CN1255549A - Human protein and its coding sequence, preparing process and application - Google Patents

Abstract

The present invention discloses a new human gene nucleotide sequence, that is, the cDNA sequence of human Rab18. Said protein is the homolog of murinus Rab18. The present invention also relates to the polypeptide coded with said nucleotide sequence, the application of said polynucleotide and polypeptide, and the process for preparing said polynucleotide and polypeptide.

Description

A kind of new people's albumen and encoding sequence thereof, and method for making and purposes

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 Rab18, this albumen is the homologue of mouse Rab18.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, and (Nature 1989,341 (6239): 209-214) for NKXD and SAK/L.In addition, in the Ypt/Rab protein family, conservative site also has: the tryptophane before the DTAGQE district, the phenylalanine of back, DTAGQE district behind about 10 amino acid whose arginine and SAK/L district etc.

Ypt/Rab family member in yeast saccharomyces cerevisiae (S.cerevisiae) has 11 (Ypt1, Sec4, Ypt31, Ypt32, Ypt51, Ypt52, Ypt53, Ypt6, Ypt7, Ypt10, Ypt11) (Trends Biochem Sci1997; 22 (12): 468-472); In Mammals, the Rab albumen of having found has 40 approximately, as Rab1, and Rab3, Rab8, Rab10, Rab30 etc.1993, Yu, H., Leaf, D.S. and Moore, H.-P.H. at first reported and have separated Rab18 first from mouse neuroendocrine cell cDNA storehouse (Gene 1993,132 (2), 273-278).The same year, Marja Agterberg etc. also has been separated to the homologous gene of mouse Rab18 from albumin (albumen) the body of gland cDNA storehouse of snail (Lymnaea stagnalis), both amino acid sequence identities are 74% (Eur.J.Biochem.1993,217,241-246), above-mentioned two genes are all successfully cloned, in addition, have also suffered homologous gene/albumen of also having found Rab18 at Arabidopsis thaliana and Caenorhabditis briggsae.

Ypt/Rab protein family member's diversity prompting, different albumen may play a role in the different step of different film bubble transport pathway.Prove that Sec4 albumen has participated in the golgi body secretory vesicle, and (Cell 1987 to the transportation of plasma membrane; 49 (4): 527-538), and Rab8 is proved to be film bubble transportation (the J Cell Biol 1993 that has mediated in the polar mdck cell by the basad LHA in TGN district; 123 (1): 35-45).Rab1 and the relevant (J.Cell.Biol.1991 of vesica transportation that reaches from endoplasmic reticulum to golgi body cis face subsequently from golgi body cis face to middle golgi body, 115,31-43) Rab3A may relevant (Proc.Natl.Acad.Sci.USA 1990 in the release of neurotransmitter, 87,1988-1992).Rab4 and Rab5 have then participated in the fusion process of endosome, and have been considered to control restricted step (Cell, 1990,62, the 317-329 of endocytosis process; Proc.Natl.Acad.Sci.USA 1991,88,6313-6317; Cell, 1992,70,729-740; Cell, 1991,64,915-925; Cell, 1992,70,715-728).

Rab albumen also relates to other factor such as GDP supressor (the GDP-dissociation inhibitor that dissociates in mediation film bubble transportation, GDI), GTP/GDP exchange factor (GTP/GDP-exchangefactor, GEF), the GTP enzyme activation factor (GTPase-activating protein, GAP), the GDI displacement factor (GDI-displacement factor, acting in conjunction GDF).

