CN100543134C - Phosphokinase and application thereof - Google Patents
Phosphokinase and application thereof Download PDFInfo
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- CN100543134C CN100543134C CNB2004100542904A CN200410054290A CN100543134C CN 100543134 C CN100543134 C CN 100543134C CN B2004100542904 A CNB2004100542904 A CN B2004100542904A CN 200410054290 A CN200410054290 A CN 200410054290A CN 100543134 C CN100543134 C CN 100543134C
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Images
Abstract
The invention provides a kind of new phosphokinase-RX218 albumen, coding proteic polynucleotide of RX218 and produce the proteic method of this RX218 through recombinant technology.RX218 albumen can interact with P16, thereby can be used as drug target screening new drug.
Description
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the new phosphokinase with anti-tumor function-RX218 albumen and the proteic polynucleotide of coding RX218.The invention still further relates to these polynucleotide and proteinic method for making and purposes, and contain the proteic composition of this RX218.
Background technology
The drug target gene of " high quality " (being called for short the medicine target) is the source of new drug development.Though finishing of the Human Genome Project is the human alluring prospect for the treatment of disease of having brought, except that the high molecular weight protein medicine, gene itself might not be the medicine target.From the gene to the new drug, this chain still lacks many requisite links.Wherein, gene functional research because of its secret that can disclose human health and disease on the molecule aspect, is sought out most important Disease-causing gene, determines that can gene become the committed step of medicine target and become.
External large-scale pharmaceutical factory finds, though depend merely on the gene order data and analysis of biological information can find a large amount of potential drug target spot genes, this genoid can only be classified as " inferior quality " medicine target.The drug development personnel press for a large amount of gene functional research and are verified in the face of the inferior quality medicine target of huge amount becomes at a loss as to what to do, just can filter out new drug development reliable " high quality " target spot.So huge using value and commercial promise are being contained in functional genomics research.
Can be used as 5 of target spot at present, in 000 gene, the sequence of phosphokinase (kinase) has very big conservative property, is the drug screening gene target spot of generally acknowledging, is referred to as a class target spot with Phosphoric acid esterase (phosphatase), proteolytic enzyme (protease) and each receptoroid.Phosphokinase (kinase) with the phosphide group-transfer of ATP or GTP position to substrate gal4 amino acid residue, the catalytic proteins phosphorylation.Proteinic phosphorylation and dephosphorylation are that protein is regulated its function/active a kind of important way.As MAPK and transcription factor CREB, Jun etc. have activity, and do not have activity when the non-phosphorylating states when phosphorylation state; Transcription factor I κ B α etc. are then opposite, do not have activity when phosphorylation state, and have activity when the non-phosphorylating state.
Proteinic phosphorylation-dephosphorylation is the important step in the cell signalling process.Cell signalling is that phalangeal cell is by being positioned at after birth or born of the same parents' acceptor, the stimulation of recipient cell external information molecule, conversion through the intracellular signal transduction system of complexity, bring into play biological effect, and then almost regulating all processes of vital movement, comprise propagation, growth and the differentiation of cell, nervous activity, Muscle contraction, metabolism, tumour generation etc.Human a lot of disease is all closely related with signal transduction pathway, in inflammation (inflammation) reaction that causes at tumour necrosis factor (TNF), tumour necrosis factor activates interaction between a series of protein phosphatase kinases by bind receptor, finally activates NF-κ B and the reaction that causes inflammation.Biochemist and many companies are all disclosing the key position of signal transmission path by the interaction between the research albumen, and develop the medicine that influences the signal transmission, to reach the purpose of treatment disease.The phosphorylation that depends on the multiple protein substrate smoothly of signal transduction process comprises tyrosine phosphorylation, threonine residues phosphorylation and serine residue phosphorylation, and this process is finished by phosphokinase catalysis just.
