CN1249341A - Human protein kinase C arrestin coding sequence, its encoded polypeptide and its preparing process - Google Patents

Abstract

The present inventin discloses the coding sequence of a new II-type human protein kinase C inhibitor PKCI-2. The protein coded by said sequence is a new member in HIT family present naturally in human body and is a homolog of human hPKCI. The present invention also relates to the polypeptide coded by said nucleotide sequence, the application of said polynucleotide and polypeptide, and their preparing process.

Description

New human protein kinase C arrestin encoding sequence, 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 the encoding sequence of a kind of new II type human protein kinase C arrestin people PKCI-2, the albumen of this sequence encoding is a newcomer of naturally occurring HIT family in the human body, is the homologue of people hPKCI.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.

Protein kinase C (protein kinase C is called for short " PKC ") is the serine/threonine kinases that gang plays an important role in the signal transduction pathway in cell.After the extracellular signaling molecule acts on tyrosine kinase receptor or G protein coupled receptor on the cytolemma, activated intracytoplasmic Phospholipid hydrolase, impel the PIP on the plasma membrane ₂(4,5-bisphosphate phosphatidylinositols) are hydrolyzed to IP ₃(1,4, the 5-InsP3) and DAG (diacylglycerol).IP ₃Mobilize the endogenous calcium of cell to cytosol, make intracellular calcium concentration rise the DAG activated protein kinase C.Activated protein kinase C passes through the phosphorylation of serine/threonine residue on the target protein is changed the biological activity of target protein, and then causes cell response.Intermediate steps between PKC activation and the cell response it be unclear that.Existing evidence shows, PKC can improve ras protein-active (Berry, Eur.J.Biochem (1990) 189,205) by suppressing GAP (GTPase activatingprotein).Thereby the PKC approach also can activate another serine/threonine kinases Raf and then influence map kinase in the protein kinase cascade reaction, the active activation of RSK target gene (J.Biol.Chem. (1990) 265 for Siegel, J.N., 18472).PKC also can be by influencing transcription factor such as c-Jun, the activity of NF-kB promote gene transcription (Boyle W.J., Cell (1991) 64,573; Silvia, S, Pharmac.Ther. (1991) 51, and 71).

Protein kinase C arrestin (protein kinase C inhibitor is called for short PKCI) belongs to HIT (histidine triad) protein family.The member of this family have the conservative motif (his-x-his-x-his) that constitutes by three Histidines (Seraphin B., DNA Sequence (1992) 3,177; Robinson K, Biochem.J. (1994) 662).According to aminoacid sequence relatively, this protein family can be divided into two subtribes again.One of them is comparatively conservative at the C end, and the amino-acid sequence identity between its Mammals homologous protein is greater than 94%.The member of this subtribe that has been found that comprises people (Homo sapien) hPKCI (Brzoska PM, 1996, accession number U51004), ox (Bos taurus) PKCI-1 (Pearson JD, 1990, accession number U9405), mouse (Mus musculus) PKCI-1 (Kelein MG does not deliver sequence, accession number U60001), corn (Zea mays) ZBP14 (Simpson GG, 1994 accession number Z29643) etc.Another subtribe is representative (Ohta M with people FHIT, 1996, accession number U46922), comprise in addition fission yeast (Schizosaccaromyces pombe) dinucleotides 5 ', 5 -P1, P4 four phosphoric acid (Ap4A) asymmetric hydrolysis enzyme (Huang Y.Biochem.J. (1995) 312,925) etc.The amino acid sequence identity of this subtribe inside is about 50%, and the identity between two subtribes has only about 20%.Two subtribes have about 100 amino acid whose core areas, and bigger variation (Lima CD, Proc.Natl.Acad.Sci.USA (1996) 93,5357) is then arranged in the zone of holding near C end and N.

