CN1250096A - New human protein phosphatase subunit and its coding series and preparation - Google Patents

Abstract

The present invention provides one new kind of human gene nucleotide sequence. Specifically, the present invention provides the cDNA sequence of human IMYPNO1, and the protein is the homologue of the B-gama subunit protein in rabbit protein phosphatase 2A1 (PP2A1). The present invention also provides the polypeptide encoded by the nucleotide sequence and the application and production method of this polynucleotides and polypeptides.

Description

New human protein phosphatase subunit, its encoding sequence 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 the cDNA sequence of people IMYPNO1, this albumen is the homologue of the B-gama protein subunit of rabbit protein phosphatase 2A1 (protein phosphatase 2A1 is called for short " PP2A1 ").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.

Extensively exist complicated signal pipeline in the biology.Each cell from extraneous received signal, passes through the bio-chemical pathway of a series of complexity by surperficial acceptor then, and signal is delivered in the born of the same parents from plasma membrane.This series of biologic chemistry route also is used to help to coordinate the cell each several part and carries out various special and complicated function (Physiol Rev.1993 Oct simultaneously; 73 (4): 673-699.Review.).And proteinic phosphorylation and dephosphorylation are exactly one of most important bio-chemical pathway (Bioorg Med Chem.1997 Sep wherein; 5 (9): 1751-1773.Review.).

Proteinic phosphorylation and dephosphorylation are catalytic by protein kinase and protein phosphatase institute respectively, wherein protein kinase is a very huge family, comprise proteinic serine/threonine kinase generally, with proteinic Tyrosylprotein kinase two big classes, quantity is very surprising, has only just found not following 1000 kinds more than in eukaryote.The classification and the protein kinase of protein phosphatase are quite similar, also corresponding proteinic serine/threonine phosphoesterase and the proteinic tyrosine-phosphatase of being divided into, but quantity is wanted much less than protein kinase, amounts in eucaryon and the prokaryotic organism and has only 15 kinds of (Physiol Rev.1993 Oct; 73 (4): 673-699.Review.).Although quantity is great disparity so, yet proteinic phosphorylation balance is kept in the work that they both can coordinate jointly.

Proteinic serine/threonine phosphoesterase family is an important protein phosphoesterase family, and it is responsible for the protein thread propylhomoserin of catalysis hexamethyl phosphoric acidization and the dephosphorylation of Threonine.Traditionally, the member of this family can be according to its specificity to substrate and inhibitor, and modulated subfamily (the Eur J Biochem.1995 Jun 1 that is divided into PP1, PP2A, three structural similitudies of PP2B with metal ion is needed different; 230 (2): 766-772.).Also once relevant for the report of PP2C, but since comparatively special on its structure, often (Physiol Rev.1993 Oct is discussed separately; 73 (4): 673-699.Review.).

Protein phosphatase 2A (protein phosphatase 2A is called for short " PP2A ") is the protein serine/threonine phosphoesterase of a quasi-representative, and it extensively is present in the various biologies; In human body, it extensively is present in various organs and tissue, is the ubiquitous protein of a class.The structure of PP2A roughly is made of three subunits: the C2 subunit is a catalytic subunit, and the A subunit is conservative regulation and control subunit, and the B subunit is a regulation and control subunit not too cautious.A-C2 often is called as the core subunit, and it changes very little in various PP2A, and is then comparatively various as the B subunit of regulation and control subunit, can be divided three classes B, B ' and B at least ".Traditionally A-C2-B is called PP2A1, A-C2-B ' is called PP2A0.Studies show that in recent years can further be subdivided into three types of B-alpha, B-beta and B-gama (J Biol Chem.1996 Feb 2 again as the category-B subunit of forming PP2A1; 271 (5): 2578-2588.).

1991, people such as the scientist Mayer RE of Switzerland cloned and have identified the B-alpha of two kinds of human PP2A1 and the gene of B-beta subunit (GENEBANK ACCESSION No.gb|M64929 ﹠amp; Gb|M64930) (Biochemistry 1991 Apr 16; 30 (15): 3589-3597).1994, people such as Zolnierowicz S cloned and have identified gene (GENEBANK ACCESSION No.gb|U09355) (the Biochemistry.1994 Oct 4 of the B-gama subunit of a kind of rabbit PP2A1; 33 (39): 11858-11867.).Further studies show that, also exist higher homology between them.

