CN1249345A - Human gene coding sequence, its encoded polypeptide and its preparing process - Google Patents

Abstract

The present invention discloses a new human Rab10 cDNA sequence. The protein coded by said sequence belongs to Yap/Rab family and is the homolog of mouse Rab10. 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 encoding 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 Rab10, this albumen belongs to Yap/Rab family, is the homologue of mouse Rab10.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.

In eukaryotic cell, the transportation of film bubble is the important way of intracellular organic matter transportation.The endocytosis of cell, exocytosis, the matter transportation process between endoplasmic reticulum and the golgi body, between each cyst membrane of golgi body, and golgi body forms lysosomal process all by all kinds of transport vesicle mediations.Transport vesicle produces from donor membrane in the mode of sprouting, then by with the fusion of special receptor film, with the substance transportation that comprises in the vesicle in another organoid or realize the interchange of material inside and outside the cell.

The conjugated protein Ypt/Rab albumen of the low-molecular-weight GTP/GDP of one class plays an important role in regulating film bubble transportation.Ypt/Rab albumen and Ras albumen belong to the conjugated protein superfamily of GTP/GDP.There are four conservative region GXXXXGKS/Ts relevant with the GTP/GDP keying action in they on protein structure, DTAGQE, NKXD and SAK/L (Pai, E.F., Nature, 341 (6239): 209-214, (1989)).In addition, in the Ypt/Rab protein family, conservative site also has: the tryptophane before the DTAGQE district, the arginine of about 10 the amino acid distances in back, DTAGQE district and the phenylalanine behind the SAK/L district etc.

Ypt/Rab family member in yeast (S.cerevisiae) has 11 (Ypt1, Sec4, Ypt31, Ypt32, Ypt51, Ypt52, Ypt53, Ypt6, Ypt7, Ypt10, Ypt11) (Thomas Lazer, Trends Biochem Sci, 22 (12): 468-472, (1997)); In Mammals, the Rab albumen of having found has 40 approximately, as Rab1, and Rab3, Rab8, Rab10, Rab30 etc.Nineteen ninety, Chavrier etc. have separated Rab10 (Philippe Chavrier, Mol Cell Biol, 10 (12): 6578-6585,1990) first from Madin-Darby dog kidney epidermic cell cDNA storehouse.1992, Lisa A.Elferink etc. also was separated to the homologous gene of dog Rab10 in rat from borwn rat (Rattus norvegicus) brain cDNA storehouse, and both amino acid sequence identities are 99% (Elferink, L.A, JBiol Chem, 267 (9): 5768-5775,1992).

The different albumen of Ypt/Rab protein family member's diversity prompting may play a role in the different step of different film bubble transport pathway.Sec4 albumen has participated in transportation (the Salminen A of golgi body secretory vesicle to plasma membrane, Cell, 49 (4): 527-538,1987), and Rab8 is proved to be film bubble transportation (the Huber LA that has mediated in the polar mdck cell by the basad LHA in TGN district, J Cell Biol, 123 (1): 35-45,1993).Which kind of approach and step that Rab10 has participated in the transportation of film bubble it be unclear that, but the Position Research in CHO and bhk cell shows to mouse Rab10 albumen, Rab10 is distributed in the far-end of all districts of nuclear and golgi body, prompting Rab10 may be similar with Sec4, participated in later steps (the Chen Yih-Tai of Secretory Pathway, Proc NatlAcad Sci USA:90 (14): 6508-65 12,1993).

Rab albumen also relates to other factor such as GDI (GDP-dissociationinhibitor in mediation film bubble transportation, the GDP supressor of dissociating), GEF (GTP/GDP-exchange factor, the GTP/GDP exchange factor), GAP (GTPase-activating protein, GTP enzyme activation albumen), the acting in conjunction of GDF (GDI-displacementfactor, GDI replaces the factor).

The working cycle of Rab albumen in the transportation of mediation film bubble is as follows: (a) in tenuigenin, GDI combines with Rab-GDP, has suppressed Rab albumen and has non-selectively combined with film.(b) Rab-GDP carries out isoprenylation to terminal two halfcystines of Rab PROTEIN C under the effect of (geranylgeranyltransferase is called for short GGTase) of geranyl geranyl transferring enzyme.Isoprenylation be Rab combine with plasma membrane the institute necessary.GGTase comprises two component: component A and claims Rab to escort albumen (Rab escort protein again, be called for short REP), this albumen is with the Rab protein binding of isoprenylation not and with it passs catalyst component B, and catalyst component B is responsible for the isoprenylation of catalysis halfcystine.The Rab albumen of isoprenylation is being positioned on the specific donor membrane under the GIP effect.When Rab-GDP was incorporated on the donor membrane, GDI disintegrated down from Rab under the GDF effect (AndresD.A., Cell, 73 (6): 1091-1099,1993).(c) under the GEF katalysis,, GTP activated Rab thereby having displaced the GDP on the Rab.(d) transport vesicle produces from donor membrane in the mode of sprouting.(e) (perhaps also having the acting in conjunction of the effector molecule on the receptor membrane) under the mediation of GTP-Rab, transport vesicle combines with receptor membrane, and GTP issues unboiled water in GAP catalysis and separates.(f) GDI combines with Rab on the receptor membrane, and Rab is discharged in the tenuigenin again.(PeterNovick，Curr?Opin?Cell?Biol，9(4)：496-504，1997)。

