CN1289524C - Human telomerase active inhibitor protein and use thereof - Google Patents
Human telomerase active inhibitor protein and use thereof Download PDFInfo
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- CN1289524C CN1289524C CN 200410017051 CN200410017051A CN1289524C CN 1289524 C CN1289524 C CN 1289524C CN 200410017051 CN200410017051 CN 200410017051 CN 200410017051 A CN200410017051 A CN 200410017051A CN 1289524 C CN1289524 C CN 1289524C
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Abstract
The present invention provides an MCRS2 protein which is a novel telomerase active inhibitor protein, polynucleotide for encoding the MCRS2 protein and a method for preparing the MCRS2 protein by a recombination technology. The present invention also discloses the applications of the MCRS2 protein and the coding sequence of the MCRS2 protein. The MCRS2 protein has the functions of leading chromosome telomere of hepatoma carcinoma cells SMMC-7721 to be shortened and inhibiting telomerase activity.
Description
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to new microspherule protein 2 MCRS2 (microspherule protein 2) and encoding sequence thereof.The invention still further relates to the purposes and the preparation of MCRS2 polynucleotide and polypeptide.
Background technology
Existing 80% the tumour cell that studies show that has telomerase activation, suppresses telomerase activation and can make tumour cell old and feeble also deathward.
LPTS is a kind of known human telomerase arrestin.The LPTS assignment of genes gene mapping is in No. 8 karyomit(e) 8p23 section of people, and this section lacks in multiple malignant cell medium-high frequency.Studies show that LPTS expression amount in liver cancer tissue and hepatoma cell line is extremely low or detect less than, so relevant [C.Liao of LPTS with liver cancer, M.J.Zhao, H.Song, K.Uchida, K.K.Yokoyama, T.P.Li, Identification of the gene for a novelliver-related putative tumor suppressor at a high-frequency loss of heterozygosityregion of chromosome 8p23 in human hepatocellular carcinoma.Hepatology 32 (2000) 721-727].
Studies show that further the LPTS gene can suppress the growth of tumour cell, and can suppress the Telomerase vigor.In tumour cell, express LPTS, cause that the cell telomere shortens, and then cause cell aging, apoptosis [C.Liao, M.J.Zhao, J.Zhao, D.Jia, H.Song, Z.P.Li, Over-expression of LPTS-L inhepatocellular carcinoma cell line SMMC-7721 induces crisis.World J Gastroenterol.8 (2002) 1050-1052.; X.Z.Zhou, K.P.Lu, The Pin2/TRF1-interacting protein PinX1 is apotent telomerase inhibitor.Cell 107 (2001) 347-359.].Therefore, LPTS is a very important Telomerase arrestin, seek the albumen that mutually combines with LPTS and help understanding the LPTS mechanism of action, finds the new albumen that acts on telomerase activation inhibition approach, and can work in coordination with LPTS and in oncotherapy, play a role.Yet the albumen relevant with the Telomerase arrestin found of this area is very limited up to now, does not more have to find to interact with LPTS and has the albumen of inhibition of telomerase.
Therefore, this area presses for the new telomerase activity inhibition protein of exploitation.
Summary of the invention
The purpose of this invention is to provide a kind of new telomerase activity inhibition protein MCRS2 with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, novel isolated MCRS2 polypeptide is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ IDNO:2 aminoacid sequence.
Preferably, this polypeptide is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the function that suppresses telomerase activation by (a) polypeptides derived.
More preferably, this polypeptide is to have 1-475 position or 1-162 amino acids polypeptide of sequence among the SEQ ID NO:2.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide contain a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people MCRS2 polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in 1-475 position among the SEQ ID NO:2 or the 1-162 position.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 173-1597 position among the SEQ ID NO:1; (b) has the sequence of 1-1953 position among the SEQ ID NO:1; Or (c) has a sequence of 173-658 position among the SEQ ID NO:1.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been 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, provide preparation to have the method for the polypeptide of people MCRS2 protein-active, this method contains: (a) under the proteic condition of suitable expressing human MCRS2, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people MCRS2 protein-active.
In a fifth aspect of the present invention, provide and above-mentioned people MCRS2 polypeptid specificity bonded antibody.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people MCRS2 polypeptide active is provided, and the compound that suppresses people MCRS2 polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people MCRS2 polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of MCRS2 in the test sample, it comprises: sample is contacted with the proteic specific antibody of MCRS2, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample MCRS2 albumen.
In a eighth aspect of the present invention, a kind of disease relevant with people MCRS2 polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.
In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.For example, polypeptide of the present invention can be used to prepare the medicine that is used for the treatment of tumour, be used to prepare the composition that suppresses telomerase activation, be used to screen the agonist that promotes people MCRS2 polypeptide active, perhaps screening suppresses the antagonist of people MCRS2 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people MCRS2 of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.