Yet, up to the present, also do not have the report of the B-gama subunit gene of human PP2A1 among relevant the present invention.

An object of the present invention is to provide a kind of new polynucleotide sequence, this polynucleotide sequence coding mankind, with the B-gama subunit homologous albumen of rabbit PP2A1, of the present inventionly be named as IMYPNO1 with B-gama subunit homologous gene rabbit PP2A1.

Another object of the present invention provides a kind of new albumen, and this albumen is named as IMYPNO1 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 IMYPNO1.

The present invention also provides this people IMYPNO1 encoding sequence and proteic application.

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 IMYPNO1 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 91-1377 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 91-1377 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 91-1377 position.

In another aspect of this invention, provide a kind of isolating IMYPNO1 protein polypeptide, it comprises: have 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, the method that provides a kind of generation to have the polypeptide of IMYPNO1 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of IMYPNO1 protein-active operationally is connected in expression regulation sequence, form the IMYPNO1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 91-1377 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 IMYPNO1;

(c) be fit to express under the condition of IMYPNO1 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with IMYPNO1 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1492 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 91-1377 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 " IMYPNO1 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with IMYPNO1 protein-active is as 91-1377 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 91-1377 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 91-1377 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 SEQ ID NO.3 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 91-1377 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 91-1377 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.3 with people IMYPNO1 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 " IMYPNO1 protein polypeptide " refers to have the SEQ IDNO.4 polypeptide of sequence of IMYPNO1 protein-active.This term also comprises having and the variant form IMYPNO1 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 IMYPNO1 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 IMYPNO1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-IMYPNO1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises IMYPNO1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of IMYPNO1 polypeptide.Usually, this fragment have the IMYPNO1 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 IMYPNO1 albumen or polypeptide.The difference of these analogues and natural IMYPNO1 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 IMYPNO1 polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of IMYPNO1 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 IMYPNO1 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 IMYPNO1 that encodes.

The present invention also comprises the method that detects the IMYPNO1 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 IMYPNO1 polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.

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 IMYPNO1 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 IMYPNO1 gene product or fragment.Preferably, refer to that those can combine with IMYPNO1 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 IMYPNO1, comprise that also those do not influence the antibody of IMYPNO1 protein function.The present invention also comprise those can with modify or without the IMYPNO1 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 IMYPNO1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing IMYPNO1 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 IMYPNO1 function and the antibody that does not influence the IMYPNO1 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of IMYPNO1 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 IMYPNO1 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 IMYPNO1 nucleotide sequence full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally 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.Certainly, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

In an example of the present invention, the cDNA nucleotide sequence of people IMYPNO1 is so to obtain, with human brain λ gt11cDNA library (available from Clontech company) is template, synthetic forward primer A:5 '-CTGCGGGACCACAGCTATGTGAC-3 ' and reverse primer B:5 '-CAGCTGTCCTCATCAGTGCTGTG-3 ', carry out PCR, obtain the purpose fragment of 1492bp.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.

PP2A is considered to and the cell growth, and cell division cycle is relevant with the transmission of cell information, and B-gama is as the important regulating and controlling subunit of PP2A, and it necessarily plays an important role to the normal growth of biomass cells.In the present invention, the B-gama gene height homology of the PP2A1 of IMYPNO1 gene and rabbit, simultaneously also very high with the homology of mouse B-gama gene, thereby this shows that it is the homologous gene of B-gama in the people, and with rabbit B-gama, mouse B-gama has similar biological function, these functions comprise participate in RNA transcribe aspects such as processing, information acceptor and ionic channel effect, the transmission of born of the same parents' internal information, regulating cell growth, cell cycle and cell fission.

In the accompanying drawings,

Fig. 1 is the nucleotide sequence homology comparison diagram of the B-gama subunit gene of people IMYPNO1 of the present invention and rabbit PP2A1.Wherein, identical Nucleotide marks with " | ".

Fig. 2 is the nucleotide sequence homology comparison diagram of the B-gama subunit gene of people IMYPNO1 of the present invention and P of Rats P2A1.Wherein, identical Nucleotide marks with " | ".

Fig. 3 is the homology comparison diagram of aminoacid sequence of the B-gama subunit of people IMYPNO1 of the present invention and rabbit PP2A1.Wherein, identical amino acid marks with monocase between two sequences.