In view of the important even conclusive effect of signal transduction pathway in cell proliferation, differentiation and multiple disease generating process, and phosphokinase is in the extremely important status of signal transduction pathway, design and exploitation are the new drug of target spot with the phosphokinase, by adjusting, control signal transduction path treatment disease, be undoubtedly a kind of with strong points, desirable approach efficiently, have the potential prospect of the multiple disease of treatment.Comprise PKC active regulator, PKA inhibitor, ptk inhibitor and receptor-mediated calcium channel modulators etc. at kinase whose medicine at present, wherein part has entered clinical trial.But existing kinases drug target still can not satisfy human Fighting Disease far away, capture the demand of tumour, and this area still needs more special, more effective new kinases as the medicine target, thereby the approach of effective broadening treatment disease is promoted human beings'health.Therefore, the new phosphokinase of exploitation becomes pressing for of a kind of new drug development.
Summary of the invention
The purpose of this invention is to provide a kind of new phosphokinase-RX218 albumen with and fragment, analogue and derivative.
Another object of the present invention provides these proteinic polynucleotide of coding.
Another object of the present invention provides the purposes of producing these method of protein and this protein and encoding sequence.
In a first aspect of the present invention, a kind of isolating RX218 albumen is provided, it is selected from down group: have the protein of SEQ ID NO:2 aminoacid sequence, or it has conservative property variant protein matter, active fragments or the reactive derivative of kinase activity.
Preferably, this protein is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) replacement, disappearance or the interpolation of SEQ ID NO:2 aminoacid sequence through one or more (as 1-10, preferably 1-8) amino-acid residue formed, and have the phosphorylation function by (a) polypeptides derived.More preferably, this albumen has SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, a kind of isolating polynucleotide are provided, the above-mentioned RX218 albumen of its coding.
Preferably, described polynucleotide polynucleotide encoding has the protein of aminoacid sequence shown in the SEQ ID NO:2.More preferably, these polynucleotide contain the sequence of 1-1101 position among the SEQ ID NO:1.
In a third aspect of the present invention, a kind of carrier is provided, it contains the proteic polynucleotide of above-mentioned coding RX218, and is transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, a kind of proteinic preparation method is provided, this method comprises step:
(a) under expression condition, cultivate above-mentioned host cell;
(c) isolate protein from culture, described protein contains the aminoacid sequence of SEQ ID NO:2.
In a fifth aspect of the present invention, provide a kind of can with above-mentioned RX218 protein-specific bonded antibody.
In a sixth aspect of the present invention, a kind of composition (as pharmaceutical composition) is provided, it contains above-mentioned RX218 albumen or its antagonist (as antibody) and the pharmaceutically acceptable carrier of safe and effective amount (or 0.001-99wt%).
In a seventh aspect of the present invention, a kind of RX218 is provided proteic purposes, it is used as the proteic inhibitor of P16.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 has shown the PCR product electrophorogram of RX218.Wherein each swimming lane is as follows: swimming lane 1.DNA molecular weight standard product; It is template that swimming lane 2. mixes RNA with tissue, conventional RT-PCR amplified production; It is template that swimming lane 3. mixes RNA with tissue, the SMARTRT-PCR amplified production; Swimming lane 4-8. is the amplified production of template with tire brain, Hela, mixing (Hela+ tire brain+lymph), lymph, kidney cDNA library respectively.The result shows, except that RT (the comprising SMART) product of different sources, in the HELA library, mixes library (Hela+ tire brain+lymph), and the RX218 band is arranged in the kidney.
Fig. 2 has shown the proteic structure of RX218.Wherein, the amino acid of the 18-41 position of SEQ ID NO:2 constitutes ATP-binding site, and the 12-272 amino acids constitutes its kinase domain.
Fig. 3 has shown the quantitative fluorescence PCR detected result of RX218.Wherein scheming A is squamous cell lung carcinoma and corresponding normal lung tissue; Figure B is esophageal squamous cell carcinoma and corresponding normal esophageal tissue.
Fig. 4 has shown the co-immunoprecipitation result of RX218 and P16.Wherein, CP represents reference protein.After the 293T cell carries out transfection and co-immunoprecipitation, can see to exist significantly between RX218 and the P16 to interact.
Fig. 5 has shown the phosphorylation activity of RX218.Flag-RX218 can autophosphorylation, and the ATP-binding site mutant (RX218mut) of Flag-RX218 then can not.