In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people MCRS2 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated and the too high diseases associated of telomerase activation, for example illness such as cancer.The composition that suppresses telomerase activation also is provided.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
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 is a 10%SDS-PAGE electrophoresis showed MCRS2 recombinant expression protein.Wherein, 6 * His-MCRS2, GST-MCRS2-N and GST are respectively the recombinant protein of escherichia coli expression, and through affinity column chromatography.Glue dyes through coomassie brilliant blue staining liquid behind the electrophoresis.
Fig. 2 has shown antagonism MCRS2 specificity detection of antibodies.Wherein, the FLAG-MCRS2 albumen of 293T cell expressing is as positive control, and the protein extract of the 293T cell of transfection empty carrier is as negative control.
Fig. 3 has shown MCRS2 albumen and LPTS is proteic mutually combines.A.MCRS2 and the experiment of LPTS co-immunoprecipitation.293T cell transient transfection FLAG-MCRS2 expression plasmid, cell pyrolysis liquid is with LPTS antibody mediated immunity precipitation, with FLAG antibody test immunoprecipitation complex, wherein, " input " represents positive control, promptly shows the FLAG-MCRS2 albumen in the preceding cell pyrolysis liquid of LPTS co-immunoprecipitation.B, C.GST-pulldown detect MCRS2 and LPTS in external combining.D. coomassie brilliant blue staining GST, GST-LPTS albumen.E. co-immunoprecipitation and GST pulldown detection MCRS2 combines with the body of hTERT is interior.
Fig. 4 has shown that transfection MCSR2 or MCRS2-N shorten the telomere of SMMC-7721 cell.Wherein, A, the cell of the cell .B. stably express MCRS2-N of stably express MCRS2.
Fig. 5 has shown the activity of the N end of MCRS2 or MCRS2 at the vitro inhibition Telomerase.Wherein, the TRAP methods analyst shows that 6His-MCRS2 and GST-MCRS2-N have the activity of vitro inhibition Telomerase.
Embodiment
The inventor is through extensive and deep research, utilize that the yeast two-hybrid screening method obtains first with human telomerase arrestin LPTS bonded albumen MCRS2.MCRS2 combines activity with external in vivo with LPTS, and expression and localization is on entoblast and telomere.MCRS2 can make the fringes of chromosome of liver cancer cell SMMC-7721 shorten, and experiment in vitro has proved that MCRS2 albumen has inhibition of telomerase.Finished the present invention on this basis.
In the present invention, term " MCRS2 albumen ", " MCRS2 polypeptide " or " telomerase activity inhibition protein MCRS2 " are used interchangeably, and all refer to have albumen or the polypeptide of microspherule protein 2 MCRS2 aminoacid sequence (SEQ IDNO:2).They comprise the telomerase activity inhibition protein MCRS2 that contains or do not contain initial methionine.
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 polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolated M CRS2 albumen or polypeptide " is meant that the MCRS2 polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying MCRS2 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of MCRS2 polypeptide can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide 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, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of people MCRS2, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human MCRS2 albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide 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 a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, 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 " people MCRS2 polypeptide " refers to have the SEQ ID NO:2 polypeptide of sequence of people MCRS2 protein-active.This term also comprises having and variant form people MCRS2 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 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, add especially that 6His or GST etc. are convenient to the element of separation and purification and the fusion rotein that forms.This term also comprises proteic active fragments of people MCRS2 and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people MCRS2 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people MCRS2 polypeptide to obtain.The present invention also provides other polypeptide, as contains people MCRS2 polypeptide or its segmental fusion rotein.
Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people MCRS2 polypeptide.Usually, this fragment have people MCRS2 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.Especially at least 162 the amino acid whose active fragmentss of N end that contain MCRS2.
Invention also provides the analogue of people MCRS2 albumen or polypeptide.The difference of these analogues and natural human MCRS2 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " people MCRS2 albumen conservative property variation polypeptide " 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 and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
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 coding region sequence of encoding mature polypeptide 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 SEQID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and 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, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide 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 polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.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 at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO: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 MCRS2.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People MCRS2 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (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 et al.Science 1985 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 contain the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or MCRS2 albumen coded sequence, and the method that produces polypeptide 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 MCRS2 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people MCRS2 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, people MCRS2 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: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) 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 people MCRS2 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.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 P
LPromotor; 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 contains 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.
Contain 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 or Bowes melanoma 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 make MgCl
2With.If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: 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 polypeptide 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 polypeptide 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 albumen 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 people MCRS2 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism MCRS2 protein function as pharmacological agent MCRS2 protein function.The peptide molecule that can suppress or stimulate people MCRS2 protein function that can be used for seeking therapeutic value with the recombinant human MCRS2 protein screening peptide library of expressing.In addition, MCRS2 can be used for suppressing the activity of Telomerase, and then is used for the treatment of various and the too high diseases associated of telomerase activation, as various tumours.