Fig. 4 is the homology comparison diagram of aminoacid sequence of the B-gama subunit of people IMYPNO1 of the present invention and P of Rats P2A1.Wherein, identical amino acid marks with monocase between two sequences.

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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone and the mensuration of the cDNA sequence of IMYPNO1

1. primer amplification

With human brain λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A:5 '-CTGCGGGACCACAGCTATGTGAC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B:5 '-CAGCTGTCCTCATCAGTGCTGT-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.A, the right PCR condition of B primer be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 66 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR fragment that electrophoresis detection obtains is the purpose sheet segment length 1492bp of primer to amplifying with A, B.

2.PCR the order-checking of product

This section pcr amplification product is connected with pGEM-T carrier (Promega), transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque with double-stranded nested type disappearance test kit (Pharmacia) to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then.Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 1492bp, detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 91-1377 position Nucleotide.

Derive the aminoacid sequence of IMYPNO1 according to the full length cDNA sequence that obtains, totally 428 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.

Embodiment 2

Homology comparison and structural research

Full length cDNA sequence and proteins encoded thereof with IMYPNO1 carry out nucleic acid and albumen homology retrieval with BLAST software in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database.Found that, it and delivered and confirmed as between the gene of B-gama subunit of PP2A1 and had height homologous sequence, and the albumen that the present invention relates to has the aminoacid sequence of the B-gama subunit family high conservative of PP2A1.Further retrieval shows, there is high homology in it with the B-gama subunit gene (nucleic acid and albumen searching number are respectively gb|U09355 and sp|P50410) of rabbit (Oryctolagus cuniculus) PP2A1 that come forth on nucleic acid and protein level, 92% identity (identity) of wherein having an appointment between their nucleotide sequence (Fig. 1), and 93% identity, 94% similarity (Fig. 3) are arranged more between its protein sequence.In addition, also there is high homology in the present invention with the B-gama subunit gene (nucleic acid and albumen searching number are respectively dbj|D38261 and pir ‖ S65686) of the mouse PP2A1 that come forth on nucleic acid and protein level, 90% identity (Fig. 2) of wherein on average having an appointment between their nucleotide sequence, and 93% identity, 94% similar (Fig. 4) are arranged also between its protein sequence.Therefore this gene is confirmed to be the homologous gene of the B-gama subunit gene of rabbit and mouse PP2A1.

Again because the B-gama subunit gene of rabbit and mouse PP2A1 has been accredited as the B-gama family member, and particularly there are two groups of respectively total characteristic sequences of B-gama subunit gene family of the PP2A1 of 15 amino acid compositions in the conservative significant sequence of strictness that has B-gama family in the protein sequence of IMYPNO1 in the aminoacid sequence of IMYPNO1---

EFDYLKSLEIEEKIN; With

N(A/G)H(T/A)YHINSIS(L/I/V/M)NSD.

[annotating: " (L/I/V/M) " expression optional amino acid from these 4 amino acid in this sequence].

The sequence fragment that meets above-mentioned pattern in albumen of the present invention is: EFDYLKSLEIEEKIN (103-117 position among the SEQID NO.4), NGHTYHINSISVNSD (194-208 position among the SEQ ID NO.4).Therefore this has confirmed that further IMYPNO1 of the present invention is the B-gama subunit gene of a kind of human PP2A1 newly.According to the structures shape function, the intimate principle of structural similitude can be inferred the function of IMYPNO1 from these similar genes or proteic function.

Discover, the function of proteinic serine/threonine phosphoesterase family surprising extensively, it has almost contained transcribing and processing of metabolism adjusting, the effect that participates in information acceptor and ionic channel, RNA, and the growth of cell, division and differentiation (Biochemistry.1994 Oct 4; 33 (39): 11858-11867.).

Why proteinic serine/threonine phosphoesterase has so many function, is because it has participated in most important signal pathway in the organism, just proteinic reversible phosphorylation approach.In cell, exist the enzyme and the signaling molecule that can change its functional status in a large number by phosphorylation and dephosphorylation, so just provide a considerable amount of, forward as feedback inhibition and reverse regulatory site, it participates in the chemical fundamentals of various cell functions and Here it is.In fact, increasing data shows that PP2A has participated in potassium ion circulation equilibrated regulation and control (Adv Exp Med Biol.1996; 396:209-215.Review.); Participated in cell deformation (the Semin Cancer Biol.1995 Aug that causes by tumour virus and viral protein thereof; 6 (4): 229-237.Review.); PP2A also with Bloom Syndrom (BS), Alzheimer ' s desease relevant (the Indian JExp Biol.1996 Apr of clinical disease such as (AD); 34 (4): 298-301.Review., FASEB be Dec J.1995; 9 (15): 1570-1576.Review.).