Fig. 6 has shown that RX218 is the inhibitor of P16, can influence the G1 phase that P16 causes to block.(B) can see the retardation (the G1 phase rises to B:71.35% from A:45.19%) of obvious G1 phase after the U20S transit cell has dyed the P16 gene, this effect disappearance (E) after the corotation RX218.On the contrary, corotation P16 and RX218m (ATP-binding site mutant) are (F) then to the not obviously influence of retardation of G1 phase of P16.
Embodiment
The inventor is extensive studies through going deep into, and has isolated the full-length cDNA of new people's phosphokinase RX218 first, and its coding contains 367 amino acid whose RX218 albumen.RX218 albumen contains the structural domain of phosphokinase, and phosphorylation experiment confirm RX218 is phosphokinase really.Yeast two-hybrid experiment and co-immunoprecipitation experimental result have confirmed to have direct interaction between RX218 and the P16 really.Finished the present invention on this basis.
In the present invention, term " phosphokinase RX218 " or " RX218 albumen " or " RX218 polypeptide " are used interchangeably, all refer to the to have RX218 Argine Monohydrochloride sequence basically protein of (SEQ ID NO:2).They comprise the RX218 albumen that contains or do not contain initial methionine.These terms also comprise the RX218 albumen that contains or do not contain signal peptide.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and protein, but same polynucleotide or protein then are separation and purification as separating from native state with in other materials that exist.
As used herein, " isolating RX218 albumen is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can go out RX218 albumen with purified technology of protein (especially HPLC) separation and purification of standard.
Protein of the present invention can be recombinant protein, natural protein, synthetic protein, preferred recombinant protein.Protein of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, protein of the present invention can be glycosylated, maybe can be nonglycosylated.Protein of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of RX218, derivative and analogue.As used herein, term " fragment ", " derivative " are meant biological function or the active protein that keeps natural RX218 albumen of the present invention identical basically with " analogue ".Protein fragments of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted protein of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has the protein of substituted radical, or (iii) mature protein and another compound (such as the compound that prolongs the protein transformation period, polyoxyethylene glycol for example) merges formed protein, or (iv) additional aminoacid sequence is fused to this protein sequence and the protein that forms (as leader sequence or secretion sequence or be used for this proteinic sequence of purifying or proenzyme sequence, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " RX218 albumen " refers to have the protein of the SEQ ID NO.2 sequence of RX218 protein-active.This term also comprises having and variant form RX218 albumen identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and (being generally in 20, preferably is in 10 to add one or several at C-terminal and/or N-terminal, more preferably be in 5) amino-acid residue as, in the art, when replacing, can not change proteinic function usually with the close or similar amino-acid residue of performance.Again such as, add one or several amino-acid residues at C-terminal and/or N-terminal and also can not change proteinic function.This term also comprises proteic active fragments of RX218 and reactive derivative.
This proteinic variant form comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with coded protein of the DNA of RX218DNA hybridization and polypeptide or the protein that utilizes the proteic antiserum(antisera) of anti-RX218 to obtain.The present invention also provides other protein, as comprises RX218 albumen or its segmental fusion rotein.Except whole length protein almost, the present invention has also comprised the soluble fragments of RX218 protein sequence.Usually, this fragment have the RX218 protein sequence at least about 10 continuous amino acid residues, usually at least about 30 continuous amino acid residues, preferably at least about 50 continuous amino acid residues, more preferably at least about 80 continuous amino acid residues, best at least about 100 continuous amino acid residues.
Invention also provides RX218 proteic analogue.These analogues and the proteic difference of natural RX218 can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps have both at the same time.These protein 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 natural L-amino acid residue (as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that protein of the present invention is not limited to the above-mentioned representational protein that exemplifies.
(the not changing primary structure usually) form of modification comprises: interior or external proteinic chemically derived form such as the acetylize or carboxylated of body.Modify and also to comprise glycosylation, as those in proteinic synthetic and processing or further carry out glycosylation modified and protein that produce in the procedure of processing.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by protein 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 protein that has been improved its proteolysis performance or optimized solubility property by modifying.