The proteic working cycle of Rab is as follows in the transportation of mediation film bubble: (a) in tenuigenin, GDI combines with Rab-GDP, has suppressed Rab albumen and has non-selectively combined with film.(b) Rab-GDP makes the isoprenylation of terminal two halfcystines of Rab PROTEIN C under the effect of geranyl geranyl transferring enzyme (geranylgeranyltransferase abbreviates " GGTase " as).It is necessary that isoprenylation is that Rab combines with plasma membrane.GGTase comprises two component: component A and claims Rab to escort albumen (Rab escort protein again, 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.(Cell 1993 on the specific donor membrane being positioned under the GIP effect for the Rab albumen of isoprenylation; 73 (6): 1091-1099).When Rab-GDP was incorporated on the donor membrane, GDI disintegrated down from Rab under the GDF effect.(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 under 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.(Peter?Novick，1997)

Reports such as Culine S. in 1993, Rab2 shows as overexpression in Sezary syndrome patient's peripheral blood lymphocytes, and this prompting Rab2 may have an effect in tumorigenic immune response, and (LeukLymphoma 1998; 28 (5-6): 515-522).Sezary syndrome is a class cutaneous T cell lymphoma disease, and symptom shows as breaks up T completely _HAccumulation (the Cancer Res 1992 of memory cell in skin histology and blood, lymphoglandula; 52 (11): 3083-3088).

Yet, before the present invention, do not have report about human Rab18 gene.

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 Rab18.

Another object of the present invention provides a kind of new people's Rab/Ypt family member, and this albumen is named as people Rab18 albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's Rab18 polypeptide.

The present invention also provides this people's the Rab18 nucleotide sequence 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 Rab18 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 30-650 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 30-650 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 30-650 position.

In another aspect of this invention, provide a kind of isolating people Rab18 protein polypeptide, it comprises: have polypeptide or its conservative property variation 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 Rab18 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people Rab18 protein-active operationally is connected in expression regulation sequence, form people Rab18 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 30-650 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 Rab18;

(c) under the condition that is fit to expressing human Rab18 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people Rab18 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 818 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 30-650 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 Rab18 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with RABPA's 18 protein-actives is as 30-650 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 30-650 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 30-650 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 30-650 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 30-650 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.4 sequence of people Rab18 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 Rab18 albumen or polypeptide " refers to have the SEQID NO.4 polypeptide of sequence of people Rab18 protein-active.This term also comprises having and the variant form human efficient lymphocyte chemotactic factor 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 Rab18 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people Rab18 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people Rab18 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people Rab18 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people Rab18 polypeptide.Usually, this fragment have people Rab18 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 Rab18 albumen or polypeptide.The difference of these analogues and natural human Rab18 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.

In the present invention, " people Rab18 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala

His(H)	Asn；Gln；Lys；Arg	Arg
			Ile(I)	Leu; Val; Met; Ala; Phe; Nor-leucine	Leu
Leu(L)	Nor-leucine; Ile; Val; Met; Ala; Phe	Ile
			Lys(K)	Arg；Gln；Asn	Arg
Met(M)	Leu；Phe；Ile	Leu
			Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
Pro(P)	Ala	Ala
			Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
			Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
			Val(V)	Ile; Leu; Met; Phe; Ala; Nor-leucine	Leu

The present invention also comprises people Rab18 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people Rab18 in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of people Rab18 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 Rab18.

The present invention also comprises the method that detects people Rab18 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 Rab18 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.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.

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 Rab18 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 Rab18 gene product or fragment.Preferably, refer to that those can combine with people Rab18 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 Rab18, comprise that also those do not influence the antibody of people Rab18 protein function.The present invention also comprise those can with modify or without the people Rab18 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 Rab18 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human Rab18 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 Rab18 function and the antibody that does not influence people Rab18 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people Rab18 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 Rab18 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 Rab18 Nucleotide 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 with its human cloning carrier, changes cell over to again, 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 the present invention, the cDNA nucleotide sequence of people Rab18 is so to obtain, with people's liver λ gt11cDNA library (available from Clontech company) is template, with two pairs of oligonucleotide is primer---A1:5 '-AGCTGGGCTCGGAGCGGAACG-3 ' is a forward primer, oligonucleotide A2:5 '-TCGATGGAGTGGGGATT (T/C) CTTGG-3 ' is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.

Studies show that Rab/Ypt family 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.Human Rab18 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 the accompanying drawings,

Fig. 1 is the homology comparison diagram of people Rab18 albumen of the present invention (below) and mouse Rab18 albumen (top) aminoacid sequence.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.Among the figure, what mark with underscore is that GTP-is in conjunction with territory and effector structural domain (effector domain).Mark with italic the 44th and 102 F and W are amino-acid residues extremely conservative in Rab albumen, but also not really conservative in other GTP-relevant with Rab are conjugated protein.