1993, Culine S. etc. was reported in that Rab2 shows as overexpression in Sezary syndrome patient's the peripheral blood lymphocytes, and prompting Rab2 may have an effect in tumorigenic immune response.Sezary syndrome is a class cutaneous T cell lymphoma disease, and symptom shows as breaks up T completely _HThe accumulation of memory cell in skin histology and blood, lymphoglandula.(Culine?S.，CancerRes，52(11)：3083-3088，1992；DummerR.，Leuk?Lymphoma，28(5-6)：515-522，1998)

Yet not about the report of human Rab10 gene, also nobody discloses remarkable Rab10 gene order before the present invention.

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding Rab/Ypt family, Rab/Ypt family member of the present invention is named as people Rab10.

Another object of the present invention provides a kind of new people's Rab/Ypt family member, and this albumen is named as people Rab10 albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's Rab/Ypt family member.

The present invention also provides this people's the Rab10 gene order and the application of 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 Rab10 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 78-680 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 78-680 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 78-680 position.

In another aspect of this invention, provide a kind of isolating people Rab10 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 Rab10 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people Rab10 protein-active operationally is connected in expression regulation sequence, form people Rab10 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 78-680 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 Rab10;

(c) under the condition that is fit to expressing human Rab10 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people Rab10 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 716 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 78-680 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 Rab10 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with RABPA's 10 protein-actives is as 78-680 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 78-680 position Nucleotide of SEQ ID NO.3 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.3 in 78-680 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 among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 78-680 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 78-680 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 Rab 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 Rab10 protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people Rab10 protein-active.This term also comprises having and the variant form human efficient lymphocyte chemotactic factor identical function, SEQ IDNO.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 Rab10 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 Rab10 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people Rab10 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people Rab10 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people Rab10 polypeptide.Usually, this fragment have people Rab10 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 Rab10 albumen or polypeptide.The difference of these analogues and natural human Rab10 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 Rab10 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of people Rab10 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 Rab10 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 coding people Rab10.

The present invention also comprises the method that detects people Rab10 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 Rab10 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 Rab10 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 Rab10 gene product or fragment.Preferably, refer to that those can combine with people Rab10 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 Rab10, comprise that also those do not influence the antibody of people Rab10 protein function.The present invention also comprise those can with modify or without the people Rab10 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 Rab10 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human Rab10 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 Hvbridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people Rab10 function and the antibody that does not influence people Rab10 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people Rab10 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 Rab10 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 Rab10 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 people Rab10 is so to obtain, with people's liver λ gr11cDNA library (available from Clontech company) is template, with two pairs of oligonucleotide is primer---A1 (SEQ IDNo.1) is a forward primer, oligonucleotide A2 (SEQ ID No.2) is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.

Film bubble transportation in the adjustable eukaryotic cell of Rab/Ypt family member.Rab/Ypt family member's diversity prompting different members plays a role in the different approaches different step of film bubble transportation.Human Rab10 of the present invention provides the new way to the research of effect in regulating the transportation of film bubble of Rab/Ypt family different members.

1993, Culine S. etc. was reported in that Rab2 shows as overexpression in Sezary syndrome patient's the peripheral blood lymphocytes, and prompting Rab2 may have an effect in tumorigenic immune response.Human Rab10 of the present invention provides the new way to the research of Sezary syndrome.

In the accompanying drawings,

Fig. 1 is the homology comparison diagram of the nucleotide sequence of people Rab10 of the present invention and dog Rab10.Wherein, be people Rab10 above, be dog Rab10 below, and identical Nucleotide mark with " | ".". " above sequence is used to mark the Nucleotide that is numbered 10 multiple.

Fig. 2 is the homology comparison diagram of the aminoacid sequence of people Rab10 of the present invention and dog Rab10.Wherein, identical amino acid marks with the abbreviation of amino acid monocase between two sequences.

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:Cold Spring HarborLaboratory 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 Rab10

1. primer amplification

With people's liver λ gt11cDNA library (available from Clontech company) is template, with two pairs of oligonucleotide is primer---A1:5 '-AGTGAGGAGT TGGCCGTAGT GAG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide A2:5 '-GGGTAGTGGA TGGCAACTGA TGG-3 ' (SEQ ID NO:2) is a reverse primer, carries out PCR.The PCR condition of A1/A2 be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.Electrophoresis detection obtains the purpose fragment of about 700bp.