In the present invention, " RX218 conservative property variant protein matter " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino-acid residue and is formed protein at the most best.These conservative property variant protein matter are preferably carried out the amino acid replacement according to table 1 and are produced.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The proteinic coding region sequence of encoding mature can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature protein of coding SEQ ID NO:2 comprise: the proteinic encoding sequence of an encoding mature; The encoding sequence of mature protein and various additional code sequence; Encoding sequence of mature protein (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded protein " can be to comprise these proteinic polynucleotide of coding, also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, it is encoded polypeptide or proteinic fragment, analogue and the derivative of identical aminoacid sequence with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded protein matter in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 60%, preferably at least 70%, more preferably at least 80%, the polynucleotide of at least 90% homology best.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1% Ficoll, 42 ℃ etc.; Or (3) only in the homology between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the protein of interfertile polynucleotide encoding has identical biological function and activity with the mature protein shown in the SEQ IDNO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding RX218.
Protein among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
RX218 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and obtain relevant sequence.Also can be directly method amplification by RT-PCR obtain relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention protein (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Use method (Saiki, the etal.Sci ence1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or RX218 albumen coded sequence, and produce method of protein of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the RX218 albumen of reorganization.In general following steps are arranged:
(1). with the proteic polynucleotide of coding RX218 of the present invention (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, RX218 albumen polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: the expression vector based on T7 of expressing in bacterium; The pMSXND expression vector of in mammalian cell, expressing and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains RX218 encoding histone dna sequence dna and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: protoplasm body coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the protein of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant protein in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the protein of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The RX218 albumen of reorganization is of use in many ways.These purposes include, but is not limited to: be used to screen antibody, protein or other part that promotes or resist the RX218 protein function.The protein molecule that can suppress or stimulate the RX218 protein function that can be used for seeking therapeutic value with the reorganization RX218 protein screening protein library of expressing.
On the other hand, the present invention also comprises the RX218 coding DNA or the protein of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into RX218 albumen or fragment.Preferably, refer to that those can combine with RX218 albumen 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 RX218, comprise that also those do not influence the antibody of RX218 protein function.The present invention comprises that also those can be with modification or without the protein bound antibody of the RX218 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; 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 RX218 albumen of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing RX218 albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Each antibody-like of the present invention can utilize proteic fragment of RX218 or functional zone, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize protein 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 proteic unmodified form of RX218 bonded antibody; 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).
Utilize RX218 albumen of the present invention,, can filter out with RX218 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.Usually, set up and to be applicable to the molecule and the cell levels screening model of high flux screening and to carry out correlative study work such as high flux screening.Use E.coli or Bacula virus expression system, cloning and expression tyrosine-phosphatase active fragments, the separation and purification recombinant protein, and use these recombinases, set up the molecular level screening model that is applicable to high flux screening.By to the crude extract that derives from traditional herbal medicine in a large number and the screening of pure compound, seek effective reactive site or pure compound.Active guidance separating monomer from effective reactive site.Use high flux screening and obtain micromolecular inhibitor, detect RX218 is suppressed effect, determine the specificity of micromolecular inhibitor RX218.And the micromolecular inhibitor of using the high flux screening acquisition detects cell levels inhibition effect.
RX218 albumen of the present invention and antibody, inhibitor, agonist or antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
The present invention also provides a kind of pharmaceutical composition, and it contains RX218 albumen of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.These compositions can be used for suppressing the activity of p16.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.1 nanogram/kg body weight-Yue 10 mg/kg body weight.In addition, protein of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that RX218 albumen with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 1 microgram/sky, and in most of the cases is no more than about 10 mg/kg body weight, and preferably this dosage is about 1 microgram/sky-Yue 0.5 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Whether having the proteic method of RX218 in a kind of test sample is to utilize the proteic specific antibody of RX218 to detect, and it comprises: sample is contacted with the RX218 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample RX218 albumen.
