CN1264739A - Human protein and its coding sequence, preparing process and application - Google Patents

Abstract

The present invention discloses a new member in Ena/VASP gene family, which is named as human neonatal brain (HNB). The cDNA coding sequence of said new HNB protein, the polypeptide coded with the sequence, the process for preparing said HNB protein by recombination technique and the application of said HNB protein are also disclosed.

Description

New people's albumen and encoding sequence thereof, and method for making and purposes

The present invention relates to a kind of new polynucleotide, by the polypeptide of this polynucleotide encoding, the application of these polynucleotide and polypeptide, and the production of described polynucleotide and described polypeptide.More particularly, polypeptide of the present invention is accredited as a newcomer of Ena/VASP protein family by deduction.

People such as Gertler in nineteen ninety-five in fruit bat, find and the clone obtained the Ena gene (Gertler1995, Genes Dev.9,521-533); In the same year, people such as Haffner have found the VASP gene in mouse; Ena gene of fruit bat and the VASP gene of mouse belong to the Ena/VASP protein family the member (Haffner 1995, EMBOJ.14,19-27).1996, people such as Gertler separated in mouse again and have obtained two newcomers of Ena/VASP protein family, and Mena and Evl (Frank B.Gertler 1996, Cell, 87,227-239).1997, people such as Shoichiro Ohta separation obtained a new rat gene RNB6 (Shoichiro Ohta 1997, Biochem ﹠amp; Biophys Res.Commun., 237,307-312).Member Ena, VASP, Mena, the Evl of RNB6 and Ena/VASP protein family all have higher homology, are all the member of Ena/VASP protein family, and have similar biological function.

Yet, before the present invention, also do not have to disclose or reported the human protein that belongs to Ena/VASP family.

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding Ena/VASP protein family, the new Human genome of the present invention is named as the HNB gene.

Another object of the present invention provides a kind of new Ena/VASP protein family member, and this albumen is named as HNB albumen.

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

The invention still further relates to the application of this people HNB protein nucleic acid sequence and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the active polypeptide of people HNB protein, shows at least 70% homology from the nucleotides sequence of Nucleotide 33-1289 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 33-1289 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 33-1289 position.

In another aspect of this invention, provide a kind of isolating HNB protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ IDNO.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, the method that provides a kind of generation to have the polypeptide of HNB protein-active, this method comprises:

(a) nucleotide sequence that coding is had an active polypeptide of HNB protein operationally is connected in expression regulation sequence, form the HNB protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 33-1289 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 HNB;

(c) be fit to express under the condition of HNB protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HNB protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1352 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 33-1289 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 " HNB albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with HNB protein-active is as 33-1289 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 33-1289 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 33-1289 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 33-1289 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 33-1289 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.3 sequence of people HNB albumen 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 " HNB protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of HNB protein-active.This term also comprises having and variant form people HNB albumen 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 HNB 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 HNB protein D NA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-HNB protein polypeptide to obtain.The present invention also provides other polypeptide, as comprises HNB protein polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of HNB protein polypeptide.Usually, this fragment have HNB protein polypeptide 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 HNB albumen or polypeptide.The difference of these analogues and natural HNB protein 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 can also comprise 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 HNB 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 HNB protein polypeptide encoding sequence or its segmental antisense sequences.This antisense sequences can be used for suppressing the proteic expression of HNB in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of HNB pyrenoids nucleotide sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample the proteic nucleic acid molecule of coding HNB.

The present invention also comprises the method that detects HNB pyrenoids 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 can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of HNB protein polypeptide.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 HNB protein D NA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into HNB protein gene product or fragment.Preferably, refer to that those can combine with HNB protein 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 molecule of HNB protein, comprise that also those do not influence the antibody of HNB protein function.The present invention also comprise those can with modify or without the HNB protein 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 HNB protein gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing HNB albumen or its has 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.Jmmunol.6:292,1976; People such as Hammerling, InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the HNB protein function and the antibody that does not influence the HNB protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of HNB protein gene product, obtains by immunological technique.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 HNB protein 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 HN 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, polynucleotide total length of the present invention is 1352 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 33-1289 position Nucleotide.These polynucleotide are so to obtain, with human brain λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer A1:5 '-CTCAGGGTTCCCTGTGCTGCCAC-3 ' and reverse primer B1:5 '-GGGCTTCTGGCTGTCCTCTGTCG-3 ' carry out PCR, obtain the purpose fragment of 1352bp.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.

According to homology result relatively, the member of the Ena/VASP protein family of nucleotide sequence of the present invention and encoded protein matter sequence and different sources has shown significant homology, therefore, this shows that it is a newcomer of Ena/VASP protein family, and has the more proteic critical functions of Ena/VASP protein family.