2.PCR the order-checking of product

The pcr amplification product A1/A2 that as above obtains is connected with pGEM-T carrier (Promega), transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque with double-stranded nested type disappearance test kit (Pharmacia) to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then.Check order with disappearance of SequiTherm EXCELTMDNA sequencing kit (Epicentre Technologies) to brachymemma successively, at last with computer software splicing order, obtain full length cDNA sequence, be total to 716bp, detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 78-680 position Nucleotide.

Derive the aminoacid sequence of people Rab10 according to the full-length gene group sequence that obtains, totally 200 amino-acid residues, its aminoacid sequence sees SEQ ID NO:4 for details.

Embodiment 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with people Rab10 of the present invention carry out nucleic acid and albumen homology retrieval with BLAST in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database.Found that they and Rab/Ypt family gene and proteins encoded thereof have homology widely, with PCGENE software relatively, find it and dog Rab10 gene about the identity on nucleic acid and the protein level reaches 96% and 99.5% respectively (Fig. 1 and Fig. 2), its albumen and dog Rab10 albumen only differ an amino acid.In addition, with other members such as the Rab2 of Rab/Ypt family, Sec4, Ypt1 etc. also have higher homology (data are unlisted).

The amino acid conserved regions is analyzed discovery, there are four conservative region GXXXXGKS/Ts relevant with the GTP/GDP keying action in people Rab10 albumen of the present invention with its homologue on protein structure, DTAGQE, NKXD and SAK/L, therefore, people Rab10 albumen of the present invention can be included into the conjugated protein superfamily of GTP/GDP.In addition, people Rab10 albumen of the present invention also has Ypt/Rab protein family special conservative site as the tryptophane before the DTAGQE district, the arginine of about 10 the amino acid distances in back, DTAGQE district and the phenylalanine behind the SAK/L district etc.

Therefore people Rab10 gene of the present invention is the homologue of dog Rab10 gene in human body, is considered to constitute a member of Rab/Ypt family, and the function that can infer human Rab10 of the present invention from the member's of this family gene or proteic function.

The Rab/Ypt family member has regulated and control the film bubble transportation in the eukaryotic cell.Rab/Ypt family member's diversity prompting different members plays a role in the different approaches different step of film bubble transportation.Sec4 albumen has participated in transportation (the Salminen A of golgi body secretory vesicle to plasma membrane, Cell, 49 (4): 527-538,1987), and Rab8 is proved to be the film bubble transportation (HuberLA 1993) that has mediated in the polar mdck cell by the basad LHA in TGN district.Which kind of approach and step that Rab10 has participated in the transportation of film bubble it be unclear that, but the Position Research in CHO and bhk cell shows to mouse Rab10 albumen, Rab10 is distributed in the far-end of all districts of nuclear and golgi body, prompting Rab10 may be similar with Sec4, participated in the later steps (Yih-Tai of Secretory Pathway, Chen, 1993 (the same)).Human Rab10 of the present invention provides the new way to the research of effect in regulating the transportation of film bubble of Rab/Ypt family different members.

In addition, 1993, Culine S. etc. was reported in that Rab2 shows as overexpression in Sezary syndrome patient's the peripheral blood lymphocytes, and prompting Rab2 may have an effect in tumorigenic immune response.Human Rab10 of the present invention provides the new way to the research of Sezary syndrome.

People Rab10 of the present invention is used for further functional study except can be used as Yap/Rab 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 Rab10 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor Rab10 and the N end of Rab8 are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor Rab10, 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 Rab10 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people Rab10 or the overexpression that suppresses people Rab10.People Rab10 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people Rab10 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 Rab10 in intestinal bacteria

In this embodiment, with the cDNA sequence of coding people Rab10 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people Rab10cDNA as inserting fragment.

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

5 '-TCAGGGATCC ATGGCGAAGA AGACGTACG-3 ' (SEQ ID NO.5), this primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the people Rab10 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-TTGGGTCGAC TCAGCAGCAT TTGCTCTTC-3 ' (SEQ ID NO.6), this primer contains the part encoding sequence of restriction enzyme site, translation termination and the people Rab10 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, 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, 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 Rab10 has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people Rab10 from solution.Wash-out people Rab10. can be with several method sex change protein precipitation from Guanidinium hydrochloride from post with 6M Guanidinium hydrochloride (pH5.0).Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.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 22.5KDa.

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 Rab10 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with the cDNA sequence of coding people Rab10 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people Rab10 cDNA as inserting fragment.