Major advantage of the present invention is: RX218 of the present invention is a kind of new phosphokinase, with P16 interaction is arranged, therefore can be used as the screening that drug target carries out micromolecular compound, foundation is at the medicaments sifting model of RX218, searching can be regulated the micromolecular compound of RX218 kinase activity, for the diagnosis of disease, treatment bring new way.
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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The screening of embodiment 1, RX218 gene:
From, the cDNA library of having set up without any functional annotation or note incomplete " newly " gene synthetic by the est sequence the Genbank, using modern information biology PFAM/Profile pattern preferentially screens phosphokinase, Phosphoric acid esterase, proteolytic enzyme, single membrane receptor of crossing, predict a new gene without any functional annotation, contain the phosphokinase structural domain, called after RX218.
The acquisition of embodiment 2, RX218 gene:
In order to obtain the total length of RX218 gene, synthesize following primer, mixing RNA with the extractive tissue of ordinary method is template, increases by conventional RT-PCR method.
Upstream primer: 5 ' GGCCAATCCGGCCATGGATGACGCTGCTGTCCTCAAGC3 ' (SEQ ID NO:3)
Downstream primer: 5 ' GGCCTCTAAGGCCTCACTGGGCCCGCGTCTCTGG3 ' (SEQID NO:4)
This contains SfiI shuttle back and forth cloning site, initiator codon and terminator codon to primer, is the encoding sequence of RX218 between this restriction enzyme site.
Utilize this that primer PCR amplification has been obtained the band (Fig. 1, swimming lane 2 and 3) that a size is about 1100bp, this fragment is reclaimed, is cloned into the full length sequence that order-checking behind the carrier obtains the RX218 gene, 1104bp (SEQ ID NO:1) altogether.
Embodiment 3, RX218 sequential analysis and location
The complete coding region that comprises the RX218 gene in the sequence of the 1104bp that embodiment 1 obtains, the protein of forming by 367 amino acid (SEQ ID NO:2) of encoding.Wherein, the 18th～41 amino acid constitutes ATP-binding site, and the 12-272 amino acids constitutes kinase domain (Fig. 2).
EST information according to RX218 is located in human chromosome 5q22.3.Coding region and rat, the mouse RX218 gene order of people RX218 are carried out the homology comparison, find to have higher homology.
Only contain an exon in the The sequencing results demonstration RX218 gene order.Because most genes all are made of jointly intron and exon, for the RX218 gene of getting rid of the RT-PCR acquisition is the result that template is polluted, further in the cDNA library of tire brain, Hela, mixing (Hela+ tire brain+lymph), lymph, nephridial tissue, utilize primer (SEQ ID NO:3 and 4), verify that by conventional PCR method the result is shown in the 4th～8 swimming lane among Fig. 1.As can be seen from the figure, in Hela, kidney and mixing cDNA library, all detect the existence of RX218 gene.Therefore, this has got rid of RX218 is possible of pseudogene, and the RX218 sequence that the clone obtains is the new gene that does not still have any functional annotation at present that is made of an exon really.
The tissue distribution of embodiment 4, RX218
Real-time fluorescence quantitative PCR (Q-PCR) is at present gene to be carried out quantitative analysis sensitivity, accurate technique the most.Made up surplus in the of about 300 the frozen tissue storehouse of tumor tissues and corresponding healthy tissues thereof with ordinary method, comprised 18 kinds of tumor types.In order further to determine to the frozen tissue storehouse of preparation,, to carry out qualitative, quantitative research by the expression of RX218 gene in various tumor tissues by the Q-PCR technology.
In the present embodiment, adopt each total tissue RNA of Trizol single stage method extracting, and through behind the dnase digestion, as the template of RT.Subsequently, design and synthesize following primer, utilized the fluorescent probe of FAM-TAMRA mark to implement Q-PCR,
Upstream primer: 5 ' TCAAGAAGATGCTGCGTATCCA3 ' (SEQ ID NO:5)
Downstream primer: 5 ' TGTGGTAGATGAGGTCCTTGCA3 ' (SEQ ID NO:6)
Probe sequence: 5 ' FAM-CACCGCGTCAACTTCCCACGCT3 '-TAMRA (SEQ ID NO:7)
Part of test results sees the following form and Fig. 3.