On the other hand, the present invention also comprises people MCRS2 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people MCRS2 gene product or fragment.Preferably, refer to that those can combine with people MCRS2 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 MCRS2, comprise that also those do not influence the antibody of people MCRS2 protein function.The present invention also comprise those can with modify or without the people MCRS2 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 MCRS2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human MCRS2 albumen 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 prepare (see people such as Kohler,
Nature256; 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 MCRS2 protein function and the antibody that does not influence people MCRS2 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people MCRS2 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people MCRS2 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).
The proteic antibody of anti-people MCRS2 can be used in the immunohistochemistry technology, detects the people MCRS2 albumen in the biopsy specimen.
Antibody among the present invention can be used for treating or prevention and the relevant disease of people MCRS2 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people MCRS2 or activity.
Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people MCRS2 albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, and the exchange by disulfide linkage is incorporated into toxin on the antibody.
The production of polyclonal antibody can choose MCRS2 albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Utilize albumen of the present invention,, can filter out with MCRS2 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor 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.
Polypeptide of the present invention can be directly used in disease treatment, for example, is used for and the too high diseases associated of telomerase activation, for example treatment of cancer aspect.When using MCRS2 albumen of the present invention, also can use other tumor therapeutic agents simultaneously, as cis-platinum, TNF etc.
The present invention also provides a kind of pharmaceutical composition, and it contains MCRS2 polypeptide of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.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 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that MCRS2 albumen with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 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.
The proteic polynucleotide of people MCRS2 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of MCRS2 of the proteic nothing expression of MCRS2 or unusual/non-activity.The MCRS2 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic MCRS2 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the MCRS2 transgenosis to cell.The method that structure carries the recombinant viral vector of MCRS2 gene is found in existing document (Sambrook, et al.).Recombinant human MCRS2 gene can be packaged in the liposome in addition, and then is transferred in the cell.
Suppress the oligonucleotide (comprising sense-rna and DNA) of people MCRS2 mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people MCRS2 obtains.During screening, must carry out mark to people MCRS2 protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people MCRS2 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people MCRS2 protein level that is detected in the test can be with laying down a definition the importance of people MCRS2 albumen in various diseases and be used to the disease of diagnosing MCRS2 albumen to work.
Whether having the proteic method of MCRS2 in a kind of detection test sample is to utilize the proteic specific antibody of MCRS2 to detect, and it comprises: sample is contacted with the MCRS2 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample MCRS2 albumen.
The proteic polynucleotide of MCRS2 can be used for the diagnosis and the treatment of MCRS2 protein related diseases.Aspect diagnosis, the proteic polynucleotide of MCRS2 can be used for detecting the proteic expression of MCRS2 MCRS2 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of MCRS2 as the MCRS2 dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of MCRS2 albumen and also can detect the proteic transcription product of MCRS2.
The sudden change that detects the MCRS2 gene also can be used for the disease of diagnosing MCRS2 albumen relevant.The form of MCRS2 protein mutation comprises that the point mutation compared with normal wild type MCRS2 dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of MCRS2 prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritancein Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from the human liver cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it contains is 1757 bases, and its open reading frame is positioned at the 173-1597 position, and the coding total length is 475 amino acid whose people MCRS2 albumen (SEQ ID NO:2).Tumour cell in view of 80% has telomerase activation, can make tumour cell old and feeble also deathward and suppress telomerase activation, therefore MCRS2 albumen can be treatment and the too high diseases associated of telomerase activation, for example disease such as cancer provides new treatment approach, thereby has great application prospect.
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 clone of embodiment 1:MCRS2 albumen cDNA
Use the interaction protein of conventional yeast-two hybrid technique screening LPTS, the cDNA insertion pGilda carrier (available from Clontech company) with coding LPTS is used to screen human brain cDNA (Clontech), the working specification that method provides according to company.Checked order in the positive colony checking back that screens, and this sequence and GenBank database are compared.
According to sequence information, the design primer carries out polymerase chain reaction (PCR), PCR reaction primer is: P1 (SEQ ID NO:3), P2 (SEQ ID NO:4) is a template with the cDNA library plasmid (GIBCO BRL company product) of liver, and pcr amplification obtains the MCRS2 gene cDNA.The PCR reaction conditions is 25 μ l volumes, 94 ℃ of pre-sex change in 4 minutes, circulating reaction be 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 2 minutes, carry out 35 circulations altogether.The PCR product reclaims purifying with the low melting-point agarose gel.
Through complete sequence order-checking (measuring) by Bo Ya company, the cDNA total length that obtains is 1953bp (SEQ ID NO:1), comprised a complete protein-coding region (173-1597 position), the protein of forming by 475 amino acid of having encoded (SEQ ID NO:2).
The proteic recombinant expressed and purifying of embodiment 2, MCRS2
(a) MCRS2 is proteic recombinant expressed
With the MCRS2 nucleotide sequence that obtains among the embodiment 1, ((Novagen company) obtains expression vector 6 * His-MCRS2 to 6 * His-fusion expression vector pET-30 that the same enzyme of packing into after EcoRI and XhoI enzyme are cut is cut.