The initial member ox PKCI-1 of first subtribe is isolating from the ox brain as a heat-staple calcium binding protein, there is stronger PKC to suppress active external, be considered to most possible body interior PKC supressor (Mcdonald JR, Biochem.J (1985) 232,559-567; Mcdonald JR, Biochem.J (1987) 242,659-705).Rane etc. import chicken embryo Sensory neurone with this albumen, find that intracellular calcium current is blocked, for ox PKCI-1 PKC restraining effect in vivo provides evidence (Rane SG, Neuron (1989) 3 (2), 239).Yet one to endogenous thermally-stabilised and thermally labile albumen in the research of the effect relative size that total PKC is active in suppressing, Fraser infers that ox PKCI-1 may not be the PKC physiological regulation factor (Fraser ED in vivo, FEBS Lett (1991) 294 (3), and 285).Simpson etc. have separated a cDNA (Simpson CG with ox PKCI homologous gene from corn in 1994, Biochimica et Biophysica Acta 1222 (1994) 306), functional study to its expressed proteins ZBP14 shows, this albumen has only very weak PKC to suppress activity and its restraining effect and another PKC arrestin 14-3-3 and has synergistic effect (Robinson K, Biochem.J. (1995) 307 (ptl) 267).1996, utilize yeast two-hybrid system, Brzoska etc. have separated a people's PKCI-1 cDNA (Brzoska PM, Genomics (1996) 36,151), its expressed proteins can also be had an effect with the complementary albumin A TDC of AT telangiectasis ataxia (ataxia-telangictasia group D complementation) except can having an effect with the regulation domain of PKC.This prompting hPKCI-1 may play an important role in the signal conduction of ionization radiation induction.(Brzoska?PM，Proc.Natl.Acad.Sci.USA(1995)92，7824)。

The line-up of delegates of second subtribe of HIT family-human FHIT utilized the exon trapping method to separate (the Ohta M., Cell (1996) 84.587) that obtains in 1996 from the clay that covers human chromosome 3P14.2 disappearance district by Ohta etc.Because the disappearance of this gene in multiple JEG-3, infer its may be a tumor suppressor gene (SozziG., Cell (1996) 85,17; Virgilio L, Proc.Natl.Acad.Sci.USA (1996) 93, and 9770), yet the evidence of its tumor suppression function of definite proof also insufficient (Mao L., Journal of the National Institute (1998) 90 (6), 412).FHIT external have dinucleotides 5 ', 5 -P1, P4 triphosphoric acid asymmetric hydrolysis enzymic activity (Barnes LD, Biochemistry (1996) 35, and 11529), Lima etc. have also reported activity (the Lima CD of the ADP lytic enzyme of hPKI, Science (1997a) 278,286).Utilization crystalline diffraction and substrate analogy method, Lima etc. have carried out catalyst mechanism and substrate The Characteristic Study to FHIT and hPKCI, proved the HIT family member be nucleotide hydrolysis enzyme or nucleotidyl transferase (Lima CD, 1997a).However, HIT family substrate in vivo is still unknown, and its biological function is also treated further research.

But before the present invention, still nobody finds new people hPKC encoding sequence or polypeptide of the present invention.

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding HIT family, people hPKCI homologue name of the present invention is people PKCI-2

Another object of the present invention provides a kind of new HIT family member albumen, and this albumen is named as people PKCI-2.

Another purpose of invention provides a kind of recombinant technology that utilizes and produces the described new proteic method of people PKCI-2.

The invention still further relates to the application of this people PKCI-2 nucleotide sequence and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people PKCI-2 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 106-492 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 106-492 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence contains among the SEQ ID NO.3 nucleotide sequence from Nucleotide 106-492 position.

In another aspect of this invention, provide a kind of isolating people PKCI-2 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, provide a kind of generation to have the method for the polypeptide of people PKCI-2 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people PKCI-2 protein-active operationally is connected in expression regulation sequence, form people PKCI-2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 106-492 position among described nucleotide sequence and the SEQ ID NO.3;

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

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

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

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 532 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 106-492 position Nucleotide.