The Q-PCR partial results
| Sample RX numbering | Pathological diagnosis | MT/N ratio |
| RX-T-F-0041 | Esophageal squamous cell carcinoma | 51.77832917 |
| RX-T-F-0042 | Esophageal ulcer type squamous cell carcinoma | N does not detect, and MT CT value is 33.9 |
| RX-T-F-0045 | Oesophagus cap type squamous cell carcinoma | N does not detect, and MT CT value is 33 |
| RX-T-F-0179 | Esophageal ulcer type squamous cell carcinoma | N does not detect, and MT CT value is 37 |
| RX-T-F-0005 | Squamous cell lung carcinoma | 35.30533646 |
| RX-T-F-0002 | Squamous cell lung carcinoma | N does not detect, and MT CT value is 31 |
| RX-T-F-0150 | The low differentiation of lung squama shape cell carcinoma | N does not detect, and MT CT value is 31 |
N: healthy tissues; MT: tumor tissues.
CT: fluorescent signal reaches the PCR cycle number of threshold value.
From the MT/N ratio of The above results as can be seen, RX218 gene or obviously increase than healthy tissues in the expression of oesophagus, squamous cell lung carcinoma; Perhaps do not express specific oesophagus, the squamous cell lung carcinoma of being expressed in its healthy tissues.Can see that from the Q-PCR amplification curve amplification curve of tumor tissues has tangible ascendant trend after 30 circulations, and corresponding healthy tissues there is not this variation.This same explanation RX218 gene expression in oesophagus, squamous cell lung carcinoma is increased, with the significant difference (Fig. 3) of related normal tissue.
The interactional albumen of embodiment 5, screening and RX218
In the present embodiment, adopt the Gal4 yeast two-hybrid system of Clontech company to screen, determine to have interactional albumen with RX218.Method is as follows: the RX218 gene of sequence verification is shuttled back and forth by the SfiI cloning site transfer to transformed fusion plasmid pGBKT7 carrier (available from Clontech company, referring to US6770446, US6376652) on,, successively Hela, lymph and tire brain library are screened on a large scale as bait (bait) with this by conversion method (transformation) and dot matrix (mating).
As a result, these two kinds of methods have all obtained the P16 positive colony.This shows that RX218 and P16 albumen have interaction.
The interaction of embodiment 6, checking RX218 and p16
Because yeast two-hybrid system itself may produce false positive, therefore utilize co-immunoprecipitation technology (Coimmunoprecipitation, Co.IP) the further interactively of checking RX218 and P16 in mammalian cell.
In the multiple clone site of pcDNA3.1 (Invitrogen company), add the SfiI site with ordinary method,, obtain to have respectively the pcDNA3.1 carrier of above-mentioned tag respectively flag-tag and myc-tag being introduced SfiI site N end.The RX218 (embodiment 2) and the P16 gene of pcr amplification are cloned into the pcDNA3.1 carrier for expression of eukaryon that has flag-tag and myc-tag that has built respectively by the SfiI site.Then, with the 293T cell of above-mentioned recombinant vectors transfection routine.Cultivate after 24 hours, centrifugal collecting cell, add the cracking of lysis liquid after, sample on the centrifugal collection supernatant.Utilize the monoclonal antibody (available from Sigma company) of anti-flag, anti-myc respectively, detect the proteic expression of RX218, P16 by the Westen-blot method.
The results are shown in Figure 4.As can be seen from the figure, RX218 and P16 protein expression are all right, expression amount stable.
Then, behind anti-flag antibody and Protein G immunoprecipitation, coprecipitated product electrotransfer behind the SDS-PAGE electrophoresis is hybridized with the capable protein immunoblot of enzyme mark anti-myc antibody to nitrocellulose membrane.Can see and exist interaction (Fig. 4, swimming lane 2) between RX218 and the P16.