Contain 162 amino acid whose cDNA fragments of MCRS2N end (corresponding to 173-658 position among the SEQ ID NO:1) and pack among the GST-fusion expression vector pGEX-4T1 (Amersham company) the plasmid called after GST-MCRS2-N of acquisition by EcoRI and XhoI restriction enzyme site into.
With the plasmid transformation escherichia coli BL21-DE3 that builds, obtain MCRS2/BL21-DE3 then, MCRS2-N/BL21-DE3 transforms bacterial strain.Transform bacterial strain after amplification for the positive that obtains, extract plasmid and identify, and prove conclusively by dna sequencing with corresponding restriction enzyme.
MCRS2/BL21-DE3 through identifying, MCRS2-N/BL21-DE3 transforms bacterial strain, be inoculated in the LB substratum (every liter of substratum contains the 10g Tryptones, 5g yeast extract, 10g NaCl) according to 1: 20 ratio, cultivate about 3 hours when OD600 is 0.6-0.8 for 37 ℃, add IPTG to final concentration 0.5mM, abduction delivering 3～4 hours, 6000rpm then, 10 minutes centrifugal collection thalline are used for following protein separation.
(b) separation and purification:
6 * His-MCRS2 albumen: centrifugal collection thalline, be resuspended in 30ml NTA-0 (20mmol/L Tris-HCLpH7.9,0.5mmol/L NaCL, 10% glycerine) in, add phenylmethylsulfonyl fluoride (PMSF) to 0.5mmol/L, behind the ultrasonication thalline, add Triton X-100 to 1% final concentration, 4 ℃, 13000rpm/min is centrifugal, 20min, collect supernatant, transfer on the prior 20mlNTA-0 of the using equilibrated 3S NTA resin affinity column, wash chromatography column with 30ml NTA-0 then, then use 5mlNTA-20 (20mmol/L Tris-HCl pH7.9 successively, 0.5mmol/L NaCL, 10% glycerine, 20mmol/L imidazoles), NTA-40, NTA-80, NTA-100, NTA-200 wash-out, collect elutriant, every pipe 1ml.The SDS-PAGE analyzing proteins distributes, and the Bradford method is measured protein concentration.
The GST-MCRS2-N fusion rotein:
Centrifugal collection thalline is with 10mL solution A (20mM Tris/pH 7.4,0.2mM EDTA, 1mM DTT, 0.5mMPMSF, 1M NaCl) resuspended thalline, the ultrasonic wave broken cell, each 15 seconds, 45 seconds at interval, have children outside the state plan ripple altogether 10 times, add TritonX-100 to 1% final concentration, placed on ice 30 minutes, 9,500rpm, 4 ℃ centrifugal 10 minutes, repeated centrifugation is once.With 20～30mL solution A balance gsh-agarose dress post, with the centrifugal supernatant upper prop of thalline, 20～30mL solution A is washed pillar, (20mMTris/pH 7.4 to use 20～30mL solution B then, 0.2mM EDTA, 0.1M NaCl) wash pillar, contain solution C (the 15mM reduced glutathione is in the solution B) eluted protein of the reduced glutathione of 15mM final concentration with 5mL.
The purifying protein of preparation, through 10%SDS-PAGE electrophoresis and the dyeing of coomassie brilliant blue staining liquid, the result as shown in Figure 1.
The preparation of embodiment 3, anti-MCRS2 protein antibodies
Get 6 * His-MCRS2 fusion rotein of 300 μ g purifying, be dissolved among the 0.5mL PBS, add isopyknic Freund's complete adjuvant, abundant mixing, the intracutaneous multi-point injection is in the male new zealand white rabbit of 2.0kg back.The 3rd day, once more with same protein content with the abundant mixing of Freund's complete adjuvant after, the intracutaneous multi-point injection is in the back.The 28th day, behind same protein content usefulness Freund's incomplete adjuvant mixing, the back intradermal injection was with booster immunization.After this week, blood is collected in the carotid artery bloodletting, places 3 hours for 37 ℃, and 4 ℃ of placements are spent the night so that serum is separated out fully, centrifugal collection serum, packing ,-70 ℃ of preservations.The method for detecting specificity of antibody is as follows: 1.5 μ gFLAG-MCRS2 plasmids are mixed the 293T cell that the back transfection is cultivated with 10 μ l Lipofectamine (GIBCOL BRL company product) in the Tissue Culture Dish of 60mm.Transfection method is seen the experimental implementation rules that GIBCOL BRL company provides.Collecting cell after 48 hours is with 300 μ l Triton X-100 lysates (10mM Tris (pH7.4), 150mM NaCl, 10mM EDTA, 10mM EGTA, 1%Triton X-100,1mM PMSF, 10mM DTT, 5 μ g/ml aprotinin) cracking.Take out 50 μ l lysates and add equivalent 2 * SDS sample-loading buffer, boiling water boiled 10 minutes, and the fine needle head is sheared DNA, the centrifugal precipitation of going.Get 10 μ l and be splined on 15%SDS polyacrylamide (PAGE) gel, carry out protein electrophoresis.Electrophoresis is transferred to poly(vinylidene fluoride) (PVDF) film (NEN company product) with albumen after finishing.Carry out immunoblotting with MCRS2 antibody, use two crosslinked anti-(Santa Cruz company products) of horseradish peroxidase (HRP) to hybridize again.Detect with chemical illuminating reagent at last, pressed the X-ray sheet 1 minute.
