CN1277260A - Human actin related protein subunit and its code sequence - Google Patents

Abstract

The present invention provides one new kind of human Arp2/3 protein complex p16 Arc subunit encoding cDNA sequence, and the cDNA encoded protein is a member of P16-Arc family. The present invention also relates to the polypeptide encoded by the nucleotide sequence and the application and production process of the polynucleotide and polypeptide.

Description

A kind of human actin related protein subunit and encoding sequence thereof

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 Arp2/3 albumen composition p16-Arc subunit (Arp2/3 proteincomplex subunit p16-Arc)-2 (abbreviating ARC16-2 as), this cDNA encoded protein is the member of p16-Arc family.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.

Actin muscle provides power and framework as the main component of cytoskeleton for motion, metamorphosis, born of the same parents' inner structure of cell.The polymerization of Actin muscle can drive the projection of migratory cell leading edge.Actin muscle is necessary for the shrinkability structure that contains myosin in the cell (as the branch dehiscence furrow of division of cytoplasm) assembling and function, in addition, Actin muscle is in guaranteeing cyto-architectural integrity, and regulate aspect cell adhesion and the film dynamic change relevant closely.

Recent research shows, based on the assembling of the structure of Actin muscle with keep and depend on actin associated protein (Actin-related protein probably, abbreviate " Arp " as) activity (the see review of 2/3 mixture, Machesky, L.M.et al.Curr.Opon.Cell Biol.1999,11:117-121).

The Arp2/3 mixture is conservative in eukaryote, it in Acanthamaoeba by the profilin agarose affinity chromatography identified (Machesky, L.M.et al.J.Cell.Biol.1994,127:107-115).In yeast and Mammals, the Arp2/3 mixture is made up of 7 subunits, and proportion of composing is 1: 1 (Machesky, L.M.et al.J.Cell.Biol.1994,127:107-115; Welch, M.D.et al.J.Cell Biol.1997,138:375-384; Winter, D.et al.Curr.Biol.1997,7:519-529; Mullins, R.D.et al.J.Cel Biol.1997,136:331-343; Machesky, L.M.et al.Biolchem.J.1997,328:105-112).In different species, title is also different owing to vary in size for 7 subunits, is hereinafter referred to as Arp2 in Mammals, Arp3, p41-Arc, p34-Arc, p21-Arc, p20-Arc, p16-Arc (Welch, M.D.et al.J.Cell Biol.1997,138:375-384).The title of ARC-16 subunit in other biology is respectively Arc15p (Saccharomyces cerevisiae), arc15p (Schizosaccharomyecespombe), p14 (Acanthamoeba and Dictyostelium) (see review, Machesky, L.M.et al.Curr.Opon.Cell Biol.1999,11:117-121).

Yet, before the application, also do not have to separate or disclose nucleotide sequence and the aminoacid sequence of remarkable ARC16-2.

An object of the present invention is to provide a kind of new polynucleotide, these polynucleotide are named as people ARC16-2, one of subunit of its coding people Arp2/3 mixture ARC16-2, and the function of this subunit is relevant with the function of Arp2/3 mixture.

Another object of the present invention provides a kind of new people ARC16-2 albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's ARC16-2 polypeptide.

The present invention also provides this people's the ARC16-2 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 ARC16-2 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 8-466 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 8-466 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 8-466 position.

In another aspect of this invention, provide a kind of isolating people ARC16-2 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 ARC16-2 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people ARC16-2 protein-active operationally is connected in expression regulation sequence, form people ARC16-2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 8-466 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 ARC16-2;

(c) under the condition that is fit to expressing human ARC16-2 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people ARC16-2 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 536 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 8-466 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 ARC16-2 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people ARC16-2 protein-active is as 8-466 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 8-466 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 8-466 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 8-466 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 8-466 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 ARC16-2 identical function.These variant forms comprise (but being not limited to): several (are generally 1-30, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 30 to hold interpolation 5 ' and/or 3 ', preferably be in 20, more preferably being 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 ARC16-2 protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people ARC16-2 protein-active.This term also comprises having and variant form people ARC16-2 identical function, SEQ IDNO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-40, 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 ARC16-2 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 ARC16-2 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people ARC16-2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people ARC16-2 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people ARC16-2 polypeptide.Usually, this fragment have people ARC16-2 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 ARC16-2 albumen or polypeptide.The difference of these analogues and natural human ARC16-2 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 ARC16-2 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	Leu
Leu(L)	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	Leu

The present invention also comprises people ARC16-2 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people ARC16-2 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 ARC16-2 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 ARC16-2.