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 Rab18

1. primer amplification

With people's liver λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-AGCTGGGCTCGGAGCGGAACG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide A2:5 '-TCGATGGAGTGGGGATT (T/C) CTTGG-3 ' (SEQ IDNO:2) is that (annotate: what " (T/C) " expression was used among the primer A2 is the mixture of two kinds of primers to reverse primer, they are T or C individually on this position), carry 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 base pair more than 800.

2.PCR the order-checking of product

Above-mentioned pcr amplification product A1/A2 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.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 818bp, detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 30-650 position Nucleotide.

Derive the aminoacid sequence of people Rab18 according to the full-length gene group sequence that obtains, totally 206 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 Rab18 of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that with Rab/Ypt family gene and proteins encoded thereof to have homology widely, with PCGENE software relatively, finder Rab18 and the identity of mouse Rab18 gene on protein level are up to 99% (Fig. 1).In addition, the Rab10 genes with other species also has higher homology.There are four conservative region GXXXXGKS/Ts relevant with the GTP/GDP keying action in amino acid conserved regions analysis revealed people Rab18 of the present invention albumen with its homologue on protein structure, DTAGQE, and (Nature 1989 for NKXD and SAK/L; 341 (6239): 209-214) (the structural domain I-IV that marks among Fig. 1), therefore, people Rab18 albumen of the present invention can be included into the conjugated protein superfamily of GTP/GDP.In addition, the same with mouse Rab18 albumen, people Rab also has identical effector structural domain (Fig. 1).In addition, people Rab18 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 of back, DTAGQE district distance and (Fig. 1) such as phenylalanines behind the SAK/L district.

Therefore people Rab18 gene of the present invention is the homologue of mouse Rab18 gene in human body, be a member of Rab/Ypt family, so the function that can infer human Rab18 of the present invention from the member's of this family gene or proteic function, and, have or identical functions similar to the high homology prompter Rab18 of mouse Rab18 to mouse Rab18.

Ypt/Rab protein family member's diversity prompting, different albumen may play a role in the different step of different film bubble transport pathway.Studies show that Sec4 albumen has participated in the golgi body secretory vesicle, and (Cell 1987 to the transportation of plasma membrane; 49 (4): 527-538), and Rab8 is proved to be film bubble transportation (the J Cell Biol 1993 that has mediated in the polar mdck cell by the basad LHA in TGN district; 123 (1): 35-45).Rabl and the relevant (J.Cell.Biol.1991 of vesica transportation that reaches from endoplasmic reticulum to golgi body cis face subsequently from golgi body cis face to middle golgi body, 115,31-43) Rab3A may relevant (Proc.Natl.Acad.Sci.USA 1990 in the release of neurotransmitter, 87,1988-1992).Rab4 and Rab5 have then participated in the fusion process of endosome, and have been considered to control restricted step (Cell, 1990,62, the 317-329 of endocytosis process; Proc.Natl.Acad.Sci.USA 1991,88,6313-6317; Cell, 1992,70,729-740; Cell, 1991,64,915-925; Cell, 1992,70,715-728).

For the polar epidermic cell is arranged, produce in its top and bottom side with the composition of keeping different albumen and lipid for the function of this class cell be vital (J.Cell.Biol.1990,111,987-1000).Compare with nonpolar cell, whether epidermic cell has unique top and bottom side drive access (apical andbasolateral transport pathway), thereby probe into and exist special Rab family member just to become an interesting problem in this class cell.To discovering of mouse, in epidermic cell, there be the Rab protein D DRab17 and the Rab18 of two epidermic cell specifically expressings.Wherein, Rab18 than enrichment, and can further be located in intensive tubule place, top in the renal glomerulus epidermic cell; Yet, it should be noted that Rab18 all have in the top of pancreas epidermic cell and bottom side expression (J.Cell.Sci.1994,107,3437-3448).In sum, Rab18 plays a role aspect the cell polarity producing and keep probably.