Polynucleotide of the present invention can be rna form and dna form, and DNA comprises cDNA, genomic dna and synthetic DNA, and DNA can be two strands or strand, if strand can be coding strand or noncoding strand

In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, term " people PKCI-2 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people PKCI-2 protein-active is as 106-492 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 106-492 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 106-492 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 106-492 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 106-492 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 PKCI-2 identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " people PKCI-2 protein polypeptide " refers to have the SEQ IDNO.4 polypeptide of sequence of people PKCI-2 protein-active.This term also comprises having and the variant form human efficient lymphocyte chemotactic factor identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people PKCI-2 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 people PKCI-2 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people PKCI-2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people PKCI-2 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of people PKCI-2 polypeptide.Usually, this fragment have people PKCI-2 peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of people PKCI-2 albumen or polypeptide.The difference of these analogues and natural human PKCI-2 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue can also comprise having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

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

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

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer.Probe molecule has 8-100 of people PKCI-2 polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people PKCI-2.

The present invention also comprises the method that detects people PKCI-2 nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of people PKCI-2 polypeptide.Primer length is generally 20-50 Nucleotide.

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

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

On the other hand, the present invention also comprises people PKCI-2DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people PKCI-2 gene product or fragment.Preferably, refer to that those can combine with people PKCI-2 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people PKCI-2, comprise that also those do not influence the antibody of people PKCI-2 protein function.The present invention also comprise those can with modify or without the people PKCI-2 gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people PKCI-2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human PKCI-2 or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people PKCI-2 function and the antibody that does not influence people PKCI-2 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people PKCI-2 gene product, obtains by immunological technique.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people PKCI-2 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

People PKCI-2 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 obtain the very long fragment of sequence.

In one embodiment of the invention, polynucleotide total length of the present invention is 532 Nucleotide, and its detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 106-492 position Nucleotide.These polynucleotide are so to obtain, with human brain λ gt11cDNA library (available from Clontech company) is template, with oligonucleotide A1:5 '-AAGATGGCGGCACGTGGTGCTG-3 ' (SEQ ID NO:1) is forward primer, A2:5 '-AAGCATCCAGAGTCTGGTTGTCC-3 ' (SEQ ID NO:2) is a reverse primer, carry out PCR, obtain the purpose fragment of 532bp.Identify the full length cDNA sequence that obtains SEQ ID NO:3 after checking order.

Polynucleotide provided by the invention and mouse PKCI height homology, and the albumen of polynucleotide encoding provided by the invention and people hPKCI albumen, ox PKCI-1 albumen, mouse PKCI albumen, the proteic sequence of corn ZBP14 also have higher homology degree respectively.Because the albumen of polynucleotide encoding of the present invention and above-mentioned its homologue have the conservative motif of his-x-his-x-his, therefore the coded albumen of polynucleotide people PKCI-2 provided by the invention can be included into the HIT protein family again.Given this, the invention provides the newcomer of a HIT protein family, and the new way to this family's research is provided.

People PKCI-2 albumen of the present invention has and the interactional activity of PKC regulation domain at the intravital homologous protein people of people hPKCI albumen, can infer that therefore people PKCI-2 albumen of the present invention may have the effect of PKC physiologically active in the control agent.In view of PKC is relating to the cell growth, play an important role in the cell signaling process of differentiation (Nishizuka Y, Science (1986) 233, and 305; Nishizuka Y, Nature (1988) 334,661), the undesired activation of PKC all has and relates to (Bradshaw D, Agents Actions (1993) 38,135) in multiple disease.The present invention carries out the research method the function of PKC in cell except providing a kind of, and new way that the caused disease of the undesired activation of multiple PKC is studied and the novel method that this type of disease is treated also are provided.

Especially, the nearest PKC that studies show that plays a significant role in the allos desensitization of opiate receptor, and receptor desensitization is considered to the tolerance of opioid drug and the concrete molecular mechanism of habituation.(Zhang L, J.Biol.Chem, (1996) 271 (19), 11449) are therefore of the present invention proteicly to be provided the tolerance of opioid drug and the new way of habituation Study on Molecular Mechanism with the interactional activity of PKC.

