CN1257927A - Human protein and its coding sequence, preparing process and usage - Google Patents

Abstract

The present invention discloses a new human protein and its nucleotide coding sequence, that is, the cDNA sequence of human ARL5. The protein coded by said sequence is the homolog of mouse ARL5. The polypeptide coded by said nucleotide sequence, the application of said polynucleotide and polypeptide, and the process for preparing said polynucleotide and said polypeptide are also disclosed.

Description

A kind of new human protein 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 protein and nucleotide coding sequence thereof.More particularly, the present invention relates to the cDNA sequence of people ARL5, this albumen is the homologue of mouse ARL5.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.

ADP ribosylation factor (ADP ribosylation factor abbreviates " ARF " as) is that molecular weight is guanine-nucleotide-binding protein (Trends Biochem.Sci.1995,20, the 147-150 about 20KD; Cell.Signal.1992.4.367-399; Prog.Nucleic Acid Res.Mol.Biol.1993,45,47-65; Nature1994,372,704-708).Mammiferous ARF family has six members at least, and promptly ARF1 to ARF6 has then found three members at least in yeast.ARF family has also comprised highly homology of some and ARF in addition, but lacks the active albumen of Toxins,exo-, cholera cofactor; In addition, different with ARF is that these albumen depend on magnesium ion and phosphatide when combining with GTP, so these albumen are collectively referred to as ARL albumen (ARF sample albumen, ARF-like protein).In addition, the proteic aminoterminal of ARF can be by myristoylization, and the alcoholization of ARF cardamom is considered to help the ARF effect, does not then find these characteristics in ARL.

ARF high conservative in the Mammals, wide spectrum are expressed in each tissue.Can be divided three classes according to amino-acid sequence, size and gene structure: one, ARF1-3; Two, ARF4 and ARF5; Three, ARF6.The member of ARF family is included into RAS superfamily (Biochemestry usually, 1991,30,4637-4648), yet their general and other members of RAS family only have slight homology, and different with other Ras family member is that their carboxyl terminal lacks cysteine residues, thereby can't become the substrate of isoprenylation.

When ARF1 is found at first, is that the activity as a kind of ADP ribosyltransferase to Toxins,exo-, cholera has synergistic activator, and therefore gains the name.Kahn, R, A and Gilman, A, G respectively at from rabbit liver and ox meninx, extracted in 1984 and 1986 the ARF factor (J.Biol.Chem1984,259,6228-6234 J.Biol.Chem 1986,261,7906-7911).1987 and 1988, Tsai etc. from the ox brain, extract again a kind of film ARF (mARF) and two kinds of solubility ARF (sARF1,2) (Proc.Natl.Acad.Sci U.S.A.1987,84,5139-5142 J.B.C.1988,263,1768-1772).After this scientist carries out ARF cDNA clone by hybridization in succession, and with oligonucleotide as probe, found the different AR F of different animals.1988, Sewell, J, L etc. cloned bARF1 (bovineARF1) from bovine adrenal, and from the yeast genes storehouse, be cloned into yARF2 (yeast ARF2) (Pro.Natl.Acad.Sci.USA1988,85,4620-4624).The same year, Price etc. in bovine retina, clone obtain bARF2 (Proc.Natl.Acad.Sci.USA, 1988,85,5488-5491).1989, Peng, Z, Calver etc. obtained hARF (human ARF1) (Biofactor 1989,2,45-49).Simultaneously, Bobak etc. also from people's think-tank, obtained hARF1 and another factor hARF3 (Proc.Natl.Acad.Sci.USA, 1989,86,6101-6105).Nineteen ninety, Stearn, T etc. obtained yARF1 (Mol.Cell.Biol, 1994,138 (1-2), 157-166).1991, Kahn, R, clones such as A obtain hARF4 (J.Biol.Chem.1991,266,2606-2614).In the same year, Price, Tsai etc. have obtained in the family latter two factors A RF5,6 (J.Biol.Chem.1991,266,2772-2777).

