CN1246529A - Coding sequence of human translation initiation factor subunit, its encoded polypeptide and its preparing process - Google Patents

Abstract

The present invention relates to the delta-subunit HeIF-2Bd of new human protein translation initiation factor 2B. Its cDNA coding sequence, the polypeptide coded with said sequence, and a method for preparing the delta-subunit by reformation are disclosed. The application of said delta-subunit is also disclosed.

Description

The encoding sequence of human translation initiation factor subunit, its encoded polypeptides and preparation method

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to δ subunit and this proteic cDNA sequence of coding of human protein translation initiation factor 2B.The invention still further relates to the production method of described polynucleotide sequence and described polypeptide, and the application of these polynucleotide and polypeptide.

In mammalian cell, translation initiation factor 2B (eIF-2B is called guanylic acid exchange factor (GEF) again) has critical regulating and controlling effect to polypeptide chain synthetic initial.In proteinic translation process, at first form a ternary complex of forming by initial tRNA, GTP and eIF-2, transfer to then on the 40S ribosomal subunit.Along with the formation of 80S initiation complex, GTP is hydrolyzed, and eIF-2 is released with the form of eIF-2-GDP binary complex.This binary complex is stable under physiological condition, and does not have the function activity.The new round circulation of translation initiation requires the participation of eIF-2B, because the affinity of eIF-2 and GDP than with the high 100-400 of affinity of GTP doubly, so need eIF-2B catalysis and eIF-2 bonded GDP and GTP to exchange, so that regenerate eIF-2-GTP and participate in translation initiation (J.Biol.Chem.268 (1993), the 7603-7606 of a new round; Annu.Rev.Biochem.60 (1991), 717-755).Therefore, eIF-2B plays an important role in the regulation process of proteinic translation initiation.

Mammiferous eIF-2B is made up of five subunit α, β, γ, δ, ε, and its molecular weight is respectively 29,39,54,66, about 84kDa.The cDNA sequence of each subunit is in the news in succession in mouse and the rabbit.1994, people such as Nigel design redundant oligonucleotide in proper order according to the δ subunit peptides of eIF-2B and carry out PCR, obtain RNA from rabbit liver, further again guidance separates the δ subunit full length cDNA sequence that obtains eIF-2B from Reticulocyte cDNA storehouse (Biochim.Biophy.Acta. (1994) 121 (2), 207-210).People such as Henderson separate subsequently and are cloned into the δ subunit cDNA order of eIF-2B of mouse (J.Biol.Chem. (1994) 269 (48), 30517-30532).Yet before the present invention, the δ subunit of the human translation initiation factor 2B that relates among the application was not disclosed.

An object of the present invention is to provide a kind of new polynucleotide, this polynucleotide encoding translation initiation factor 2B newcomer's of family δ subunit, the δ subunit called after HeIF-2Bd of translation initiation factor 2B of the present invention.

Another object of the present invention provides the δ subunit of a kind of new human translation initiation factor 2B, and this albumen is named as HeIF-2Bd.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce the δ subunit of described new human translation initiation factor 2B.

The invention still further relates to the δ subunit gene of this human translation initiation factor 2B 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 HeIF-2Bd protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 20-1582 position among described nucleotide sequence and the SEQ ID NO.9; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.9 in from the nucleotide sequence hybridization of Nucleotide 20-1582 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.10.More preferably, this sequence has among the SEQ ID NO.9 nucleotide sequence from Nucleotide 20-1582 position.

In another aspect of this invention, provide a kind of isolating HeIF-2Bd protein polypeptide, it comprises: have polypeptide or its active fragments of SEQ ID NO.10 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.10 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 HeIF-2Bd protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of HeIF-2Bd protein-active operationally is connected in expression regulation sequence, form the HeIF-2Bd protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 20-1582 position among described nucleotide sequence and the SEQ ID NO.9;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of HeIF-2Bd;