People hPKCI has the interactional activity with ATDC because the albumen of polynucleotide encoding provided by the invention and people PKCI albumen have 62% sequence identity, the invention provides the new way that the signal transduction pathway of inherited disease telangiectasis ataxia AT and associated ionization radiation induction is studied.

In the accompanying drawings,

Fig. 1 is the homology comparison diagram of the nucleotide sequence of people PKCI-2 of the present invention and people PKCI.Wherein, identical Nucleotide marks with " | ".

Fig. 2 is the homology comparison diagram of the nucleotide sequence of people PKCI-2 of the present invention and mouse PKCI.Wherein, identical Nucleotide marks with " | ".

Fig. 3 is the homology comparison diagram of people PKCI-2 of the present invention and the proteic aminoacid sequence of people PKCI.Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Fig. 4 is the homology comparison diagram of people PKCI-2 of the present invention and the proteic aminoacid sequence of mouse PKCI.Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

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

Embodiment 1

The clone and the mensuration of the cDNA sequence of people PKCI-2

1. primer amplification

With human brain λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-AAGATGGCGGCACGTGGTGCTG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide A2:5 '-AAGCATCCAGAGTCTGGTTGTCC-3 ' (SEQ ID NO:2) is a reverse primer, carries out PCR.The PCR condition of A1/A2 be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, A1/A2 is the purpose fragment of 532bp.

2.PCR the order-checking of product

With pcr amplification product A1/A2 and the pGEM-T that as above obtains ^TMCarrier (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 532bp, detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 106-492 position Nucleotide.

Derive the aminoacid sequence of people PKCI-2 according to the full length cDNA sequence that obtains, totally 128 amino-acid residues, its aminoacid sequence sees SEQ ID NO:4 for details

Embodiment 2

. people PKCI-2 homology is relatively

Utilize blastn and blastp program that Non-redundant GenBank+EMBL+DDBJ+PDB sequences and Non-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR database are carried out the homology retrieval.Find according to the homology result for retrieval, polynucleotide provided by the invention and people PKCI gene and mouse PKCI have the sequence identity (identity) (Fig. 1 and Fig. 2) about 50%, and the albumen of polynucleotide encoding provided by the invention and people hPKCI albumen, ox PKCI-1 albumen, mouse PKCI albumen, the proteic sequence identity of corn ZBP14 is 61% (Fig. 3) respectively, 62%, 60% (Fig. 4), 52%.In addition, reached about 75% (Fig. 3 and 4) with hPKCI albumen, the proteic similarity of mouse PKCI.

Again because the conservative motif of the total his-x-his-x-his of the albumen of polynucleotide encoding provided by the invention and above-mentioned its homologue.In albumen of the present invention corresponding to motif be His Leu His Ile His (112-116 position), therefore determined that the more coded albumen of polynucleotide people PKCI-2 provided by the invention belongs to the HIT protein family.Given this, the invention provides the newcomer of a HIT protein family, and the new way to this family's research is provided.

People PKCI-2 albumen of the present invention has and the interactional activity of PKC regulation domain at the intravital homologous protein people of people hPKCI albumen, can infer that therefore people PKCI-2 albumen of the present invention may have the effect of PKC physiologically active in the control agent.In view of PKC relating to cell growth, play an important role in the cell signaling process of differentiation (Nishizuka, 1985,1986), the undesired activation of PKC all has in multiple disease and relates to (Bradshaw, 1993).The present invention carries out also providing the research method new way that the caused disease of the undesired activation of multiple PKC is studied and the novel method that this type of disease is treated except providing a kind of to the function of PKC in cell.

Especially, the nearest PKC that studies show that plays a significant role in the allos desensitization of opiate receptor, and receptor desensitization is considered to the tolerance of opioid drug and the concrete molecular mechanism of habituation (Li Zhang, 1996).Therefore, proteic and the interactional activity of PKC of the present invention provide the tolerance of opioid drug and the new way of habituation Study on Molecular Mechanism.