The present invention also comprises the method that detects people ARC16-2 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 ARC16-2 polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 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 ARC16-2 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 ARC16-2 gene product or fragment.Preferably, refer to that those can combine with people ARC16-2 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 ARC16-2, comprise that also those do not influence the antibody of people ARC16-2 protein function.The present invention also comprise those can with modify or without the people ARC16-2 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 ARC16-2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human ARC16-2 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 ARC16-2 function and the antibody that does not influence people ARC16-2 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people ARC16-2 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 ARC16-2 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 ARC16-2 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 ARC16-2 is so to obtain, with human brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer--A:5 '-TCCCGCCATGGCCCGGAACACGC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B:5 '-GTCAATCCCTTCTTCACCAGCCTG-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 60 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains is about the purpose fragment (SEQ ID NO:3) of 500bp.

The Arp2/3 mixture can be accelerated speed (Mullins, R.D.et al.Proc.Natl.Acad.Sci.USA 1998, the 95:6181-6186 of Actin muscle fibril nucleation; Welch, M.D et al.Science 1998,281:105-108), can promote the branch of Actin muscle fibril, (Mullins, R.D.et al.Proc.Natl.Acad.Sci.USA1998,95:6181-6186), (Mullins, R.D.etal.Proc.Natl.Acad.Sci.USA 1998,95:6181-6186) to add cap can also for the slower end of Actin muscle fibril prolongation.As the subunit of Arp2/3 mixture, ARC16 must and keep with the assembling of Actin muscle structure, and the performance of Actin muscle function is relevant.

People ARC16-2 of the present invention helps the research of people to the function separately of Actin muscle and actin associated protein, also helps the interactional pattern between them of studying.In addition, because ARC16-2 of the present invention has the natural acid sequence that is derived from the people, therefore, compare, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour with the albumen of the same clan that derives from other species.

In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence of people ARC16-2 of the present invention and people ARC16.Wherein, identical amino acid marks with " * " below two sequences, and similar amino acid marks with ": ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York: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 ARC16-2

1. primer amplification

With human brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer--A:5 '-TCCCGCCATGGCCCGGAACACGC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B:5 '-GTCAATCCCTTCTTCACCAGCCTG-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 60 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The length that electrophoresis detection obtains is about PCR purpose Segment A/B of 500bp.

2.PCR the order-checking of product

Above-mentioned pcr amplification product A/B is connected with pGEM-T carrier (Promega), and transformed into escherichia coli JM103 extracts plasmid with QIAprep Plasmid test kit (QIAGEN), uses SequiThermEXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to inserting fragment, with computer software splicing order, obtains full length cDNA sequence at last, is total to 536bp, and detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 8-466 position Nucleotide.

Derive the aminoacid sequence of people ARC16-2 according to the full length cDNA sequence that obtains, totally 152 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 ARC16-2 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 ARC16 (gb|AF006088) of people etc. higher homology, identity being arranged respectively on nucleotide level is 82%.Homologous protein is more also supported the correct of this sequence, ARC16-2 derivation albumen and people's ARC16 (sp|O15511) identity position 69% on amino acid levels, similarity is 85% (both homologies is relatively seen Fig. 1), p16-Arc (gi|4093171) with Dictyostelium discoideum, the AR16 of Caenorhabditis elegans (sp|P91167), the AR16 of Schizosaccharomyces pombe (sp|Q10316), AR16 (sp|P40518) identity of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) reaches 38% respectively, 33%, 26%, 28%, similarity reaches 62% respectively, 55%, 48%, 46%.

According to the structures shape function, the Biological Principles that the structural similitude function is relevant can be inferred the biological function of ARC16-2 of the present invention according to the function of ARC16 gene in the known different plant species and proteins encoded thereof.

Because ARC16 is one of subunit of Arp2/3 mixture, the function of its function and Arp2/3 mixture is closely related.