The result as shown in Figure 2, in transfection in the cell of FLAG-MCRS2, detect a narrow spectrum protein band, and in transfection in the cell of empty carrier, do not detect protein band, this explanation has obtained the polyclonal antibody of single-minded identification MCRS2.
The interactional experimental verification of embodiment 4, MCRS2 and LPTS
With the difference transfection 293T cell of various plasmids, transfection method is cultivated collecting cell after 48 hours as described in the embodiment 3.Cell lysis buffer [20mM Tris-HCl, pH 7.5,150mM NaCl, 1mMEDTA, 0.5%Nonidet-P40, and the 1mM Phenylmethylsulfonyl chloride (phenylmethyl sulfonyl fluoride, PMSF), 1mM aprotinin (aprotinin) and 1mM leupeptin (leupeptin)] at 4 ℃ of cracking 0.5h., centrifugal collection lysate supernatant is standby.
External synthetic
35S-MCRS2 albumen uses the rabbit reticulocyte lysate TNT of system test kit (Promega company).
Protein mutually combine the experiment be above-mentioned cell pyrolysis liquid or
35Add corresponding recombinant protein or antibody in the albumen of S-Met mark, place 2h for 4 ℃, next add 15 μ l albumin A pearls or gsh-sepharose 4B (Amersham), place 2h for 4 ℃.Pearl is resuspended in (50mM Tris-HCl, pH 6.8,100mM DTT, 2%SDS, 20% glycerine, 0.2mg/ml bromjophenol blue) in the 30 μ l albumen sample-loading buffers with lysis buffer washing 3-4 time.10 μ l samples are transferred to poly(vinylidene fluoride) (PVDF) film (NEN company product) after the SDS-PAGE electrophoretic separation, detect with corresponding antibody, and detection method is as described in the embodiment 3.
(1) co-immunoprecipitation experiment
Prepare cell pyrolysis liquid with expressing the plasmid transient transfection 293T cell of FLAG-MCRS2, cultivating after 48 hours, in cell pyrolysis liquid, add anti-LPTS antibody, carry out co-immunoprecipitation.In the immunoprecipitation complex that adds anti-LPTS antibody, detect FLAG-MCRS2 albumen, do not detect FLAG-MCRS2 albumen (Fig. 3 A) in the adding preimmune serum immunoprecipitation complex in contrast, therefore prove that MCRS2 is a bonded with LPTS in vivo.
(2) GST-pull down experiment
This test is used to verify that MCRS2 and LPTS are in external combining.Method is as follows: at external synthetic
35Add the GST-LPTS recombinant protein in the S-MCRS2 albumen, shown in Fig. 3 B,
35S-MCRS2 can combine external with GST-LPTS, with GST albumen debond in contrast.Therefore prove MCRS2 and LPTS external also be bonded.In order to determine the zone of LPTS and MCRS2 effect, with GST-LPTS-N (comprising 132 amino acid of LPTS albumen n end), GST-LPTS-C (comprising LPTS PROTEIN C end 133-328 amino-acid residue), and the GST-LPTS fusion rotein joins in the cell pyrolysis liquid of transient expression FLAG-MSCR2, carries out GST-pull down experiment.The result shows that the N end of LPTS can both combine (Fig. 3 C) with MCRS2 with the C end.The above-mentioned albumen of escherichia coli expression purifying is seen Fig. 3 D.
(3) co-immunoprecipitation experiment
This test is used to verify that MCRS2 and hTRET are in external combining.FLAG-MCRS2 and GFP-hTERT expression plasmid transient transfection in the 293T cell, are carried out co-immunoprecipitation with anti-FLAG antibody, use GFP antibody test GFP-hTERT then.Shown in Fig. 3 E, in the FLAG immunoprecipitation complex, can detect GFP-hTERT, this illustrates that MCRS2 and Telomerase mixture combine in vivo.
Embodiment 5, cell in vitro experimental results show that MCRS2 can cause the tumour cell telomere to shorten.