In recent years, from different plant species, cloned some codings and the proteic cDNA of ARF homologous in succession, and be named as ARL-like or ARL.They comprise ARL1 and the ARL2 of fruit bat, people's ARL1, ARL2, ARL3, ARL4 ^*And ARL5 ^*, ARL1, the ARL3 of rat, ARL4, ARL5, the ARL4 of mouse ^*(Proc.Natl.Acad.Sci.USA, 1991,88,3120-3124; Proc.Natl.Acad.Sci.USA, 1993,90,8952-8956; J.Biol.Chem., 1994,269,15683-15688; J.Biol.Chem., 1994,269,18937-18942; J.Biol.Chem., 1995,270,21-24; J.Cell.Sci., 1996,109,209-220; Mark " ^*" person's still unexposed report before application), although more existing ARL studies show that out tissue or/and the expression specificity of differentiation, yet on the whole, still unclear to the function of ARL.

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding ARF family, ARF family member of the present invention is named as people ARL5.

Another object of the present invention provides a kind of new people's ARF family member, and this albumen is named as people ARL5 albumen.

A further object of the present invention provides a kind of proteic method of ARL5 of utilizing recombinant technology to produce described new people.

The present invention also provides the purposes of this people ARL5 protein polypeptide and nucleotide sequence.

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 ARL5 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 17-556 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 17-556 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 17-556 position.

In another aspect of this invention, provide a kind of isolating people ARL5 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 ARL5 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people ARL5 protein-active operationally is connected in expression regulation sequence, form people ARL5 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 17-556 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 ARL5;

(c) under the condition that is fit to expressing human ARL5 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people ARL5 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 849 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 17-556 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 ARL5 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people ARL5 protein-active is as 17-556 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 17-556 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 17-556 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 17-556 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 17-556 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 ARL5 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 ARL5 protein polypeptide " refers to have the SEQ IDNO.4 polypeptide of sequence of people ARL5 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 ARL5 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 ARL5 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people ARL5 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people ARL5 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people ARL5 polypeptide.Usually, this fragment have people ARL5 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 ARL5 albumen or polypeptide.The difference of these analogues and natural human ARL5 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 ARL5 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 ARL5 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people ARL5 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 ARL5 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 ARL5.

The present invention also comprises the method that detects people ARL5 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 ARL5 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 ARL5 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 ARL5 gene product or fragment.Preferably, refer to that those can combine with people ARL5 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 ARL5, comprise that also those do not influence the antibody of people ARL5 protein function.The present invention also comprise those can with modify or without the people ARL5 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 ARL5 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human ARL5 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 ARL5 function and the antibody that does not influence people ARL5 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people ARL5 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 ARL5 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 ARL5 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 about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

In one embodiment of the invention, the cDNA nucleotide sequence of people ARL5 is so to obtain, with people's liver λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-GGTCCGCTGCCCGAGAATGGG-3 ' is a forward primer, oligonucleotide A2:5 '-GTCTTCCTCTCCCTTTGTACTTAC-3 ' is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQID NO.3 after the order-checking.

ARF is an other structure incitant, when ARF with promptly have activity after GTP combines, activate PLD; Behind the hydrolysis GTP, the mixture inactivation becomes ARF-GDP.The function of ARF may relate to the protein transportation (J.Biol.Chem.1992 from endoplasmic reticulum to golgi body cis face, 267,13053-13061), golgi body cis face in-plant transportation (Cell, 1992,70,69-79), the exocytosis of vesica (FEBS Lett, 1993,121-124) with the transportation of nuclear intracellular vesicle (Nature, 1992,358, aspect such as 512-514).It not only regulates accumulation process (Proc.Natl.Acad.Sci.USA, 1992,89, the 6408-6412 of the early stage coatmer of secretion (coatomer); J.Biol.Chem, 1993,268,12083-12089), and be effective instrumentality of the clathrin (clathrin coat) that is gathered in trans-Golgi network.

In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence of people ARL5 albumen of the present invention (hARL5) and mouse ARL5 albumen (mARL5).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with ": ".

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

Embodiment 1

The clone and the mensuration of the cDNA sequence of people ARL5

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 '-GGTCCGCTGCCCGAGAATGGG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide A2:5 '-GTCTTCCTCTCCCTTTGTACTTAC-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 850bp.

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.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 849bp, detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 17-556 position Nucleotide.

Derive the aminoacid sequence of people ARL5 according to the full-length gene group sequence that obtains, totally 179 amino-acid residues, its aminoacid sequence sees SEQ ID NO:4 for details.