Because PKCI-2 provided by the invention and people PKCI protein sequence height homology and people hPKCI have the interactional activity with ATDC, the invention provides the new way that the signal transduction pathway of inherited disease telangiectasis ataxia AT and associated ionization radiation induction is studied.

People PKCI-2 of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor PKCI-2 can also merge or exchange fragment with other members of this family, to produce new albumen, as the N end of inventor PKCI-2 and the N end of people PKCI are exchanged, to produce the albumen that new activity is higher or have new features.

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

In addition, inventor PKCI-2 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people PKCI-2 or the overexpression that suppresses people PKCI-2.People PKCI-2 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people PKCI-2 disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3

The expression of people PKCI-2 in intestinal bacteria

The cDNA sequence of coding people PKCI-2 is used corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, with the pcr amplification product among human brain λ gt11cDNA library (available from Clontech company) or the embodiment 1 is that template increases, with the synthetic fragment of inserting.

5 ' Oligonucleolide primers sequence is 5 '-CCTAGTCGACATGG GAATTGAAGT GGCCA-3 ' (SEQ ID NO.5).

This primer sequence contains the restriction enzyme site of SalI restriction enzyme, and what connect is 19 Nucleotide of the people PKCI-2 encoding sequence that begun by initiator codon;

3 ' end primer sequence is 5 '-ATCGAAGCTTTCAA CCTGGAGGCC ACTGG-3 ' (SEQ IDNO.6).

This primer sequence contains the restriction enzyme site of HindIII restriction enzyme, the encoding sequence of translation termination and people PKCI-2.

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

With HindIII and SalI digestion pQE-9 carrier, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people PKCI-2 has correctly been inserted carrier.

Incubated overnight (O/N) contains the clone of required construction positive transformant 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 people PKCI-2 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people PKCI-2 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.At first, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein can be incorporated in second post from nickel-chelate column, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, and the molecular weight that identifies expressing protein is 14KDa.

This external application ordinary method checks order to the amino acid of expressing proteic N end and each 10 amino acid length of C end, finds consistent with the sequence of SEQ ID NO:4.

Embodiment 4

The expression of people PKCI-2 in eukaryotic cell (Chinese hamster ovary celI strain)

The cDNA sequence of coding people PKCI-2 is used corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end, with the pcr amplification product among human brain λ gt11cDNA library (available from Clontech company) or the embodiment 1 is that template increases, with the synthetic fragment of inserting.

5 ' Oligonucleolide primers sequence is 5 '-CGTAAAGCTTATGG GAATTGAAGT GGCCA-3 ' (SEQ ID NO.7),

This primer sequence contains the restriction enzyme site of HindIII restriction enzyme, and what connect is 19 Nucleotide of the people PKCI-2 encoding sequence that begun by initiator codon;

3 ' end Oligonucleolide primers sequence is 5 '-ATTCGAATTCTCAA CCTGGAGGCC ACTGG-3 ' (SEQ ID NO.8)

This primer sequence contains the restriction enzyme site of EcoRI restriction enzyme, the encoding sequence of translation termination and people PKCI-2.

The restriction enzyme site of restriction enzyme 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 (f1ori), 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 EcoRI digestion pcDNA3 carrier, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, the cDNA fragment of sequence verification people PKCI-2 has correctly been inserted carrier.

Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mM TrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

SDS-PAGE glue with 12% carries out electrophoresis, and the molecular weight that identifies expressing protein is 14K Da.

This external application ordinary method checks order to the amino acid of expressing proteic N end and each 10 amino acid length of C end, finds consistent with the sequence of SEQ ID NO:4.