With MCRS2 stable gene transfection liver cancer cell SMMC-7721, and the stable transfected cells strain of screening expression FLAG-MCRS2 and FLAG-MCRS2-N (162 amino-acid residues of N end that comprise MCRS2) (Fig. 4 A, B).Method is as follows, in six orifice plates, according to every hole 2 * 10
5Cell concentration is inoculated the SMMC-7721 liver cancer cell in the 2mL perfect medium, 37 ℃ of overnight incubation; Preparation solution A: dissolve the ultrapure DNA of 1.5 μ g cell transfecting levels (FLAG-MCRS2 and FLAG-MCRS2-N) in the substratum of 100 μ L serum-free antibiotic-frees; The preparation solution B: 5 μ L LipofectAMIN (GIBCO BRL company product) reagent is diluted in the substratum of 100 μ L serum-free antibiotic-frees.Mixed solution A, B, room temperature was placed 45 minutes, formed liposome particles.Use PBS successively, the substratum rinse cell of serum-free antibiotic-free.In forming the pipe of liposome particles, add the substratum of 0.8mL serum-free antibiotic-free, be added to behind the mixing gently on the cell of rinse, 37 ℃ of transfections 5 hours.In each hole, add the perfect medium that contains 10% new-born calf serum.After the transfection 24 hours, be changed to fresh perfect medium.Divide the bottle back to add the G418 of proper concn by 1: 10 cell then, screening has the cell of Neo gene resistance.The resistant cell that obtains is FLAG-MCRS2 and the strain of FLAG-MCRS2-N stable transfected cells.Measure the length of cell telomere then.Adopt the length of TRF (terminal restrictionfragment) Southern technical Analysis telomere.The genome DNA extraction of cell is come out, after getting 10 μ gDNA and cutting, separate through 0.7% agarose with HinfI and RsaI enzyme.Glue closes 30min at 0.5M NaOH/1.5M NaCl sex change 30min in 0.5M Tris-HCl/3M NaCl (pH7.5), carry out the Southern engram analysis.Probe is
32The end-labelled strand of P (TTAGGG)
6DNA.
The contrast that experimental results show that the transfection empty carrier is consistent with its telomere length of cell, does not shorten.And the stable cell line of expression FLAG-MCRS2 and FLAG-MCRS2-N, along with constantly going down to posterity, their telomere is constantly reducing (Fig. 4 A and B).Illustrate that MCRS2 can suppress the synthetic of telomere in cell paste.
Reorganization MCRS2 albumen with escherichia coli expression carries out the vitro inhibition telomerase activity.6 * His-MCRS2 and GST-MCRS2-N are joined in the cell pyrolysis liquid, hatched 10 minutes for 4 ℃, carry out telomerase activity then.Adopt common TRAP (telomeric repeat amplification protocol) method to detect telomerase activation.This method is a kind of activity test method of telomerase of PCR-based technology.Concrete operations are as follows: culturing cell is washed one time with PBS, centrifugal collecting cell; (1). with lavation buffer solution (10mM Hepes-KOH (pH7.5), 1.5mMMgCl
2, 10mM KCl, 1mM DTT (dithiothreitol (DTT))) and wash cell once more one time; (2) ice-cold lysis buffer (10mM Tris-HCl (pH 7.5), 1mM MgCl
2, 1mM EGTA, 0.1mM PMSF, 5mM mercaptoethanol, 0.5%CHAPS, 10% glycerine) and re-suspended cell, put 30 minutes on ice, 4 ℃ of high speed centrifugations 30 minutes, the supernatant cell extract can be used to measure telomerase activation.The supernatant extract can be-70 ℃ of preservations, and molten still maintenance of freezing that telomerase activation can stand repeatedly stablizes; (3). add in the reaction tubes: 1 μ L Ts primer (0.1 μ g/ μ L), 1 μ L cell extract supernatant, 0.25 μ L 10mM dNTP, 42 μ L reaction buffer (20mM Tns-HCl (pH8.3), 1.5mM MgCl
2, 63mM KCl, 0.005%Tween-20,1mM EGTA, 0.1mg/mL BSA).25 ℃ of extensions 30 minutes, 90 ℃ of deactivations 3 minutes; (4) add 1 μ L Cx primer, 0.5 μ L (2U) Taq enzyme; Carry out the PCR reaction: 94 ℃ of 2min; 94 ℃ of 40sec, 50 ℃ of 40sec, 72 ℃ of 1min, 30cycles; (5) the PCR reaction product is carried out the non-sex change gel electrophoresis of 10%PAGE, and damping fluid is 0.5 * TBE, voltage 100V, 6～8 hours; 6.PAGE glue silver dyes colour developing, takes pictures.
The Ts primer: 5 '-AATCCGTCGAGCAGAGTT-3 ' (SEQ ID NO:5)
The Cx primer: 5 '-(CCCTTA)
3CCCTAA-3 ' (SEQ ID NO:6)
Experimental result is seen Fig. 5.Experimental results show that 6 * His-MCRS2 and GST-MCRS2-N have inhibition of telomerase, and present concentration dependent, promptly telomerase activation descends with the increase of MCRS2 protein concentration.Contrast GST albumen does not suppress active.