Embodiment 2

Homology relatively

With full length cDNA sequence and the proteins encoded thereof of people ARL5 of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with BLAST.Found that they and ARF family gene and proteins encoded thereof have homology widely, with BLAST software relatively, find it and the rat ARL5 gene identity (being also referred to as homogeny) on protein level up to 99% (Fig. 1), with other most of members' of ARF family identity and similarity also about 50% and 70% (data do not provide) respectively.To amino acid conserved regions analysis revealed, cDNA encoded protein of the present invention has the sequence of an ARF family characteristic with ProSite software.This sequence is shown as in ARF family: (H/R/Q/T) X (F/Y/W/I) X4AX2GX2 (L/I/V/M) X2 (G/S/A) (L/I/V/M/F) X (W/K) (L/I/V/M),

Wherein, (H/R/Q/T) wait a seed amino acid of representing among H, R, Q, the T, X represents arbitrary amino acid, and subscript is represented amino acid no.In known ARF family member, only there are 4 not have above-mentioned characteristic sequence; In protein sequence of the present invention, the sequence that meets above-mentioned feature is 150HQWHIQACCALTGEGLCQGLEWM.In addition, in protein sequence of the present invention, also found two GTP binding sites--- ₆₅WDIGGQ ₇₀With ₁₂₅NKQD ₁₂₉, these two GTP binding sites also are high conservative in ARF family.Comprehensive above-mentioned phenomenon, it is the another newcomer in human body of people ARF family that gene of the present invention is determined, and name is people ARL5 in view of the above, and according to the structures shape function, the Biological Principles that the structural similitude function is close can be inferred the function of human ARL5 of the present invention from other member's of ARF family function.

ARF1 is that the activity as a kind of ADP ribosyltransferase to Toxins,exo-, cholera has synergistic activator found at first.Discover that ARF is an other structure incitant, when ARF with promptly have activity after GTP combines; Behind the hydrolysis GTP, the mixture inactivation becomes ARF-GDP.Guanylic acid exchanger (GEPs) impels ARF to combine with GTP.And GTP enzyme activation albumen (GAPs) promotes the GTP hydrolysis.As if ARF influences the activity with membrane-bound Starch phosphorylase D (PLD) usually, yet be not like this in yeast.In yeast saccharomyces cerevisiae, PLD mainly regulates and control the formation of protospore film--and this is the prerequisite that gemma forms, increase a C-myc epi-position at the ARF1 carboxyl terminal, obtain zymic ARF1-myc ARF2 mutant, though the formation of protospore film is weakened, this is not to be because the affected cause of catalytic activity of PLD, and in fact ARF does not activate the activity (Mol.Biol.Cell.1998 of PLD in the yeast, 9 (8), 2025-2036).The function of ARF may relate to the protein transportation (J.Biol.Chem.1992 from endoplasmic reticulum to golgi body cis face, 267,13053-13061), golgi body cis face in-plant transportation (Cell, 1992,70,69-79), the exocytosis of vesica (FEBS Lett, 1993,121-124) with the transportation of nuclear intracellular vesicle (Nature, 1992,358, aspect such as 512-514).It not only regulates accumulation process (Proc.Natl.Acad.Sci.USA, 1992,89, the 6408-6412 of the early stage coatmer of secretion (coatomer); J.Biol.Chem, 1993,268,12083-12089), and be effective instrumentality of the clathrin (clathrin coat) that is gathered in trans-Golgi network.Experiment in vitro confirms that fungi brefeldin A (brefeldin A) can suppress AP-1 (Trends Cell Biol, 1992,2,293-297; J.Biol.Chem.1992,117,171-1179) and β-COP and Golgi membrane (Cell1991,64, combination 1183-1195).Known AP-1 is the part of clothing, probably by playing a major role in the accumulation process of clothing with the interaction of clathrin and triangle proteoplast; β-COP then is a subunit of coatmer, and it is that vesica sprouts necessary in the early stage Secretory Pathway.Brefeldin A goes up GDP suppresses ARF to the conversion of GTP function by stoping ARF.(J.Biol.Chem.1996，271(4)，2162-2170)。

ARF is inferred the fusion (J.Biol.Chem.1992 of participation endoplast-endoplast, 267,13047-13052), yet some experiments have simultaneously then been negated this hypothesis (Mol.Cell.Biolchem.1994,135,159-164), there is experiment to find that ARF is also nonessential to the fusion of endosomal vesicle (endosomal vesicle) simultaneously, but 5 '-inhibition that O-(3-thio triphosphates) (GTP gamma S) etc. merges endosomal vesicle must rely on (the J.Biol.Chem.1995 Jun 9 that exists of ARF, 270 (23), 13693-13697)