(c) be fit to express under the condition of HeIF-2Bd protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HeIF-2Bd protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1196 Nucleotide, and its detailed sequence is seen SEQ ID NO.9, and wherein open reading frame is positioned at 20-1582 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 " HeIF-2Bd albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with HeIF-2Bd protein-active is as 20-1582 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.9.This degenerate sequence is meant, is arranged in the encoder block 20-1582 position Nucleotide of SEQ ID NO.9 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.9 in 20-1582 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.10 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition with SEQ ID NO.9 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 20-1582 position.This term also comprise with SEQ ID NO.9 in from the nucleotide sequence homology at least 70% of Nucleotide 20-1582 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.9 sequence of people HeIF-2Bd 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 " HeIF-2Bd protein polypeptide " refer to have the HeIF-2Bd protein-active, SEQID NO.10 polypeptide of sequence.This term also comprises variant form δ subunit identical function, SEQ ID NO.10 sequence that has with human translation initiation factor 2B.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 HeIF-2Bd and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of HeIF-2Bd DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-HeIF-2Bd polypeptide to obtain.The present invention also provides other polypeptide, as comprises HeIF-2Bd polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of HeIF-2Bd polypeptide.Usually, this fragment have the HeIF-2Bd 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 HeIF-2Bd albumen or polypeptide.The difference of these analogues and natural HEIF-2BD polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

The present invention also comprises the antisense sequences of HeIF-2Bd polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of HeIF-2Bd in the cell.

The present invention also comprises a kind of probe molecule, and this molecule has 8-100 of HeIF-2Bd 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 the HEIF-2BD that encodes.

The present invention also comprises the method that detects the HeIF-2Bd 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 HeIF-2Bd 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 various carriers.

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 HEIF-2BD 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 HeIF-2Bd gene product or fragment.Preferably, refer to that those can combine with HEIF-2BD 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 HeIF-2Bd, comprise that also those do not influence the antibody of HeIF-2Bd protein function.The present invention also comprise those can with modify or without the HeIF-2Bd 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, United States Patent (USP) NO.10,946,778); 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 HeIF-2Bd gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing HeIf-2Bd 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.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 the HeIF-2Bd function, also comprises the antibody that does not influence the HeIF-2Bd function.Each antibody-like of the present invention can utilize the fragment or the functional zone of HeIF-2Bd gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.With the unmodified form bonded antibody of HeIF-2Bd gene product, can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli); 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).

In the present invention, the cDNA nucleotide sequence of HeIF-2Bd is so to obtain: with human brain λ gt11cDNA library (available from Clontech company) is template, with two pairs of oligonucleotide is primer---A1:5 '-GAGCCTAGGACTGAGGGCGATGG-3 ' (SEQ ID NO.1) and A2:5 '-TGCCTTGCAGCAGGTGATTCAGG-3 ' (SEQ ID NO.2) they are forward primer, oligonucleotide B1:5 '-CTGCAGGAATCAGCAGGTAGGAG-3 ' (SEQ ID NO.3) and B2:5 '-TCACTGGTCACTGCTCTTGACTCG-3 ' (SEQ ID NO.4) are reverse primer, carry out PCR, obtain the purpose fragment of 1180bp (A1/B1) and 920bp (A2/B2) respectively.The Computer Analysis of order-checking back, splicing obtains the cDNA sequence that total length is 1582bp (SEQ ID NO.9).

Find by the homology retrieval, the δ subunit gene sequence height homology of known translation initiation factor 2B in the cDNA sequence of HeIF-2Bd among the present invention and other species (as rabbit, mouse), and novel polypeptide of the present invention has the height homologous aminoacid sequence with the δ subunit family of translation initiation factor 2B.The same with the δ subunit of the rabbit of the known δ subunit family that belongs to translation initiation factor 2B and mouse eIF-2B, HeIF-2Bd gene of the present invention also belongs to eIF-2B family and has similar function.

It is initial that known eIF-2B (being called the guanylic acid exchange factor again) participates in protein translation, and the regulation and control translation process, this proteic not normal meeting cause protein translation and regulate and control undesired.EIF-2B is made up of five subunit α, β, γ, δ, ε, and HeIF-2Bd of the present invention is the homologue of δ subunit.Therefore the cDNA of the HeIF-2Bd that is separated to of the present invention can be used to detect the protein translation that the inactivation with eIF-2B homologue of the present invention causes specifically and regulate and control not normal.In addition, HeIF-2Bd of the present invention is for protein translation and regulate and control not normal treatment a kind of approach is provided.