The member of Ena/VASP protein family all combines with profilin by their GP5 fancy structure, controls the motion of cell by the gathering of modulate actin microfilament.The most of members that discover Ena/VASP family express gradually in brain and strengthen all in biological embryo development procedure, and reach the climax in postnatal first day in biology, then just gradually reduce.RNB6 albumen to rat studies show that it is not only expressed in brain, and also expresses in spleen, thymus gland and testis.The member of Ena/VASP protein family mainly comes the neural growth of control center (shoichiro Ohta 1997, Biochem ﹠amp by the adjusting to the neurocyte motion; BiophysRes.Commun.237,307-312).

In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence of people HNB of the present invention (HNBP) and mouse Evl albumen (EVLP).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Fig. 2 is the homology comparison diagram of the aminoacid sequence of people HNB of the present invention (HNBP) and rat RNB6 albumen (RNB6P).

Fig. 3 is people HNB of the present invention, mouse Evl albumen and 3 kinds of proteic amino acid sequence homologous comparison diagrams of rat RNB6 albumen.Wherein, identical amino acid marks with " * " below sequence, 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:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone and the mensuration of the proteic cDNA sequence of HNB

1. primer amplification

With human brain λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-CTCAGGGTTCCCTGTGCTGCCAC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B1:5 '-GGGCTTCTGGCTGTCCTCTGTCG-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, 70 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains is about the purpose fragment of .4Kb.

2.PCR the order-checking of product

With pcr amplification product and the pGEM-T that as above obtains ^Carrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).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 1352bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 33-1289 position Nucleotide.

Derive the proteic aminoacid sequence of HNB according to the full length cDNA sequence that obtains, totally 418 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.

Embodiment 2

Homology relatively

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 proteic full length cDNA sequence of HNB and proteins encoded thereof with BLAST.Found that it and Ena/VASP protein family member have shown higher homology, as use the PCGENE software analysis that the Evl albumen of it and mouse has shown 93.9% identity and 97.5% similarity (Fig. 1) on protein level; And for example, the RNB6 albumen of it and rat has shown 93.6% identity and 96.9% similarity (Fig. 2) on protein level.

Specifically all there is the proline(Pro) fancy structure of forming by six amino acid a: Gly-Pro-Pro-Pro-Pro-Pro (GP5) in Ena/VASP protein family member's the aminoacid sequence; This proline(Pro) fancy structure is considered to Ena/VASP protein family member's characteristic sequence, it may combine with profilin the member of Ena/VASP protein family, regulate play an important role in the cell movement process of controlling the Actin muscle mediation (Shoichiro Ohta 1997, Biochem ﹠amp; Biophys Res.Commun., 237,307-312).The sequence fragment that meets above-mentioned pattern in albumen of the present invention is: Gly-Pro-Pro-Pro-Pro-Pro (183-188 position among the SEQ ID NO.4).In addition, Ena/VASP protein family member's amino acid composition also contains two structural domain EVH1 and EVH2 (Fig. 3).Also contain two corresponding with it structural domains in the proteic aminoacid sequence of people HNB of the present invention, they be respectively 224-412 position among 1-114 position and the SEQ ID NO 4 among the SEQ ID NO 4 (FrankB.Gertler 1996, Cell, 87,227-239).This has shown that further HNB albumen of the present invention also is a member in the Ena/VASP protein family, and have to the Ena/VASP protein family in the similar function of other members.

The member of Ena/VASP protein family all combines with profilin by their GP5 fancy structure, by the actinmicrofilament accumulative being regulated the motion of controlling cell.

Northern and immune Research show that in fruit bat and mouse embryo development procedure, the expression in brain strengthens gradually respectively for VASP albumen of the Ena of fruit bat, mouse and the RNB6 albumen of rat, and reaches the climax in postnatal first day, then just gradually reduces.Tissue distribution discovers that the RNB6 albumen of rat is not only expressed in brain, and also expresses in spleen, thymus gland and testis.Histochemical studies shows that VASP albumen, RNB6 albumen and Ena albumen mainly come the neural growth of control center (shoichiro Ohta 1997, Biochem ﹠amp by the adjusting to the neurocyte motion; Biophys Res.Commun.237,307-312).

The member of HNB albumen of the present invention and Ena/VASP protein family all has higher homology, it is the newcomer of Ena/VASP protein family, therefore think that it also has the function with above-mentioned each protein similar, promptly plays an important role in the neural growth course in biological embryo center.

People HNB 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 HNB can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor HNB and the N end of rat RNB6 are exchanged, to produce the albumen that new activity is higher or have new features.

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

Coding HNB proteic cDNA sequence is a template with the pcr amplification product A1/B1 among the embodiment 1, use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, with the synthetic fragment of inserting.