The effect of ARL is also very unclear at present.1991, Tamkun, J.W. etc. report title, and ARL albumen and the ARF of fruit bat has many similar character, however the former lacks the activity of ARF fully; The ARL gene of a recessive mutation has been discovered in transgenation, and this sudden change has caused the phenotype of fruit bat zygotic lethality, this hinted in the fruit bat ARL may be a critical function gene (Proc.Natl.Acad.Sci.USA, 1991,88,3120-3124).Other studies show that the specific phenomenon in the ARL expression.Can't be detected in fibroblast as rat ARL4, then content is abundant to the cell of adipocyte sample (adipocyte-like) phenotype breaking up, the expression of ARL6 starts from the 6th day (J.Biol.Chem.1994 of 3T3-L1 clone differentiation, 269 (22), 15683-15688), the function of this explanation ARL4 may be relevant with the adipocyte sample phenotype of 3T3-L1 cell.1996, Lowe, S.L. wait discovering in rat, in the normal rat kidney ARL1 link to each other with golgi body (J.Cell Sci.1996,109 (ptl), 209-220), in to yeast ARF1 (yARF1) research, also found similar phenomenon (J.Biol.Chem.1997 recently, 272 (49), 30998-31005), this prompting ARL also may equally with ARF play a role in the vesica transportation.

New GTP enzyme relevant with Ras in 1996, by utilization PCR, the clone comes out from the adipocyte cDNA storehouse of mouse.This albumen has the characteristic feature of ARF, and the most relevant with ARL1 (ARF sample albumen), is named as ARL5.To the research of rat ARL5 still not deeply, relevant report claim its mRNA the detected tissue of most of rats (as heart, skeletal muscle, fat, liver, kidney, lung, spleen, intestines, thymus gland) content is lower in, and brain, intestines, the highest (Biochim Biophys Acta 1996 Jul31 of content in the thymus gland, 1308 (1), 1-6).Relevant people's ARL5 yet there are no open report.About they in vivo role still remain further research.

In sum, new albumen of the present invention and plays a role in some other cell function probably in the transport pathway of vesica (as vesica transhipment of exocytosis, endocytosis and golgi body inside or the like).The present invention also provides new approach for further studying the effect and the mechanism of ARL in organism (especially human body) in addition.

People ARL5 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 ARL5 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end with inventor ARL5 exchanges with the proteic N end of the ARL5 of other animals, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor ARL5, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family, such as the ARL5 albumen of mouse).

In addition, inventor ARL5 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people ARL5 or the overexpression that suppresses people ARL5.People ARL5 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people ARL5 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 ARL5 in intestinal bacteria

In this embodiment, with the cDNA sequence (pcr amplification product among the embodiment 1) of coding people ARL5 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people ARL5cDNA as inserting fragment.

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

5′-TCAGGGATCCATGGGAATTCTCTTCACTAG-3′(SEQ?ID?NO.5)，

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 20 Nucleotide of the people ARL5 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5i-TTGGgtcgacTCATCTAATCTTAAGTCGT-3i(SEQ?ID?NO.6)，

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people ARL5 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 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 ARL5 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 ARL5 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people ARL5 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 21KDa.

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 ARL5 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with the cDNA sequence (pcr amplified fragment among the embodiment 1) of coding people ARL5 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people ARL5cDNA as inserting fragment.

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

5′-TCAGGGATCCATGGGAATTCTCTTCACTAG-3′(SEQ?ID?NO.5)

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 20 Nucleotide of the people ARL5 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5i-TTGGGATATCTCATCTAATCTTAAGTCGT-3i(SEQ?ID?NO.7)

This primer contains the part encoding sequence of the restriction enzyme site of EcoRV restriction enzyme, translation termination and people ARL5.

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 EcoRV digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, and the cDNA fragment of sequence verification people ARL5 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 21KDa.