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

5′-TCAGGGATCC?ATGGACAAGG?AGTACGTGG-3′(SEQ?ID?NO.5)

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the people Rab10 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-TTGGGAGCTC?TCAGCAGCAT?TTGCTCTTC-3’(SEQ?ID?NO.7)

This primer contains the part encoding sequence of the restriction enzyme site of SacI restriction enzyme, translation termination and people Rab10.

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 (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With BamHI and XbaI digestion pcDNA3 carrier, 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 ApaI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification people Rab10 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 50mMTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 23KDa.

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, and concrete grammar is 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 Rab10 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 encoding sequence, its encoded polypeptides and preparation method be the sequence number (iii): information (i) sequence signature of 7 (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:AGTGAGGAGT TGGCCGTAGT GAG 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:2GGGTAGTGGA TGGCAACTGA TGG 23 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 716bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3 1 AGTGAGGAGT TGGCCGTAGT GAGAGGGACC GATCCCTTGG GGCCGCCGGC GGCGAGCAGC 61 CCGAGCCGCT CCTCCCAATG GCGAAGAAGA CGTACGACCT GCTTTTCAAG CTGCTCCTGA121 TCGGGGATTC CGGAGTGGGG AAGACCTGCG TCCTTTTTCG TTTTTCGGAT GATGCCTTCA181 ATACTACCTT TATTTCCACC ATAGGAATAG ACTTCAAGAT CAAAACAGTT GAATTACAAG241 GAAAGAAGAT CAAGCTACAG ATATGGGATA CAGCAGGCCA GGAGCGATTT CACACCATCA301 CAACCTCCTA CTACAGAGGC GCAATGGGTA TCATGCTAGT ATATGACATC ACCAATGGTA361 AAAGTTTTGA AAACATCAGC AAATGGCTTA GAAACATAGA TGAGCATGCC AATGAAGATG421 TGGAAAGAAT GTTACTAGGA AACAAGTGTG ATATGGACGA CAAAAGAGTT GTACCTAAAG481 GAAAAGGAGA ACAGATTGCA AGGGAGCATG GTATTAGGTT TTTTGAGACT AGTGCAAAAG541 CAAATATAAA CATCGAAAAG GCGTTCCTCA CGTTAGCTGA AGATATCCTT CGAAAGACCC601 CTGTAAAAGA GCCCAACAGT GAAAATGTAG ATATCAGCAG TGGAGGAGGC GTGACAGGCT661 GGAAGAGCAA ATGCTGCTGA GCATTCTCCT GTTCCATCAG TTGCCATCCA CTACCC ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 200 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4 1 Met Ala Lys Lys Thr Tyr Asp Leu Leu Phe Lys Leu Leu Leu Ile 16 Gly Asp Ser Gly Val Gly Lys Thr Cys Val Leu Phe Arg Phe Ser 31 Asp Asp Ala Phe Asn Thr Thr Phe Ile Ser Thr Ile Gly Ile Asp 46 Phe Lys Ile Lys Thr Val Glu Leu Gln Gly Lys Lys Ile Lys Leu 61 Gln Ile Trp Asp Thr Ala Gly Gln Glu Arg Phe His Thr Ile Thr 76 Thr Ser Tyr Tyr Arg Gly Ala Met Gly Ile Met Leu Val Tyr Asp 91 Ile Thr Asn Gly Lys Ser Phe Glu Asn Ile Ser Lys Trp Leu Arg106 Asn Ile Asp Glu His Ala Asn Glu Asp Val Glu Arg Met Leu Leu121 Gly Asn Lys Cys Asp Met Asp Asp Lys Arg Val Val Pro Lys Gly136 Lys Gly Glu Gln Ile Ala Arg Glu His Gly Ile Arg Phe Phe Glu151 Thr Ser Ala Lys Ala Asn Ile Asn Ile Glu Lys Ala Phe Leu Thr166 Leu Ala Glu Asp Ile Leu Arg Lys Thr Pro Val Lys Glu Pro Asn181 Ser Glu Asn Val Asp Ile Ser Ser Gly Gly Gly Val Thr Gly Trp196 Lys Ser Lys Cys Cys ( 2 ) SEQ ID NO.5 ( i )

(A) length: 29 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:TCAGGGATCC ATGGCGAAGA AGACGTACG 29 (2) SEQ ID NO.6

(A) length: 29 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:TTGGGTCGAC TCAGCAGCAT TTGCTCTTC 29 (2) SEQ ID NO.7

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.7TTGGGAGCTC TCAGCAGCAT TTGCTCTTC 29

Claims (14)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people Rab10 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 78-680 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 78-680 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 has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 78-680 position.

4. isolating people Rab10 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 Rab10 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people Rab10 protein-active operationally is connected in expression regulation sequence, form people Rab10 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 78-680 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 Rab10;

(c) under the condition that is fit to expressing human Rab10 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people Rab10 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 78-680 position among the SEQ ID NO.3.

12. energy and the described people Rab10 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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