In addition, particularly importantly PP2A has participated in control (the Physiol Rev.1993 Oct of cell growth and cell cycle; 73 (4): 673-699.Review.).For example found once that PP2A can regulate and control transition process (the Semin Cancer Biol.1995 Aug of Africa xenopus cell from the G2 phase to the M phase of eucaryon; 6 (4): 203-209.Review.); The PP2A that finds overexpression in yeast again can cause cell can't enter cell division phase, or postpones arrival (the Ciba Found Symp.1992 of division stage; 170:130-140.Review).Comparatively significantly example be a kind of maturation promoting factor of in frog's egg, finding (maturation-promoting factor, MPF), it is actually a kind of protein kinase (Physiol Rev.1993 Oct; 73 (4): 673-699.Review.), and, can infer to exist a kind of what is called " to delay maturation factor " that its essence is proteinic phosphoesterase, and has the splitted of delaying effect by proteic phosphoesterase and the protein kinase character of antagonism mutually.This shows, the decision that proteinic phosphoesterase is really direct or indirect the growth and the fragmentation condition of cell.And B-gama plays a part the whole phosphoesterase of guiding to finish required function (Adv Enzyme Regul.1994 as one of regulation and control subunit of PP2A; 34:199-224.Review.), also just should have the function of equal importance with the corresponding proteins phosphoesterase.

As previously mentioned, IMYPNO1 has the function identical or close with the B-gama family member, PP2A under promptly also guiding participates in various cell signal approach, and influence ionic and circulate, and may be relevant with treatment of diseases such as " Bloom syndromes ", " Alzheimer ' s disease ".In addition, IMYPNO1 also may participate in the regulation and control of cell growth and cell cycle, and becomes one of cell growth factor with critical function and cell division factor.

In addition, the present invention also has even more important meaning.Because at nature, the kind of protein kinase will be far longer than the kind of albumen esterase, and the work why they can be coordinated mutually, exist B-alpha like this because resemble the B subunit of PP2A to a great extent, B-beta, the multiple regulation and control subunit form of B-gama, and the differential expression of these three kinds of subunits can be regulated and control the different Substratspezifitaet of PP2A generation, make same PP2A produce different activity, so these three kinds of subunits are actually closely-related (Physiol Rev.1993 Oct; 73 (4): 673-699.Review.).The B-alpha of human PP2A1 and B-beta subunit were just cloned and had been identified as far back as 1991, but because the vacancy of B-gama makes the research of this respect to carry out.And IMYPNO1 of the present invention has just in time filled up this vacancy, for a brand-new situation is opened in the research work of human PP2A1.If consider PP2A and cell growth and splitted relation, we more can pass through therapy gene or medicine, the concentration ratio of three kinds of B subunits of adjusting PP2A is controlled the enzyme of PP2A and is lived, and then the growth and the division of control cell, make people might control the development of tumour and cancer, to reach the target of final treatment cancer.

People IMYPNO1 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 IMYPNO1 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end with the PP2A1 B-game subunit of the N of inventor IMYPNO1 end and rabbit or people's B-alpha subunit exchanges, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor IMYPNO1, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment 3

The expression of IMYPNO1 in intestinal bacteria

In this embodiment, use PCR Oligonucleolide primers to increase, obtain IMYPNO1 cDNA as inserting fragment corresponding to 5 ' and 3 ' end of the cDNA sequence of coding IMYPNO1.

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

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

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the IMYPNO1 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

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

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the IMYPNO1 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 BamHI 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 (Kanr).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification IMYPNO1 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 is inoculated in the large volume substratum culturing cell to 600 optical density(OD) (OD then ₆₀₀) be 0.4-0.6, add subsequently 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 IMYPNO1 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out IMYPNO1 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps from nickel-chelate column, isolate purifying protein.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 49kDa.

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 IMYPNO1 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, use PCR Oligonucleolide primers to increase, obtain IMYPNO1 cDNA as inserting fragment corresponding to 5 ' and 3 ' end of the cDNA sequence of coding IMYPNO1.