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 Freundis 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 Freundis 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 ARL5 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 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 human protein and encoding sequence thereof reach (iii) sequence number of method for making and purposes: information (i) sequence signature of 7 (2) SEQ ID NO.1

(A) length: 21 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:GGTCCGCTGC CCGAGAATGG G 21 (2) SEQ ID NO.2

(A) length: 24 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:2:GTCTTCCTCT CCCTTTGTAC TTAC 24 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 849bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3：GGTCCGCTGC CCGAGAATGG GAATTCTCTT CACTAGAATA TGGAGACTGT TCAATCACCA 60GGAGCACAAA GTTATCATTG TTGGGCTGGA TAATGCAGGG AAAACTACCA TTCTTTACCA 120ATTTTCTATG AATGAAGTTG TACATACATC TCCTACAATA GGAAGTAATG TAGAAGAGAT 180AGTGATTAAT AATACACGTT TCCTAATGTG GGATATTGGT GGCCAAGAAT CTCTTCGTTC 240TTCCTGGAAC ACTTACTATA CTAACACAGA GTTTGTAATA GTTGTTGTGG ACAGTACAGA 300CAGAGAGAGG ATTTCTGTAA CTAGAGAAGA ACTCTATAAA ATGTTAGCGC ATGAGGACCT 360AAGAAAAGCT GGATTGCTGA TTTTTGCTAA TAAACAAGAT GTTAAAGAAT GCATGACTGT 420AGCAGAAATC TCCCAGTTTT TGAAGCTAAC TTCTATTAAA GATCACCAGT GGCATATCCA 480GGCATGCTGT GCTCTAACTG GCGAGGGATT GTGCCAAGGA CTTGAATGGA TGATGTCACG 540ACTTAAGATT AGATGATCTC TACTGACCTC TTCTCATAGA TTTTGTATAA ATGAAGTGCT 600GGACTTTACC TGAAAGCTGC AAAAATTAAT GGTTTAGATA TATTTATAAT AAACTGATTT 660AAACTTTTTC TATAAGAAGA AAAATTAAGA CCACTTATTT GAAAACAAAG ATGAAGTCTC 720ACCTTCCAGT TTGCTTTCTC ATTAGTTTTT TCCAAAGTAA GTTATTGAAG CTGTGATTGA 780CATTTTTCTC ATAATGAATC CTCTCAGGAC ATTGTGTAGC CTATGGTAAG TACAAAGGGA 840GAGGAAGAC 849 ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 179 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4：Met Gly Ile Leu Phe Thr Arg Ile Trp Arg Leu Phe Asn His Gln 15Glu His Lys Val Ile Ile Val Gly Leu Asp Asn Ala Gly Lys Thr 30Thr Ile Leu Tyr Gln Phe Ser Met Asn Glu Val Val His Thr Ser 45Pro Thr Ile Gly Ser Asn Val Glu Glu Ile Val Ile Asn Asn Thr 60Arg Phe Leu Met Trp Asp Ile Gly Gly Gln Glu Ser Leu Arg Ser 75Ser Trp Asn Thr Tyr Tyr Thr Asn Thr Glu Phe Val Ile Val Val 90Val Asp Ser Thr Asp Arg Glu Arg Ile Ser Val Thr Arg Glu Glu 105Leu Tyr Lys Met Leu Ala His Glu Asp Leu Arg Lys Ala Gly Leu 120Leu Ile Phe Ala Asn Lys Gln Asp Val Lys Glu Cys Met Thr Val 135Ala Glu Ile Ser Gln Phe Leu Lys Leu Thr Ser Ile Lys Asp His 150Gln Trp His Ile Gln Ala Cys Cys Ala Leu Thr Gly Glu Gly Leu 165Cys Gln Gly Leu Glu Trp Met Met Ser Arg Leu Lys Ile Arg 179 ( 2 ) SEQ ID NO.5 ( i )

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.5:TCAGGGATCC ATGGGAATTC TCTTCACTAG 30 (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 TCATCTAATC TTAAGTCGT 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.7TTGGGATATC TCATCTAATC TTAAGTCGT 29

Claims (14)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people ARL5 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 17-556 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 17-556 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 17-556 position.

4. isolating people ARL5 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 ARL5 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people ARL5 protein-active operationally is connected in expression regulation sequence, form people ARL5 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 17-556 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 ARL5;

(c) under the condition that is fit to expressing human ARL5 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people ARL5 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 17-556 position among the SEQ ID NO.3.

12. energy and the described people ARL5 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.