Because the P16 gene exists high-frequency change in human tumor, comprise disappearance and sudden change, relation for the mutant of clear and definite RX218 and different P16, the inventor has cloned p16G101W again, the pathology mutant of the P16 that p16A100P, p16D74N etc. be in the news carries out finding behind transfection and the co-immunoprecipitation at the 293T cell equally, the mutant of RX218 and a plurality of P16 all has tangible interaction, and the interaction of RX218 and these mutants is stronger than wild-type.These results suggest, RX218 may be an important gene that participates in the generation of adjusting tumour.
Embodiment 7, with the phosphorylation of external phosphorylation technical study RX218
In order to determine that RX218 has kinase whose activity really, carried out the external autophosphorylation experiment of RX218 in the present embodiment.
At first made up the ATP-binding site mutant RX218m (K41A) of RX218 with conventional site-directed mutagenesis method, the Lys that is about to the 41st of SEQ ID NO:1 is replaced into Ala, thereby makes 41 among the SEQ ID NO:2 to become A by K.RX218 and mutant RX218m thereof are building up to the pcDNA3.1 carrier for expression of eukaryon that has Flag-tag, transfection 293T cell.Cultivate after 24 hours, lysing cell is collected supernatant.Behind the Protein G immunoprecipitation that has anti-flag antibody, get half and be used for the Western blotting to identify the expression of RX218; Second half uses kinase reaction damping fluid (20mM Tris/HCL pH=7.4,150mMNaCl, 10mM MnCl
2, 50 μ M ATP, 10mM MgCl
2) balance, add then 10 μ Ci γ-
32P ATP is in 30 ℃ of reaction 30min.Add equal-volume 2 * SDS-PAGE sample-loading buffer, behind 95 ℃ of sex change 5min, centrifugal sample is added 15%SDS-PAGE gradient glue electrophoresis.Electrophoresis finishes, and behind dried glue, presses the radioautograph of X-ray sheet.
The result shows that RX218 has kinase whose activity, can autophosphorylation, and after ATP-binding site sudden change with it, and its kinase whose activity also disappeared (Fig. 5).
The influence of technical objective gene pairs cell physiologicals such as embodiment 8, usefulness FACS and immunofluorescence
For the effect of further clear and definite RX218 cell cycle, whether cell cycle is influential for the overexpression of utilization FACS technology further research RX218 in the U2OS cell, and the relation of further clear and definite and P16.
Transfection has the pCDNA3.1 carrier for expression of eukaryon of P16 and RX218 gene respectively in U20S cell (ATCC), then digestion and fixed cell.When the U20S transit cell toward the 6cm culture plate dyes P16 gene 0.5ug, can see the retardation (Fig. 6-B:G1 phase rises to 69.7% from 45.9%) of tangible G1 phase, this effect disappearance after the corotation RX218 (Fig. 6-E).On the contrary, (Fig. 6-F) is then to the not obviously influence of retardation of G1 phase of p16 for corotation P16 and RX218m (ATP-binding site mutant).This shows that RX218 has restraining effect to the P16 activity, is the inhibitor of P16.Detailed data corresponding to Fig. 6 sees the following form.
RX218 is to the active restraining effect of P16
The preparation of embodiment 9, the anti-RX218 protein antibodies of rabbit
Get body weight and be one of male new zealand rabbit about 2 kilograms.Add Freund's complete adjuvant with 1mg RX218 protein sample (embodiment 6) and grind to form emulsion state, at the rabbit neck part multi-point injection.After raising two weeks, add Freund's incomplete adjuvant with 1mg RX218 protein sample and grind to form emulsion state, again at the rabbit neck part multi-point injection.After one month, add Freund's incomplete adjuvant with same method booster immunization with the 1.5mgRX218 protein sample.Behind the two weeks, add Freund's incomplete adjuvant booster immunization once more with the 1mgRX218 protein sample again.After raising two weeks, carotid artery is got blood, 4 ℃ of standing over night, and 2,000 left the heart 3 minutes.Upper serum is the anti-RX218 protein antibodies of rabbit.
Results of hybridization shows, anti-RX218 protein antibodies can with RX218 albumen and specificity combine.