5 ' Oligonucleolide primers sequence is

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

This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is 20 Nucleotide of the HNB albumen coded sequence that begun by initiator codon;

3 ' Oligonucleolide primers sequence is

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

This primer contains the restriction enzyme site of SalI restriction enzyme, the sub and proteic encoding sequence of HNB of translation termination.

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

With BamHI and SalI digestion pQE-9 carrier, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, 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).The extracting plasmid is cut with the PstI enzyme and identify to be inserted clip size and direction, and sequence verification result shows that the proteic cDNA of HNB inserts the fragment carrier of correctly packing into.

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 HNB albumen from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out HNB albumen from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.At first, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein can be incorporated in second post from nickel-chelate column, 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 45KDa.

In addition, the amino acid of expressing 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 HNB albumen in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, be template with coding HNB proteic cDNA sequence with the pcr amplification product A1/B1 among the embodiment 1, use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, with the synthetic fragment of inserting.

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

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

This primer contains the restriction enzyme site of HindIII restriction enzyme, and what connect is 20 Nucleotide of the HNB albumen coded sequence that begun by initiator codon;

3 ' end primer sequence is:

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

This primer contains the restriction enzyme site of BamHI restriction enzyme, translation termination and the proteic encoding sequence of Ena/VASP.

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

With HindIII, BamHI digestion pcDNA3 carrier, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid is cut with the PstI enzyme and identify to be inserted clip size and direction, and sequence verification result shows that the proteic cDNA of HNB inserts the fragment carrier of correctly packing into.

Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mM TrisHCl (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 45KDa.

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

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, specific as follows.Separate recombinant molecule back standby 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 deposit E na/VASP protein 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): new people's albumen and encoding sequence thereof, and (iii) sequence number of method for making and purposes: 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:CTCAGGGTTC CCTGTGCTGC CAC 23 (2) SEQ ID NO.2