The Rab18 gene of mouse is located in karyomit(e) No. 18, and in the scope that measuring error allows, the seat of Rab18 is consistent with the seat of Tw (twirler) and these two sudden changes of ax (ataxia).Tw heterozygote mouse demonstrates and shakes the head, turn-takes, inner ear is lacked of proper care, vestibular is examined and cerebellum stellate cell apoptosis (astrocytosis); Ax homozygote mouse is cerebral dysplasia then, and shows the forfeiture of the progressive coordination ability and tremble.And, some Rab albumen stably express in the allelotaxis, other are then induced at development later stage, so the sudden change of this family gene, particularly Rab18 might in the process of above-mentioned disease, work (Genomics, 1997,45,623-625).

People Rab18 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 Rab18 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the C end with the Rab18 of the C end of inventor Rab18 and mouse or other species exchanges, to produce the albumen that new activity is higher or have new features.

At the antibody of people Rab18 of the present invention, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

For example, people Rab18 nucleic acid of the present invention (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people Rab18 or the overexpression that suppresses people Rab18.People Rab18 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people Rab18 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 Rab18 in intestinal bacteria

In this embodiment, be masterplate with the PCR product among the embodiment 1, with the cDNA sequence of coding people Rab18 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people Rab18 cDNA as inserting fragment.

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

5 '-TCAGGTCGACATGGACGAGGACGTGCTAAC-3 ' (SEQ ID NO.5), this primer contains the restriction enzyme site of SalI restriction enzyme, is 19 Nucleotide of the people Rab18 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-AAGGAAGCTTTTATAACACAGAGCAATAAC-3 ' (SEQ ID NO.6), this primer contains the part encoding sequence of restriction enzyme site, translation termination and the people Rab18 of HindIII 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 and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people Rab18 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 Rab18 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people Rab18 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.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 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 4

The expression of people Rab18 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, be masterplate with the PCR product among the embodiment 1, with the cDNA sequence of coding people Rab18 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people Rab18 cDNA as inserting fragment.

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

5′-TCAGAAGCTTATGGACGAGGACGTGCTAAC-3′(SEQ?ID?NO.7)

This primer contains the restriction enzyme site of HindIII restriction enzyme, is 20 Nucleotide of the people Rab18 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-AAGGGAGCTCTTATAACACAGAGCAATAAC-3’(SEQ?ID?NO.8)

This primer contains the part encoding sequence of the restriction enzyme site of SacI restriction enzyme, translation termination and people Rab18.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (fl ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With BamHI and 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 Rab18 has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin (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 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 molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people Rab18 gene translation product with it.Found that antibody can precipitate with protein-specific of the present invention ground specifically.

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 shapes of equal value

Sequence table (1) general information: (i) applicant: Fudan University is denomination of invention (ii): a kind of new people's albumen and encoding sequence thereof reach (iii) sequence number of method for making and purposes: the information of 8 (2) SEQ ID NO.1

(i) sequence signature

(A) length: 21 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: the information of SEQ ID NO.1:AGCTGGGCTC GGAGCGGAAC G 21 (2) SEQ ID NO.2

(i) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: the information of SEQ ID NO:2TCGATGGAGT GGGGATT (T/C) CT TGG 23 (2) SEQ ID NO.3:

(i) sequence signature:

(A) length: 818bp

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

( iii ) ：SEQ ID NO.3AGCTGGGCTC GGAGCGGAAC GGGGTCAGGA TGGACGAGGA CGTGCTAACC ACCCTGAAGA 60TCCTCATCAT CGGCGAGAGT GGGGTGGGCA AGTCCAGCCT GCTCTTGAGG TTCACAGATG 120ATACGTTTGA TCCAGAACTT GCAGCAACAA TAGGTGTTGA CTTTAAGGTG AAAACAATTT 180CAGTGGATGG AAATAAGGCT AAACTTGCAA TATGGGATAC TGCTGGTCAA GAGAGGTTTA 240GAACATTAAC TCCCAGCTAT TATAGAGGTG CACAGGGTGT TATATTAGTT TATGATGTCA 300CAAGAAGAGA TACATTTGTT AAACTGGATA ATTGGTTAAA TGAATTGGAA ACATACTGTA 360CAAGAAATGA CATAGTAAAC ATGCTAGTTG GAAATAAAAT CGATAAGGAA AATCGTGAAG 420TCGATAGAAA TGAAGGCCTG AAATTTGCAC GAAAGCATTC CATGTTATTT ATAGAGGCAA 480GTGCAAAAAC CTGTGATGGT GTACAATGTG CCTTTGAAGA ACTTGTTGAA AAGATCATTC 540AGACCCCTGG ACTGTGGGAA AGTGAGAACC AGAATAAAGG AGTCAAACTG TCACACAGGG 600AAGAAGGCCA AGGAGGAGGA GCCTGTGGTG GTTATTGCTC TGTGTTATAA ACTCTGGGAA 660ATTCCATCTC TTGCATATTT GGATCCAGAT AGTTGACATC TTTCTGTATA TTAAACTCTT 720TTAACTGCTA ATTTTAGGGG ACCTTGGCAG TTTGCACATA ATTGGTTTTA TATCCATAGC 780CAGTTAAATA TTTTGCCAAG AAATCCCCAC TCCATCGA 818

(2) information of SEQ ID NO.4:

(i) sequence signature:

(A) length: 206 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

( xi ) ：SEQ ID NO.4Met Asp Glu Asp Val Leu Thr Thr Leu Lys Ile Leu Ile Ile Gly 15Glu Ser Gly Val Gly Lys Ser Ser Leu Leu Leu Arg Phe Thr Asp 30Asp Thr Phe Asp Pro Glu Leu Ala Ala Thr Ile Gly Val Asp Phe 45Lys Val Lys Thr Ile Ser Val Asp Gly Asn Lys Ala Lys Leu Ala 60Ile Trp Asp Thr Ala Gly Gln Glu Arg Phe Arg Thr Leu Thr Pro 75Ser Tyr Tyr Arg Gly Ala Gln Gly Val Ile Leu Val Tyr Asp Val 90Thr Arg Arg Asp Thr Phe Val Lys Leu Asp Asn Trp Leu Asn Glu 105Leu Glu Thr Tyr Cys Thr Arg Asn Asp Ile Val Asn Met Leu Val 120Gly Asn Lys Ile Asp Lys Glu Asn Arg Glu Val Asp Arg Asn Glu 135Gly Leu Lys Phe Ala Arg Lys His Ser Met Leu Phe Ile Glu Ala 150Ser Ala Lys Thr Cys Asp Gly Val Gln Cys Ala Phe Glu Glu Leu 165Val Glu Lys Ile Ile Gln Thr Pro Gly Leu Trp Glu Ser Glu Asn 180Gln Asn Lys Gly Val Lys Leu Ser His Arg Glu Glu Gly Gln Gly 195Gly Gly Ala Cys Gly Gly Tyr Cys Ser Val Leu 206

(2) information of SEQ ID NO.5

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO.5:

TCAGGTCGAC?ATGGACGAGG?ACGTGCTAAC?????????????????30

(2) information of SEQ ID NO.6

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO.6:

The information of AAGGAAGCTT TTATAACACA GAGCAATAAC 30 (2) SEQ ID NO.7

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: the information of SEQ ID NO.7TCAGAAGCTT ATGGACGAGG ACGTGCTAAC 30 (2) SEQ ID NO.8

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO.8AAGGGAGCTC TTATAACACA GAGCAATAAC 30

Claims (14)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people Rab18 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 30-650 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 30-650 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 30-650 position.

4. isolating people Rab18 protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation 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 Rab18 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people Rab18 protein-active operationally is connected in expression regulation sequence, form people Rab18 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 30-650 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 Rab18;

(c) under the condition that is fit to expressing human Rab18 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people Rab18 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 30-650 position among the SEQ ID NO.3.

12. energy and the described people Rab18 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 8-100 continuous nucleotide in the described dna molecular of claim 1.