Embodiment 5

Preparation antibody

The recombinant protein that as above obtains is used for immune animal to produce antibody, specific as follows.Recombinant protein separates standby with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people PKCI-2 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: Fudan University is denomination of invention (ii): new human protein kinase C arrestin encoding sequence,

Its encoded polypeptides and preparation method be the sequence number (iii): information (i) sequence signature of 8 (2) SEQ ID NO:1

(A) length: 22 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:AAGATGGCGG CACGTGGTGC TG 22 (2) SEQ ID NO:2

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：2：AAGCATCCAG AGTCTGGTTG TCC 23 ( 2 ) SEQ ID NO：3： ( i ) ： ( A ) ：532bp ( B ) ： ( C ) ： ( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：3 1 AAGATGGCGG CACGTGGTGC TGGCTGCTGG GTTGCGCGCG CGCAGAAGCC GTGGCGCCCA 61 CGGGGGTGCG CGGGGGGCAG GTCCGAGGAG CTGCAGGTGT GACTGATGGG AATTGAAGTG121 GCCAAGGCCC AGCAGGCAAC TCCTGGGGGA GCAGCCCCAA CCATCTTCTC CCGGATCCTG181 GACAAGAGCC TCCCAGCTGA CATTCTCTAT GAGGACCAGC AGTGTCTTGT GTTCCGTGAT241 GTGGCCCCTC AGGCTCCTGT GCACTTCCTG GTCATTCCTA AGAAGCCCAT TCCTCGGATT301 AGCCAGGCTG AAGAAGAAGA CCAGCAGCTT CTAGGACACC TACTCCTTGT GGCCAAGCAG361 ACAGCAAAGG CTGAGGGCCT GGGAGATGGA TACCGACTTG TGATCAACGA TGGGAAGCTG421 GGTGCACAAT CTGTGTATCA TCTGCACATT CATGTACTTG GGGGCCGGCA GCTCCAGTGG481 CCTCCAGGTT GAACCTGCCA ACTGATTAAA GGACACCAGA CTCTGGATGC TT ( 2 ) SEQ ID NO：4： ( i ) ：

(A) length: 128 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：4 1 Met Gly Ile Glu Val Ala Lys Ala Gln Gln Ala Thr Pro Gly Gly 16 Ala Ala Pro Thr Ile Phe Ser Arg Ile Leu Asp Lys Ser Leu Pro 31 Ala Asp Ile Leu Tyr Glu Asp Gln Gln Cys Leu Val Phe Arg Asp 46 Val Ala Pro Gln Ala Pro Val His Phe Leu Val Ile Pro Lys Lys 61 Pro Ile Pro Arg Ile Ser Gln Ala Glu Glu Glu Asp Gln Gln Leu 76 Leu Gly His Leu Leu Leu Val Ala Lys Gln Thr Ala Lys Ala Glu 91 Gly Leu Gly Asp Gly Tyr Arg Leu Val Ile Asn Asp Gly Lys Leu106 Gly Ala Gln Ser Val Tyr His Leu His Ile His Val Leu Gly Gly121 Arg Gln Leu Gln Trp Pro Pro Gly

(2) information of SEQ ID NO:5

(i) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:5

CCTAGTCGAC?ATGGGAATTG?AAGTGGCCA 29

(2) information of SEQ ID NO:6

(i) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:6

ATCGAAGCTT?TCAACCTGGA?GGCCACTGG 29

(2) information of SEQ ID NO:7

(i) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:7

Information (i) sequence signature of CGTAAAGCTT ATGGGAATTG AAGTGGCCA 29 (2) SEQ ID NO:8

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO:8ATTCGAATTC TCAACCTGGA GGCCACTGG 29

Claims (14)

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

Described nucleotide sequence and SEQ ID NO.3 show at least 70% homology from the nucleotides sequence of Nucleotide 106-492 position; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 106-492 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 sequence from Nucleotide 106-492 position.

4. isolating people PKCI-2 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 people PKCI-2 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people PKCI-2 protein-active operationally is connected in expression regulation sequence, form people PKCI-2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 106-492 position among described nucleotide sequence and the SEQ ID NO.3;

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

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

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

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 106-492 position among the SEQ ID NO.3.

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

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

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

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