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

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

(A) length: 1352bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNACTCAGGGTTC CCTGTGCTGC CACTTTTCAG CCATGGCCAC AAGTGAACAG AGTATCTGCC 60AAGCCCGGGC TTCCGTGATG GTCTACGATG ACACCAGTAA GAAATGGGTA CCAATCAAAC 120CTGGCCAGCA GGGATTCAGC CGGATCAACA TCTACCACAA CACTGCCAGC AACACCTTCA 180GAGTCGTTGG AGTCAAGTTG CAGGATCAGC AGGTTGTGAT CAATTATTCA ATCGTGAAAG 240GGCTGAAGTA CAATCAGGCC ACGCCAACCT TCCACCAGTG GCGAGATGCC CGCCAGGTCT 300ACGGCTTAAA CTTTGCAAGT AAAGAAGAGG CAACCACGTT CTCCAATGCA ATGCTGTTTG 360CCCTGAACAT CATGAATTCC CAAGAAGGAG GCCCCTCCAG CCAGCGTCAG GTGCAGAATG 420GCCCCTCTCC TGATGAGATG GACATCCAGA GAAGACAAGT GATGGAGCAG CACCAGCAGC 480AGCGTCAGGA ATCTCTAGAA AGAAGAACCT CGGCCACAGG GCCCATCCTC CCACCAGGAC 540ATCCTTCATC TGCAGCCAGC GCCCCCGTCT CATGTAGTGG GCCTCCACCG CCCCCCCCAC 600CCCCAGTCCC ACCTCCACCC ACTGGGGCTA CCCCACCTCC CCCACCCCCA CTGCCAGCCG 660GAGGAGCCCA GGGGTCCAGC CACGACGAGA GCTCCATGTC AGGACTGGCC GCTGCCATAG 720CTGGGGCCAA GCTGAGAAGA GTCCAACGGC CAGAAGACGC ATCTGGAGGC TCCAGTCCCA 780GTGGGACCTC AAAGTCCGAT GCCAACCGGG CAAGCAGCGG GGGTGGCGGA GGAGGCCTCA 840TGGAGGAAAT GAACAAACTG CTGGCCAAGA GGAGAAAAGC AGCCTCCCAG TCAGACAAGC 900CAGCCGAGAA GAAGGAAGAT GAAAGCCAAA TGGAAGATCC TAGTACCTCC CCCTCTCCGG 960GGACCCGAGC AGCCAGCCAG CCACCTAACT CCTCAGAGGC TGGCCGGAAG CCCTGGGAGC 1020GGAACAACTC GGTGGAGAAG CCTGTGTCCT CGATTCTGTC CAGAACCCCG TCTGTGGCAA 1080AGAGCCCCGA AGCTAAGAGC CCCCTTCAGT CGCAGCCTCA CTCTAGGATG AAGCCTGCTG 1140GGAGCGTGAA TGACATGGCC CTGGATGCCT TCGACTTGGA CCGGATGAAG CAGGAGATCC 1200TAGAGGAGGT GGTGAGAGAG CTCCACAAGG TGAAGGAGGA GATCATCGAC GCCATCAGGC 1260AGGAGCTGAG TGGGATCAGC ACCACGTAAG GGGCCGGCCT CGCTGCGCTG ATTCGTCGAG 1320CCCATCCGGC GACAGAGGAC AGCCAGAAGC CC 1352 ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 418 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4： Met Ala Thr Ser Glu Gln Ser Ile Cys Gln Ala Arg Ala Ser Val 15Met Val Tyr Asp Asp Thr Ser Lys Lys Trp Val Pro Ile Lys Pro 30Gly Gln Gln Gly Phe Ser Arg Ile Asn Ile Tyr His Asn Thr Ala 45Ser Asn Thr Phe Arg Val Val Gly Val Lys Leu Gln Asp Gln Gln 60Val Val Ile Asn Tyr Ser Ile Val Lys Gly Leu Lys Tyr Asn Gln 75Ala Thr Pro Thr Phe His Gln Trp Arg Asp Ala Arg Gln Val Tyr 90Gly Leu Asn Phe Ala Ser Lys Glu Glu Ala Thr Thr Phe Ser Asn 105Ala Met Leu Phe Ala Leu Asn Ile Met Asn Ser Gln Glu Gly Gly 120Pro Ser Ser Gln Arg Gln Val Gln Asn Gly Pro Ser Pro Asp Glu 135Met Asp Ile Gln Arg Arg Gln Val Met Glu Gln His Gln Gln Gln 150Arg Gln Glu Ser Leu Glu Arg Arg Thr Ser Ala Thr Gly Pro Ile 165Leu Pro Pro Gly His Pro Ser Ser Ala Ala Ser Ala Pro Val Ser 180Cys Ser Gly Pro Pro Pro Pro Pro Pro Pro Pro Val Pro Pro Pro 195Pro Thr Gly Ala Thr Pro Pro Pro Pro Pro Pro Leu Pro Ala Gly 210Gly Ala Gln Gly Ser Ser His Asp Glu Ser Ser Met Ser Gly Leu 225Ala Ala Ala Ile Ala Gly Ala Lys Leu Arg Arg Val Gln Arg Pro 240Glu Asp Ala Ser Gly Gly Ser Ser Pro Ser Gly Thr Ser Lys Ser 255Asp Ala Asn Arg Ala Ser Ser Gly Gly Gly Gly Gly Gly Leu Met 270Glu Glu Met Asn Lys Leu Leu Ala Lys Arg Arg Lys Ala Ala Ser 285Gln Ser Asp Lys Pro Ala Glu Lys Lys Glu Asp Glu Ser Gln Met 300Glu Asp Pro Ser Thr Ser Pro Ser Pro Gly Thr Arg Ala Ala Ser 315Gln Pro Pro Asn Ser Ser Glu Ala Gly Arg Lys Pro Trp Glu Arg 330Asn Asn Ser Val Glu Lys Pro Val Ser Ser Ile Leu Ser Arg Thr 345Pro Ser Val Ala Lys Ser Pro Glu Ala Lys Ser Pro Leu Gln Ser 360Gln Pro His Ser Arg Met Lys Pro Ala Gly Ser Val Asn Asp Met 375Ala Leu Asp Ala Phe Asp Leu Asp Arg Met Lys Gln Glu Ile Leu 390Glu Glu Val Val Arg Glu Leu His Lys Val Lys Glu Glu Ile Ile 405Asp Ala Ile Arg Gln Glu Leu Ser Gly Ile Ser Thr Thr 418 ( 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:TGCAGGATCC ATGGCCACAA GTGAACAGAG 30 (2) SEQ ID NO.6

(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.6:GTTCGTCGAC TTACGTGGTG CTGATCCCAC 30 (2) SEQ ID NO.7

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.7:TGACAAGCTT ATGGCCACAA GTGAACAGAG 30 (2) SEQ ID NO.8

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.8:GTTCGGATCC TTACGTGGTG CTGATCCCAC 30

Claims (10)

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

Show at least 70% homology from the nucleotides sequence of Nucleotide 33-1289 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 33-1289 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 33-1289 position.

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

(a) nucleotide sequence that coding is had a polypeptide of HNB protein-active operationally is connected in expression regulation sequence, form the HNB protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 33-1289 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 HNB;

(c) be fit to express under the condition of HNB protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HNB protein-active.

9. energy and the described HNB protein polypeptide of claim 4 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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