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

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

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the IMYPNO1 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-GTGAGAATTCCTAGTGCATGTCAGAGTTT-3’(SEQ?ID?NO.7)

This primer contains the part encoding sequence of the restriction enzyme site of EcoRI restriction enzyme, translation termination and IMYPNO1.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With BamHI 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, the cDNA fragment of sequence verification IMYPNO1 has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI 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 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 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mM TrisHCl (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 49kDa.

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, specific as follows.Recombinant protein 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 IMYPNO1 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.

Sequence table (1) general information: (i) applicant: Xinhuangpu-Fudan Gene Engineering Co., Ltd., Shanghai is denomination of invention (ii): new human protein phosphatase subunit, its encoding sequence and preparation method be the sequence number (iii): information (i) sequence signature of 7 (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.1CTGCGGGACC ACAGCTATGT GAC 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.2CAGCTGTCCT CATCAGTGCT GTG 23 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 1492bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3 1 CTGCGGGACC ACAGCTATGT GACTGAAGGT AACGTACGTG TTGTCTGAGA CCCCTCCGGC 61 CGGCCGCGGC GTGGGGATGC CTCGCACCGA ATGCCCTCCG AAGCTGACAT CATCTCTACC121 GTTGAGTTCA ACCACACGGG AGAGCTGCTG GCCACAGGTG ACAAGGGCGG CCGGGTCGTC181 ATCTTCCAGC GGGAACCAGA GAGTAAAAAT GCGCCCCACA GCCAGGGCGA CTACGACGTG241 TACAGCACTT TCCAGAGCCA CGAGCCGGAG TTTGACTATC TCAAGAGCCT GGAGATAGAG301 GAGAAGATCA ACAAGATCAA GTGGCTCCCA CAGCAGAACG CCGCCCACTC ACTCCTGTCC361 ACCAACGATA AAACTATCAA ATTATGGAAG ATTACCGAAC GAGATAAAAG GCCCGAAGGA421 TACAACCTGA AGGATGAAGA GGGGAAACTT AAGGACCTGT CCACGGTGAC GTCACTGCAG481 GTGCCAGTGC TGAAGCCCAT GGATCTGATG GTGGAGGTGA GCCCTCGGAG GATCTTTGCC541 AATGGCCACA CCTACCACAT CAACTCCATC TCCGTCAACA GTGACTGCGA GACCTACATG601 TCGGCGGATG ACCTGCGCAT CAACCTCTGG CACCTGGCCA TCACCGACAG GAGCTTCAAC661 ATCGTGGACA TCAAGCCGGC CAACATGGAG GACCTTACGG AGGTGATCAC AGCATCTGAG721 TTCCATCCGC ACCACTGCAA CCTCTTCGTC TACAGCAGCA GCAAGGGCTC CCTGCGGCTC781 TGCGACATGC CGGCAGCTGC CCTGTGTGAC AAGCATTCCA AGCTCTTTGA AGAGCCTGAG841 GACCCCAGTA ACCGCTCATT CTTCTCGGAA ATCATCTCCT CCGTGTCCGA CGTGAAGTTC901 AGCCACAGCG ACCGCTACAT GCTCACCCGG GACTACCTTA CAGTCAAGGT CTGGGACCTG961 AACATGGAGG CAAGACCCAT AGAGACCTAC CAGGTCCATG ACTACCTTCG GAGCAAGCTC1021 TGTTCCCTGT ACGAGAACGA CTGCATTTTC GACAAGTTTG AATGTGCCTG GAACGGGAGC1081 GACAGTTCAA TCATGACCGG GGCCTACAAC AACTTCTTCC GCATGTTCGA TCGGAACACC1141 AAGCGGGACG TGACCCTGGA GGCCTCGAGG GAAAGCAGCA AGCCCCGGGC TGTGCTCAAG1201 CCACGGCGCG TGTGCGTGGG GGGCAAGCGC CGGCGTGATG ACATCAGTGT GGACAGCTTG1261 GACTTCACCA AGAAGATCCT GCACACGGCC TGGCACCCGG CTGAGAACAT CATTGCCATC1321 GCCGCCACCA ACAACCTGTA CATCTTCCAG GACAAGGTAA ACTCTGACAT GCACTAGGTA1381 TGTGCAGTTC CCGGCCCCTG CCACCCAGCC TCATGCAAGT CATCCCCGAC ATGACCTTCA1441 CGACCGCAAT GCAAGGAGGG GAAGAAAGTC ACAGCACTGA TGAGGACAGC TG ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 428 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4 1 Met Pro Ser Glu Ala Asp Ile Ile Ser Thr Val Glu Phe Asn His 16 Thr Gly Glu Leu Leu Ala Thr Gly Asp Lys Gly Gly Arg Val Val 31 Ile Phe Gln Arg Glu Pro Glu Ser Lys Asn Ala Pro His Ser Gln 46 Gly Asp Tyr Asp Val Tyr Ser Thr Phe Gln Ser His Glu Pro Glu 61 Phe Asp Tyr Leu Lys Ser Leu Glu Ile Glu Glu Lys Ile Asn Lys 76 Ile Lys Trp Leu Pro Gln Gln Asn Ala Ala His Ser Leu Leu Ser 91 Thr Asn Asp Lys Thr Ile Lys Leu Trp Lys Ile Thr Glu Arg Asp106 Lys Arg Pro Glu Gly Tyr Asn Leu Lys Asp Glu Glu Gly Lys Leu121 Lys Asp Leu Ser Thr Val Thr Ser Leu Gln Val Pro Val Leu Lys136 Pro Met Asp Leu Met Val Glu Val Ser Pro Arg Arg Ile Phe Ala151 Asn Gly His Thr Tyr His Ile Asn Ser Ile Ser Val Asn Ser Asp166 Cys Glu Thr Tyr Met Ser Ala Asp Asp Leu Arg Ile Asn Leu Trp181 His Leu Ala Ile Thr Asp Arg Ser Phe Asn Ile Val Asp Ile Lys196 Pro Ala Asn Met Glu Asp Leu Thr Glu Val Ile Thr Ala Ser Glu211 Phe His Pro His His Cys Asn Leu Phe Val Tyr Ser Ser Ser Lys226 Gly Ser Leu Arg Leu Cys Asp Met Pro Ala Ala Ala Leu Cys Asp241 Lys His Ser Lys Leu Phe Glu Glu Pro Glu Asp Pro Ser Asn Arg256 Ser Phe Phe Ser Glu Ile Ile Ser Ser Val Ser Asp Val Lys Phe271 Ser His Ser Asp Arg Tyr Met Leu Thr Arg Asp Tyr Leu Thr Val286 Lys Val Trp Asp Leu Asn Met Glu Ala Arg Pro Ile Glu Thr Tyr301 Gln Val His Asp Tyr Leu Arg Ser Lys Leu Cys Ser Leu Tyr Glu316 Asn Asp Cys Ile Phe Asp Lys Phe Glu Cys Ala Trp Asn Gly Ser331 Asp Ser Ser Ile Met Thr Gly Ala Tyr Asn Asn Phe Phe Arg Met346 Phe Asp Arg Asn Thr Lys Arg Asp Val Thr Leu Glu Ala Ser Arg361 Glu Ser Ser Lys Pro Arg Ala Val Leu Lys Pro Arg Arg Val Cys376 Val Gly Gly Lys Arg Arg Arg Asp Asp Ile Ser Val Asp Ser Leu391 Asp Phe Thr Lys Lys Ile Leu His Thr Ala Trp His Pro Ala Glu406 Asn Ile Ile Ala Ile Ala Ala Thr Asn Asn Leu Tyr Ile Phe Gln421 Asp Lys Val ASn Ser Asp Met His ( 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.5TAGGGGATCC ATGCCCTCCG AAGCTGACA 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.6GTGAAAGCTT CTAGTGCATG TCAGAGTTT 29 (2) SEQ ID NO.7

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.7GTGAGAATTC CTAGTGCATG TCAGAGTTT 29

Claims (14)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people IMYPNO1 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 91-1377 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 91-1377 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 91-1377 position.

4. isolating IMYPNO1 protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

6. a carrier is characterized in that, it contains the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6.

8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

10. a generation has the method for the polypeptide of IMYPNO1 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of IMYPNO1 protein-active operationally is connected in expression regulation sequence, form the IMYPNO1 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 91-1377 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 IMYPNO1;

(c) be fit to express under the condition of IMYPNO1 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with IMYPNO1 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 91-1377 position among the SEQ ID NO.3.

12. energy and the described IMYPNO1 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.

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