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
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉a kind of microspherule protein 2 and application thereof
<130>041783
<160>6
<170>PatentIn version 3.1
<210>1
<211>1953
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
<221>CDS
<222>(173)..(1597)
<223>
<400>1
cccctgggcc agcaccaccc atgtacctgt cggcagagcc acagttgact gacttgctgt 60
tgagctgctg ctctttagca cggctgcatt ttgatggcag agggagcagt tatgcccaga 120
gcccagcgtg tcggcatcac cagtctgacc tgagcaccat gtgctgcacg ac atg aca 178
Met Thr
1
cgt ggc acc ggg gga act gcc cag cgt gga agg tct ggg cca gat tct 226
Arg Gly Thr Gly Gly Thr Ala Gln Arg Gly Arg Ser Gly Pro Asp Ser
5 10 15
cag ggg ctg cta gat tca tcc ctg atg gca tca ggc act gcc agc cgc 274
Gln Gly Leu Leu Asp Ser Ser Leu Met Ala Ser Gly Thr Ala Ser Arg
20 25 30
tca gag gat gag gag tca ctg gca ggg cag aag cga gcc tcc tcc cag 322
Ser Glu Asp Glu Glu Ser Leu Ala Gly Gln Lys Arg Ala Ser Ser Gln
35 40 45 50
gcc ttg ggc acc atc cct aaa cgg aga agc tcc tcc agg ttc atc aag 370
Ala Leu Gly Thr Ile Pro Lys Arg Arg Ser Ser Ser Arg Phe Ile Lys
55 60 65
agg aag aag ttc gat gat gag ctg gtg gag agc agc ctg gca aaa tct 418
Arg Lys Lys Phe Asp Asp Glu Leu Val Glu Ser Ser Leu Ala Lys Ser
70 75 80
tct acc cgg gca aag ggg gcc agt ggg gtg gaa cca ggg cgc tgt tcg 466
Ser Thr Arg Ala Lys Gly Ala Ser Gly Val Glu Pro Gly Arg Cys Ser
85 90 95
ggg agt gaa ccc tcc tcc agt gag aag aag aag gta tcc aaa gcc ccc 514
Gly Ser Glu Pro Ser Ser Ser Glu Lys Lys Lys Val Ser Lys Ala Pro
100 105 110
agc act cct gtg cca ccc agc cca gcc cca gcc cct gga ctc acc aag 562
Ser Thr Pro Val Pro Pro Ser Pro Ala Pro Ala Pro Gly Leu Thr Lys
115 120 125 130
cgt gtg aag aag agt aaa cag cca ctt cag gtg acc aag gat ctg ggc 610
Arg Val Lys Lys Ser Lys Gln Pro Leu Gln Val Thr Lys Asp Leu Gly
135 140 145
cgc tgg aag cct gca gat gac ctc ctg ctc ata aat gct gtg ttg cag 658
Arg Trp Lys Pro Ala Asp Asp Leu Leu Leu Ile Asn Ala Val Leu Gln
150 155 160
acc aac gac ctg acc tcc gtc cac ctg ggc gtg aaa ttc agc tgc cgc 706
Thr Asn Asp Leu Thr Ser Val His Leu Gly Val Lys Phe Ser Cys Arg
165 170 175
ttc acc ctt cgg gag gtc cag gag cgt tgg tac gcc ctg ctc tac gat 754
Phe Thr Leu Arg Glu Val Gln Glu Arg Trp Tyr Ala Leu Leu Tyr Asp
180 185 190
cct gtc atc tcc aag ttg gcc tgt cag gcc atg agg cag ctg cac cca 802
Pro Val Ile Ser Lys Leu Ala Cys Gln Ala Met Arg Gln Leu His Pro
195 200 205 210
gag gct att gca gcc atc cag agc aag gcc ctg ttt agc aag gct gag 850
Glu Ala Ile Ala Ala Ile Gln Ser Lys Ala Leu Phe Ser Lys Ala Glu
215 220 225
gag cag ctg ctg agc aaa gtg gga tcg acc agc cag ccc acc ttg gag 898
Glu Gln Leu Leu Ser Lys Val Gly Ser Thr Ser Gln Pro Thr Leu Glu
230 235 240
acc ttc cag gac ctg ctg cac aga cac cct gat gcc ttc tac ctg gcc 946
Thr Phe Gln Asp Leu Leu His Arg His Pro Asp Ala Phe Tyr Leu Ala
245 250 255
cgt acc gcg aag gcc ctg cag gcc cac tgg cag ctc atg aag cag tat 994
Arg Thr Ala Lys Ala Leu Gln Ala His Trp Gln Leu Met Lys Gln Tyr
260 265 270
tac ctg ctg gag gac cag aca gtg cag ccg ctg ccc aaa ggg gac caa 1042
Tyr Leu Leu Glu Asp Gln Thr Val Gln Pro Leu Pro Lys Gly Asp Gln
275 280 285 290
gtg ctg aac ttc tct gat gca gag gac ctg att gat gac agt aag ctc 1090