Embodiment 1

The clone of the cDNA of HeIF-2Bd and order-checking

1.PCR amplification

With human brain λ gt11cDNA library (available from Clontech company) is template, with two pairs of oligonucleotide is primer---A1:5 '-GAGCCTAGGACTGAGGGCGATGG-3 ' (SEQ ID NO.1) and A2:5 '-TGCCTTGCAGCAGGTGATTCAGG-3 ' (SEQ ID NO.2) they are forward primer, oligonucleotide B1:5 '-CTGCAGGAATCAGCAGGTAGGAG-3 ' (SEQ ID NO.3) and B2:5 '-TCACTGGTCACTGCTCTTGACTCG-3 ' (SEQ ID NO.4) are reverse primer, carry out PCR.A1, the right PCR condition of B1 primer 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; A2, the right PCR condition of B2 primer be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 69 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, carrying out the purpose fragment that PCR obtains with A1, B1 is 1180bp, carrying out the purpose fragment that PCR obtains with A1, B1 is 920bp.

2.PCR the order-checking of product

With this two sections pcr amplification products and pGEM-T ^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 1582bp, detailed sequence is seen SEQ ID NO:9, and wherein open reading frame is positioned at 20-1582 position Nucleotide.

Derive the aminoacid sequence of HeIF-2Bd according to the full-length gene group sequence that obtains, totally 520 amino-acid residues, its aminoacid sequence sees SEQ ID NO:10 for details.

Embodiment 2

The HeIF-2Bd homology relatively

Full-length cDNA of the present invention carries out Basic BLAST homology relatively in proper order, the database that adopts is the GenBank+EMBL+DDBJ+PDB of nonredundancy, (database numbering and title: gb|M98036|MUSJGR1AX) the homology degree is the highest to found that δ subunit with the guanylic acid exchange factor of mouse, have four sections nucleotide sequences similar with corresponding MUSJGR1AX, their similarity is respectively: 81% (1-75 position), 81% (193-252 position), 87% (458-1586 position), 85% (263-467 position).

Full-length cDNA of the present invention is translated into the protein order in proper order, find that ORF is positioned at 20-1582 position Nucleotide, 520 the amino acid whose polypeptide of a segment length of encoding.Then the amino-acid sequence that obtains is carried out Basic BLAST homology relatively, the database that adopts is that the GenBank translation CDS+PDB+SwissPort+ of nonredundancy upgrades SP+PIR, (database numbering and title: sp|P41111|E2BD_RABIT sp|Q61749|E2BD_MOUSE) the homology degree is the highest to found that δ subunit with the eIF-2B of rabbit, mouse, wherein similar to 19 amino-acid residues 100% of eIF-2Bd aminoterminal of rabbit, 94% is identical, 3/4ths orders of carboxyl terminal also have 91% similar, and 87% is identical.Therefore, this polypeptide is confirmed as the δ subunit of human translation initiation factor.

Know at present, in proteinic translation process, at first form a ternary complex of forming by initial tRNA, GTP, eIF-2, transfer to then on the 40S ribosomal subunit.Along with the formation of 80S initiation complex, GTP is hydrolyzed, thereby eIF-2 is discharged with the form of eIF-2-GDP binary complex.This binary complex is stable under physiological condition, and does not have the function activity.The new round circulation of translation initiation requires the participation of eIF-2B.Because the affinity of eIF-2 and GDP than with the high 100-400 of affinity of GTP doubly, so need eIF-2B catalysis and eIF-2 bonded GDP and GTP to exchange, regenerate eIF-2-GTP, to participate in translation initiation (J.Biol.Chem.268 (1993), the 7603-7606 of a new round; Annu.Rev.Biochem.60 (1991), 717-755).Because it is initial that eIF-2B participates in protein translation, and the regulation and control translation process, so have critical function.This proteic not normal meeting cause protein translation and regulate and control undesired.

EIF-2B is made up of five subunit α, β, γ, δ, ε, and HeIF-2Bd of the present invention is the homologue of δ subunit.Therefore the HeIF-2Bd that is separated to of the present invention participates in protein translation and regulation and control, and for protein translation and regulate and control not normal treatment a kind of approach is provided.

In addition, in eukaryotic cell, but the phosphorylation of known protein matter kinases catalysis initiation factor eIF-2, when lacking protoheme in the cell, protein kinase is activated, and makes the α subunit generation phosphorylation of eIF-2.Phosphorylation eIF-2-GDP and eIF-2B have great avidity, so in case in conjunction with just being difficult to dissociate later on, thereby make eIF-2 can not drop into new initiating process again.This phenomenon has the important physical meaning, because when content of hemachrome reduces, the synthetic meeting of oxyphorase stops, thereby has prevented the oxyphorase (this oxyphorase is volatility very) of synthetic no protoheme.EIF-2B combines with the eIF-2-GDP of phosphorylation, thereby realizes the initiation of eIF-2 is suppressed.The eIF-2B that contains HeIF-2Bd subunit of the present invention also has similar function, and it is working aspect the oxyphorase that prevents synthetic no protoheme.