The Arp2/3 mixture can be accelerated speed (Mullins, R.D.et al.Proc.Natl.Acad.Sci.USA 1998, the 95:6181-6186 of Actin muscle fibril nucleation; Welch, M.D et al.Science 1998,281:105-108).Can induce the reformulation of Actin muscle end at the surfaces A rp2/3 mixture of Listeria monocytogenes bacterium, and the Actin muscle end is formed by the Actin muscle monomer polymerization, prompting Arp2/3 mixture becomes the effect (Welch, M.D.et al.Nature 385:265-269) of nuclear dimension at Actin muscle.Have experiment to find that the increase of actin polymerization speed depends on the concentration of Arp2/3 mixture simultaneously, this phenomenon and above-mentioned supposition be conform to (Mullins, R.D.et al.Proc.Natl.Acad.Sci.USA 1998,95:6181-6186).

In addition, the Arp2/3 mixture can promote the branch of Actin muscle fibril, one end of Actin muscle fibril is fixed to the side of other fibril, form 70 ° bifurcated (Mullins, R.D.et al.Proc.Natl.Acad.Sci.USA 1998 95:6181-6186), also observes in the lamellipodia of motor cell and is similar to observed structure (Svitkina in this experiment in vitro, T.M.et al.J.Cell Biol.1997,129:397-415).

The Arp2/3 mixture adds cap for the slower end (slow-growing/pointed ends) of Actin muscle fibril prolongation, the depolymerization of prompting Actin muscle is not a simple process, but strict regulation and control, dissociating of possible Arp2/3 mixture is a prerequisite (Mullins, R.D.et al.Proc.Natl.Acad.Sci.USA 1998,95:6181-6186).Like this, on the one hand, dissociating of Arp2/3 makes the depolymerization of cell rack fast, and the adaptability of cell is stronger; On the other hand, if the Arp2/3 mixture is suppressed dissociated words, cytoskeleton more stable (Mullins, R.D.et al.Proc.Natl.Acad.Sci.USA 1998,95:6181-6186)

Discover in the body, the cell mitochondrial of Saccharomyces cerevisiae Arc15 subunit (corresponding to mammiferous p16-Arc subunit) sudden change shifts defectiveness, because its plastosome utilizes actin cytoskeleton to transfer to daughter cell (Yan from parent cell, C.et al.EMBO J.1997,16:357-3586), show that at least Arp2/3 moves relevant with plastosome along Actin muscle in Saccharomyces cerevisiae.

People ARC16-2 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 ARC16-2 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor ARC16-2 and the N end of yeast saccharomyces cerevisiae ARC16 or people ARC16 are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor ARC16-2, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

In addition, inventor ARC16-2 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people ARC16-2 or the overexpression that suppresses people ARC16-2.People ARC16-2 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people ARC16-2 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 ARC16-2 in intestinal bacteria

In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, with the cDNA sequence of coding people ARC16-2 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.5 and 6) of 3 ' end increase, obtain people ARC16-2 cDNA as inserting fragment.

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

5-CAAGGTCGACATGGCCCGGAACACGCTGT-3 ' (SEQ ID NO.5), this primer contains the restriction enzyme site of SalI restriction enzyme, is 19 Nucleotide of the people ARC16-2 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-TAGGAAGCTTTTAAACAGTCTTTCTTGCT-3 ' (SEQ ID NO.6), this primer contains the part encoding sequence of restriction enzyme site, translation termination and the people ARC16-2 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, 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 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 cDNA fragment of extracting plasmid sequence verification people ARC16-2 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 ARC16-2 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people ARC16-2 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 17kDa.

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 ARC16-2 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, with the cDNA sequence of coding people ARC16-2 use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.7 and 8) of 3 ' end increase, obtain people ARC16-2 cDNA as inserting fragment.

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

5 '-this primer of CAAGAAGCTTATGGCCCGGAACACGCTGT-3 ' (SEQ ID NO.7) contains the restriction enzyme site of HindIII restriction enzyme, is 19 Nucleotide of the people ARC16-2 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

This primer of 5 '-TAGGGGATCCTTAAACAGTCTTTCTTGCT-3 ' (SEQ ID NO.8) contains the part encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and people ARC16-2.