Discuss
The inventor has successfully cloned the full-length cDNA of RX218, analyzes according to its cDNA, and the RX218 encoded protein contains the structural domain of a phosphokinase.Use real-time fluorescence quantitative PCR technology (Q-PCR), in kinds of tumors and corresponding healthy tissues thereof, detect the expression of RX218.The result shows that the RX218 gene is expressed obviously enhancing in the squamous cell carcinoma of oesophagus, lung, and healthy tissues do not express or expression amount very low.This prompting, the RX218 gene has certain dependency in generation, the development that the specificity of oesophagus, squamous cell lung carcinoma increases with these tumours.
Yeast two-hybrid and co-immunoprecipitation experiment be proof further, and phosphokinase RX218 and p16 have direct interaction really.
The P16 gene is MTS (multiple tumor suppressor 1) gene again, it is the new antioncogene that U.S. cold spring laboratory Kamb in 1994 etc. find, directly participate in the regulation and control of cell cycle, by suppressing cyclin white matter dependent kinase 4,6 (Cyclin dependent kinase, CDKE4 and CDK6) make cell-cycle arrest in the G1 phase, negative adjusting cell proliferation and division all have a very important role in processes such as cell aging, tumour generation.The p16 assignment of genes gene mapping is in karyomit(e) 9P21, and total length 8.5kb is made up of 2 introns and 3 exons, the albumen of coding 16KD.CDKs may be the core of cell cycle regulation device, and the complex body of CDK4 and cyclin (Cyclin) is participated in the regulation and control of cell cycle G1-S conversion.P16 albumen suppresses the Cdk4 activity, finally stops cell to enter the S phase, in case p16 genetically deficient, sudden change etc. cause afunction, then can not suppress CDK4, finally causes cell to enter malignant proliferation.Therefore, there is the people that p16 is likened to braking devices in the cell cycle, in case malfunctioning then can cause the cell malignant proliferation, cause malignant tumour to take place.
In lung cancer, mammary cancer, cerebral tumor, bone tumor, skin carcinoma, bladder cancer, kidney, ovarian cancer, lymphoma and melanoma, detected at present sub-disappearance of p16 gene pure 50% or more and nonsense, missense, phase shift mutation, shown that the p16 gene is with disappearance, the formation of sudden change mode wide participation tumour.As the key negative regulatory factor of cell proliferation, will promote the understanding of human and disease healthy, the approach of effective broadening treatment disease to self to the deep understanding of p16 and mutant thereof.Yet up to now, the regulatory mechanism of p16 is still unclear.How it adjusts initial transcribing, and the biologic activity of which upstream regulation factor regulation and control p16 is arranged, and these regulatory factors are again the regulation and control what mechanism to participate in the p16 function by, and these all become pressing for the problem of solution in the further research of p16.New kinases RX218 of the present invention may be the important molecule in the signal transduction pathway, prove that first the RX218 gene has specificity to increase in the mRNA of oesophagus, squamous cell lung carcinoma expression, and RX218 and p16 have direct interaction, can suppress the caused G1 phase of p16 and block, this is a challenging result.Therefore, to the further investigation of new kinases RX218, may be activation and the mechanism of action of clear and definite P16, and then develop brand-new drug target, for the mankind defeat tumour, delay senility and bring breakthrough.
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.
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Claims (4)
1. isolating proteinic purposes, this protein is the polypeptide of SEQ ID NO:2 aminoacid sequence,
It is used to prepare the proteic inhibitor of P16.
2. purposes as claimed in claim 1 is characterized in that, described inhibitor suppresses the caused G1 phase of p16 and blocks.
3. isolating proteinic purposes, this protein is the polypeptide of SEQ ID NO:2 aminoacid sequence,
Described protein is used for setting up and the interactional medicaments sifting model of P16, carries out the screening of micromolecular compound as drug target.
4. isolating proteinic purposes, this protein is the polypeptide of SEQ ID NO:2 aminoacid sequence,
Described protein is used for screening and regulates the active micromolecular compound of described protein kinase.
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Non-Patent Citations (3)
| Title |
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| AAK29413. NCBI. 2001 |
| AAK29413. NCBI. 2001;AF348076. NCBI. 2001 * |
| AF348076. NCBI. 2001 |
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