Embodiment 3

The expression of HeIF-2Bd in intestinal bacteria

In this embodiment, two fragments that will obtain in embodiment 1 are cut with the EcoRI enzyme respectively, mix and add ligase enzyme and connect, and carry out electrophoresis after connection is finished, and identify and also reclaim the correct full length fragment that connects.Use PCR Oligonucleolide primers to increase the dna sequence dna of coding HeIF-2Bd corresponding to 5 of this dna sequence dna ' and 3 ' end, with or HeIF-2Bd as inserting fragment.

PCR reaction 5 ' Oligonucleolide primers sequence is:

5′-AGGAGTCGAC?ATGGCTGCTG?TGGCCGTGG-3′(SEQ?ID?NO.5)

This primer contains the restriction enzyme site of the restricted restriction enzyme of SalI, is 19 Nucleotide of the HeIF-2Bd encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-CTCTAAGCTT?TCACTGGTCA?CTGCTCTTG-3’(SEQ?ID?NO.6)

This primer contain the restriction enzyme site of the restricted restriction enzyme of HindIII, translation termination and the HeIF-2Bd encoding sequence.

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 extracting plasmid is cut with the BamHI enzyme and to be identified and insert clip size and direction that the cDNA fragment of sequence verification HeIF-2Bd 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 HeIF-2Bd from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out HeIF-2Bd 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.In protein binding to the second behind the purifying 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 to containing PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

Embodiment 4

The expression of HeIF-2Bd in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, two fragments that will obtain in embodiment 1 are cut with the EcoRI enzyme respectively, mix and add ligase enzyme and connect, and walk electrophoresis after connection is finished, and identify and also reclaim the correct full length fragment that connects.The dna sequence dna of coding HEIF-2BD uses the PCR Oligonucleolide primers corresponding to 5 of this dna sequence dna ' and 3 ' end to increase, and obtains HEIF-2BDC cDNA as inserting fragment.

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

5′-TAGGAAGCTT?ATGGCTGCTG?TGGCCGTGG-3′(SEQ?ID?NO.7)

This primer contains the restriction enzyme site of the restricted restriction enzyme of HindIII, is 19 Nucleotide of the HEIF-2BD encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-GGAAGATATC?TCACTGGTCA?CTGCTCTTG-3’(SEQ?ID?NO.8)

This primer contains the encoding sequence of the restriction enzyme site of the restricted restriction enzyme of EcoRV, translation termination and HEIF-2BD.

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 (Invitrogen company), 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 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 cuts evaluation with the ApaI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification HeIF-2Bd has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to carry out with Lipofectin test kit (GiBcolife), and transfection is after 48 hours, and 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 50mM TrisHCl (pH8.0) solution that contains 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 57kD.

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:10 with ordinary method.

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 or 4 is used for immune animal to produce antibody, specific 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 HeIF-2Bd gene translation product with it.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Information (i) sequence signature of sequence table (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:GAGCCTAGGA CTGAGGGCGA TGG 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: information (i) sequence signature of SEQ ID NO:2TGCCTTGCAG CAGGTGATTC AGG 23 (2) SEQ ID NO:3

(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:3CTGCAGGAAT CAGCAGGTAG GAG 23 (2) SEQ ID NO:4

(A) length: 24 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:4TCACTGGTCA CTGCTCTTGA CTCG 24 (2) SEQ ID NO:5

(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:5AGGAGTCGAC ATGGCTGCTG TGGCCGTGG 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:6CTCTAAGCTT TCACTGGTCA CTGCTCTTG 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:7TAGGAAGCTT ATGGCTGCTG TGGCCGTGG 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: the information of SEQ ID NO:8GGAAGATATC TCACTGGTCA CTGCTCTTG 29 (2) SEQ ID NO:9: (i) sequence signature:

(A) length: 1582bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：9 1 GAGCCTAGGA CTGAGGGCGA TGGCTGCTGT GGCCGTGGCT GTTCGCGAGG ACTCGGGATC 61 CGGGATGAAG GCGGATTCCC CCTGGGCCTG GGTGGGGAGG GAAATGACCA AAGAAGAAAA121 GCTGCAGCTT CGGAAGGAAA AGAAACAGCA GAAGAAGAAA CGGAAGGAAG AAAAGGGGGC181 AGAACCAGAG ACTGGCTCTG CTGTATCTGC AGCCCAATGT CAAGGCCCAA CCAGAGAACT241 GCCAGAATCG GGCATTCAGT TGGGCACTCC TCGGGAGAAA GTTCCAGCTG GTCGGAGTAA301 GGCCGAACTT CGGGCTGAGC GTCGAGCCAA GCAGGAGGCC GAGCGGGCCC TGAAACAGGC361 AAGAAAAGGG GAACAAGGAG GACCACCTCC TAAGGCCAGC CCCAGCACAG CTGAAGAAAC421 CCCCTCAGGA GTGAAGCGTC TCCCTGAGTA CCCTCAGGTT GATGACCTAC TTCTGAGAAG481 GCTTGTTAAA AAACCAGAGC GTCAACAGGT TCCTACACGA AAGGATTATG GATCCAAAGT541 CAGTCTCTTC TCTCACCTAC CCCAGTACAG CAGACAAAAC TCTCTGACCC AGTTTATGAG601 CATCCCATCC TCTGTGATCC ACCCAGCCAT GGTGCGACTC GGCCTGCAGT ACTCCCAGGG661 CCTGGTCAGT GGCTCCAATG CCCGGTGTAT TGCCCTGCTT CGTGCCTTGC AGCAGGTGAT721 TCAGGATTAC ACAACACCGC CTAATGAAGA ACTCTCCAGG GATCTAGTGA ATAAACTAAA781 ACCCTACATG AGCTTCCTGA CTCAGTGCCG TCCCCTGTCA GCGAGCATGC ACAACGCCAT841 CAAGTTCCTT AACAAGGAAA TCACCAGTGT GGGCAGTTCC AAGCGGGAAG AGGAGGCCAA901 GTCAGAACTT CGAGCAGCCA TTGATCGGTA TGTGCAAGAG AAGATTGTGC TAGCAGCTCA961 GGCAATTTCA CGCTTTGCTT ACCAGAAGAT CAGTAATGGA GATGTGATCC TGGTATATGG1021 ATGCTCATCT CTGGTATCAC GAATTCTTCA GGAGGCTTGG ACAGAGGGCC GGCGGTTTCG1081 GGTGGTAGTG GTGGACAGCC GGCCATGGCT GGAAGGAAGG CACACACTAC GTTCTCTAGT1141 CCATGCTGGT GTCCCAGCCT CCTACCTGCT GATTCCTGCA GCCTCCTATG TGCTCCCAGA1201 GGTTTCCAAG GTGCTATTGG GAGCTCATGC ACTCTTGGCC AACGGGTCTG TGATGTCACG1261 GGTAGGGACA GCACAGTTAG CCCTGGTGGC TCGAGCCCAT AATGTACCAG TGCTGGTTTG1321 CTGTGAAACA TACAAGTTCT GTGAGCGTGT GCAGACTGAT GCCTTTGTCT CTAATGAGCT1381 AGATGACCCT GATGATCTGC AATGTAAGCG GGGAGAACAT GTTGCGCTGG CTAACTGGCA1441 GAACCACGCA TCCCTACGGT TGTTGAATCT AGTCTATGAT GTGACTCCCC CAGAGCTTGT1501 GGATCTGGTG ATCACGGAGC TGGGGATGAT CCCTTGCAGT TCTGTACCTG TTGTTCTACG1561 AGTCAAGAGC AGTGACCAGT GA ( 2 ) SEQ ID NO：10： ( i ) ：