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 HindIII and BamHI 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 cDNA fragment of extracting plasmid sequence verification people ARC16-2 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 50mMTris HCl (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 17kDa.

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 ARC16-2 gene translation product with it.Found that antibody can precipitate with albumen of the present invention 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 human actin related protein subunit and encoding sequence thereof be the sequence number (iii): information (i) sequence signature of 8 (2) SEQ ID NO.1

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.1:TCCCGCCATG GCCCGGAACA CGC 23 (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:2GTCAATCCCT TCTTCACCAG CCTG 24 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 536bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( iii ) ：SEQ ID NO.3TCCCGCCATG GCCCGGAACA CGCTGTCCTC GCGCTTCCGC CGGGTGGACA TCGACGAATT 60TGACGAGAAC AAATTTGTGG ACGAGCAGGA GGAGGCGGCG GCGGCGGCGC GGACAGGCCC 120GGACCCGAGC GAGGTGGACG GGCTCCTGCG GCAAGGGGAC ATGCTTCGGG CATTCCATGC 180AGCCTTGCGG AACTCTCCCG TCAACACCAA GAATCAAGCT GTGAAGGAGC GAGCCCAGGG 240CGTGGTGCTG AAAGTGCTCA CAAACTTCAA GAGCAGTGAG ATTGAGCAGG CTGTGCAGTC 300ACTGGACAGA AACGGCGTTG ACTTGTTAAT GAAGTACATT TATAAAGGCT TTGAGAAGCC 360CACAGAAAAT AGCAGCGCAG TGTTACTCCA GTGGCACGAA AAGGCCTTAG CAGTAGGAGG 420ACTAGGCTCC ATTATAAGAG TTCTTACAGC AAGAAAGACT GTTTAAAAAA AATAAAAAGA 480CTCATGTTAC CTTGAGAAGA ATTCTGGATG CCCAGGCTGG TGAAGAAGGG ATTGAC 536 ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 152 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4Met Ala Arg Asn Thr Leu Ser Ser Arg Phe Arg Arg Val Asp Ile 15Asp Glu Phe Asp Glu Asn Lys Phe Val Asp Glu Gln Glu Glu Ala 30Ala Ala Ala Ala Arg Thr Gly Pro Asp Pro Ser Glu Val Asp Gly 45Leu Leu Arg Gln Gly Asp Met Leu Arg Ala Phe His Ala Ala Leu 60Arg Asn Ser Pro Val Asn Thr Lys Asn Gln Ala Val Lys Glu Arg 75Ala Gln Gly Val Val Leu Lys Val Leu Thr Asn Phe Lys Ser Ser 90Glu Ile Glu Gln Ala Val Gln Ser Leu Asp Arg Asn Gly Val Asp 105Leu Leu Met Lys Tyr Ile Tyr Lys Gly Phe Glu Lys Pro Thr Glu 120Asn Ser Ser Ala Val Leu Leu Gln Trp His Glu Lys Ala Leu Ala 135Val Gly Gly Leu Gly Ser Ile Ile Arg Val Leu Thr Ala Arg Lys 150Thr Val 152 ( 2 ) SEQ ID NO.5 ( i )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.5:CAAGGTCGAC ATGGCCCGGA ACACGCTGT 29 (2) SEQ ID NO.6

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.6:TAGGAAGCTT TTAAACAGTC TTTCTTGCT 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: information (i) sequence signature of SEQ ID NO.7CAAGAAGCTT ATGGCCCGGA ACACGCTGT 29 (2) SEQ ID NO.8

(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.8TAGGGGATCC TTAAACAGTC TTTCTTGCT 29

Claims (10)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people ARC16-2 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 8-466 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 8-466 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 8-466 position.

4. isolating people ARC16-2 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. a generation has the method for the polypeptide of people ARC16-2 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people ARC16-2 protein-active operationally is connected in expression regulation sequence, form people ARC16-2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 8-466 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 ARC16-2;

(c) under the condition that is fit to expressing human ARC16-2 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people ARC16-2 protein-active.

9. energy and the described people ARC16-2 of claim 4 protein polypeptide specificity bonded antibody.

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

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