lys Pro Arg Glu His Ile Ile Asp106 Cys Gly Asp Ile Val Trp Ser Leu Gly Phe Gly Ser Ser Val Pro121 Glu Lys Gln Ser Arg Cys Val Asn Ile Glu Trp His Arg Phe Arg136 Phe Gly Gln Asp Gln Leu Leu Leu Ala Thr Gly Leu Asn Asn Gly151 Arg Ile Lys Ile Trp Asp Val Tyr Thr Gly Lys Leu Leu Leu Asn166 Leu Val Asp His Thr Glu Val Val Arg Asp Leu Thr Phe Ala Pro181 Asp Gly Ser Leu Ile Leu Val Ser Ala Ser Arg Asp Lys Thr Leu196 Arg Val Trp Asp Leu Lys Asp Asp Gly Ash Met Met Lys Val Leu211 Arg Gly His Gln Asn Trp Val Tyr Ser Cys Ala Phe Ser Pro Asp226 Ser Ser Met Leu Cys Pro Val Gly Ala Ser Lys Ala Val Phe Leu241 Trp Asn Met Asp Lys Tyr Thr Met Ile Arg Lys Leu Glu Gly His256 His His Asp Val Val Ala Cys Asp Phe Ser Pro Asp Gly Ala Leu271 Leu Ala Thr Ala Ser Tyr Asp Thr Arg Val Tyr Ile Trp Asp Pro286 His Asn Gly Asp Ile Leu Met Glu Phe Gly His Leu Phe Pro Pro301 Pro Thr Pro Ile Phe Ala Gly Gly Ala Asn Asp Arg Trp Val Arg316 Ser Val Ser Phe Ser His Asp Gly Leu His Val Ala Ser Leu Ala331 Asp Asp Lys Met Val Arg Phe Trp Arg Ile Asp Glu Asp Tyr Pro346 Val Gln Val Ala Pro Leu Ser Asn Gly Leu Cys Cys Ala Phe Ser361 Thr Asp Gly Ser Val Leu Ala Ala Gly Thr His Asp Gly Ser Val376 Tyr Phe Trp Ala Thr Pro Arg Gln Val Pro Ser Leu Gln His Leu391 Cys Arg Met Ser Ile Arg Arg Val Met Pro Thr Gln Glu Val Gln406 Glu Leu Pro Ile Pro Ser Lys Leu Leu Glu Phe Leu Ser Tyr Arg421 Ile ( 2 ) SEQ ID NO：5 ( i )

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:5:CAGAGTCGAC ATGGCCAGCT TTCCCCCGAG 30 (2) SEQ ID NO:6

(A) length: 32 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:6:CCAAGTCGAC CTAAATACGA TACGAGAGAA AC 32 (2) SEQ ID NO:7

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:7:CAGAGAATTC ATGGCCAGCT TTCCCCCGAG 30 (2) SEQ ID NO:8

(A) length: 32 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO:8:CCAATCTAGA CTAAATACGA TACGAGAGAA AC 32

Claims (14)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people WSB1 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 31-1296 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 31-1296 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.4.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence comprises the nucleotide sequence of 31-1296 position among the SEQ ID NO.3.

4. isolating people WSB1 protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

6. a carrier is characterized in that, it contains the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6.

8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

10. a generation has the method for the polypeptide of people WSB1 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people WSB1 protein-active operationally is connected in expression regulation sequence, form people WSB1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 31-1296 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people WSB1;

(c) under the condition that is fit to expressing human WSB1 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people WSB1 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 31-1296 position among the SEQ ID NO.3.

12. energy and the described people WSB1 of claim 4 protein polypeptide specificity bonded antibody.

13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

14. a probe molecule is characterized in that, it contains about 8-100 continuous nucleotide in the described dna molecular of claim 1.

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