(A) length: 520 amino acid

(B) type: amino acid

( C ) ： ( ii ) ： ( xi ) ：SEQ ID NO：10 1 Met Ala Ala Val Ala Val Ala Val Arg Glu Asp Ser Gly Ser Gly 16 Met Lys Ala Asp Ser Pro Trp Ala Trp Val Gly Arg Glu Met Thr 31 Lys Glu Glu Lys Leu Gln Leu Arg Lys Glu Lys Lys Gln Gln Lys 46 Lys Lys Arg Lys Glu Glu Lys Gly Ala Glu Pro Glu Thr Gly Ser 61 Ala Val Ser Ala Ala Gln Cys Gln Gly Pro Thr Arg Glu Leu Pro 76 Glu Ser Gly Ile Gln Leu Gly Thr Pro Arg Glu Lys Val Pro Ala 91 Gly Arg Ser Lys Ala Glu Leu Arg Ala Glu Arg Arg Ala Lys Gln106 Glu Ala Glu Arg Ala Leu Lys Gln Ala Arg Lys Gly Glu Gln Gly121 Gly Pro Pro Pro Lys Ala Ser Pro Ser Thr Ala Glu Glu Thr Pro136 Ser Gly Val Lys Arg Leu Pro Glu Tyr Pro Gln Val Asp Asp Leu151 Leu Leu Arg Arg Leu Val Lys Lys Pro Glu Arg Gln Gln Val Pro166 Thr Arg Lys Asp Tyr Gly Ser Lys Val Ser Leu Phe Ser His Leu181 Pro Gln Tyr Ser Arg Gln Asn Ser Leu Thr Gln Phe Met Ser Ile196 Pro Ser Ser Val Ile His Pro Ala Met Val Arg Leu Gly Leu Gln211 Tyr Ser Gln Gly Leu Val Ser Gly Ser Asn Ala Arg Cys Ile Ala226 Leu Leu Arg Ala Leu Gln Gln Val Ile Gln Asp Tyr Thr Thr Pro241 Pro Asn Glu Glu Leu Ser Arg Asp Leu Val Asn Lys Leu Lys Pro256 Tyr Met Ser Phe Leu Thr Gln Cys Arg Pro Leu Ser Ala Ser Met271 His Asn Ala Ile Lys Phe Leu Asn Lys Glu Ile Thr Ser Val Gly286 Ser Ser Lys Arg Glu Glu Glu Ala Lys Ser Glu Leu Arg Ala Ala301 Ile Asp Arg Tyr Val Gln Glu Lys Ile Val Leu Ala Ala Gln Ala316 Ile Ser Arg Phe Ala Tyr Gln Lys Ile Ser Asn Gly Asp Val Ile331 Leu Val Tyr Gly Cys Ser Ser Leu Val Ser Arg Ile Leu Gln Glu346 Ala Trp Thr Glu Gly Arg Arg Phe Arg Val Val Val Val Asp Ser361 Arg Pro Trp Leu Glu Gly Arg His Thr Leu Arg Ser Leu Val His376 Ala Gly Val Pro Ala Ser Tyr Leu Leu Ile Pro Ala Ala Ser Tyr391 Val Leu Pro Glu Val Ser Lys Val Leu Leu Gly Ala His Ala Leu406 Leu Ala Asn Gly Ser Val Met Ser Arg Val Gly Thr Ala Gln Leu421 Ala Leu Val Ala Arg Ala His Asn Val Pro Val Leu Val Cys Cys436 Glu Thr Tyr Lys Phe Cys Glu Arg Val Gln Thr Asp Ala Phe Val451 Ser Asn Glu Leu Asp Asp Pro Asp Asp Leu Gln Cys Lys Arg Gly466 Glu His Val Ala Leu Ala Asn Trp Gln Asn His Ala Ser Leu Arg481 Leu Leu Asn Leu Val Tyr Asp Val Thr Pro Pro Glu Leu Val Asp496 Leu Val Ile Thr Glu Leu Gly Met Ile Pro Cys Ser Ser Val Pro511 Val Val Leu Arg Val Lys Ser Ser Asp Gln

Claims (14)

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

Show at least 70% homology from the nucleotides sequence of Nucleotide 20-1582 position among described nucleotide sequence and the SEQ ID NO.9; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.9 in from the nucleotide sequence hybridization of Nucleotide 20-1582 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.10.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has the sequence of Nucleotide 20-1582 position among the SEQ ID NO.9.

4. isolating HeIF-2Bd protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.10 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.10 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 HeIF-2Bd protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of HeIF-2Bd protein-active operationally is connected in expression regulation sequence, form the HeIF-2Bd protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 20-1582 position among described nucleotide sequence and the SEQ ID NO.9;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of HeIF-2Bd;

(c) be fit to express under the condition of HeIF-2Bd protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with HeIF-2Bd protein-active.

11. method as claimed in claim 10 is characterized in that, this nucleotides sequence is classified as among the SEQ ID NO.9 from Nucleotide 20-1582 position.

12. energy and the described HeIF-2Bd protein polypeptide of claim 4 specificity bonded antibody.

13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

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