Val Leu Asn Phe Ser Asp Ala Glu Asp Leu Ile Asp Asp Ser Lys Leu
295 300 305
aag gac atg cga gat gag gtc ctg gaa cat gag ctg atg gtg gct gac 1138
Lys Asp Met Arg Asp Glu Val Leu Glu His Glu Leu Met Val Ala Asp
310 315 320
cgg cgc cag aag cga gag att cgg cag ctg gaa cag gaa ctg cat aag 1186
Arg Arg Gln Lys Arg Glu Ile Arg Gln Leu Glu Gln Glu Leu His Lys
325 330 335
tgg cag gtg cta gtg gac agc atc aca ggc atg agc tct ccg gac ttc 1234
Trp Gln Val Leu Val Asp Ser Ile Thr Gly Met Ser Ser Pro Asp Phe
340 345 350
gac aac cag aca ctg gca gtg ctg cgg ggc cgc atg gtg cgg tac ctg 1282
Asp Asn Gln Thr Leu Ala Val Leu Arg Gly Arg Met Val Arg Tyr Leu
355 360 365 370
atg cgc tcg cgt gag atc acc ctg ggc aga gca acc aag gat aac cag 1330
Met Arg Ser Arg Glu Ile Thr Leu Gly Arg Ala Thr Lys Asp Asn Gln
375 380 385
att gat gtg gac ctg tct ctg gag ggt ccg gcc tgg aag ata tcc cgg 1378
Ile Asp Val Asp Leu Ser Leu Glu Gly Pro Ala Trp Lys Ile Ser Arg
390 395 400
aaa caa ggt gtc atc aag ctg aag aac aac ggt gat ttc ttc att gcc 1426
Lys Gln Gly Val Ile Lys Leu Lys Asn Asn Gly Asp Phe Phe Ile Ala
405 410 415
aat gag ggt cga cgg ccc atc tac atc gat gga cgg ccg gtg ctc tgt 1474
Asn Glu Gly Arg Arg Pro Ile Tyr Ile Asp Gly Arg Pro Val Leu Cys
420 425 430
ggc tcc aaa tgg cgc ctc agc aac aac tct gtg gtg gag atc gcc agc 1522
Gly Ser Lys Trp Arg Leu Ser Asn Asn Ser Val Val Glu Ile Ala Ser
435 440 445 450
ctg cga ttc gtc ttc ctt atc aac cag gac ctc att gcc ctc atc agg 1570
Leu Arg Phe Val Phe Leu Ile Asn Gln Asp Leu Ile Ala Leu Ile Arg
455 460 465
gct gag gct gcc aag atc aca cca cag tgaggagtgg tggcaggact 1617
Ala Glu Ala Ala Lys Ile Thr Pro Gln
470 475
cgtgggccct ctccggcctg tttcccctgc cactccagcc cccttgagct gggaactcag 1677
gctcctggaa aaacctgggc agtgggaggc tcagctgcgg gccattgatt tgagcctttg 1737
agggaggata gggctggcct ttgtgaagcc agcagaggct gagaacctca ggcttcccta 1797
gatccagagc ccctccccat cttcctctct ctaaaaacaa ccctaccccc cattgccacc 1857
ttcactcctg tgtctccagc tgattagcct cagactcttc ttttattgtt tttcttttgt 1917
aaataaaaag caccaggttc aaaaaaaaaa aaaaaa 1953
<210>2
<211>475
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>2
Met Thr Arg Gly Thr Gly Gly Thr Ala Gln Arg Gly Arg Ser Gly Pro
1 5 10 15
Asp Ser Gln Gly Leu Leu Asp Ser Ser Leu Met Ala Ser Gly Thr Ala
20 25 30
Ser Arg Ser Glu Asp Glu Glu Ser Leu Ala Gly Gln Lys Arg Ala Ser
35 40 45
Ser Gln Ala Leu Gly Thr Ile Pro Lys Arg Arg Ser Ser Ser Arg Phe
50 55 60
Ile Lys Arg Lys Lys Phe Asp Asp Glu Leu Val Glu Ser Ser Leu Ala
65 70 75 80
Lys Ser Ser Thr Arg Ala Lys Gly Ala Ser Gly Val Glu Pro Gly Arg
85 90 95
Cys Ser Gly Ser Glu Pro Ser Ser Ser Glu Lys Lys Lys Val Ser Lys
100 105 110
Ala Pro Ser Thr Pro Val Pro Pro Ser Pro Ala Pro Ala Pro Gly Leu
115 120 125
Thr Lys Arg Val Lys Lys Ser Lys Gln Pro Leu Gln Val Thr Lys Asp
130 135 140
Leu Gly Arg Trp Lys Pro Ala Asp Asp Leu Leu Leu Ile Asn Ala Val
145 150 155 160
Leu Gln Thr Asn Asp Leu Thr Ser Val His Leu Gly Val Lys Phe Ser
165 170 175
Cys Arg Phe Thr Leu Arg Glu Val Gln Glu Arg Trp Tyr Ala Leu Leu
180 185 190
Tyr Asp Pro Val Ile Ser Lys Leu Ala Cys Gln Ala Met Arg Gln Leu
195 200 205
His Pro Glu Ala Ile Ala Ala Ile Gln Ser Lys Ala Leu Phe Ser Lys
210 215 220
Ala Glu Glu Gln Leu Leu Ser Lys Val Gly Ser Thr Ser Gln Pro Thr
225 230 235 240
Leu Glu Thr Phe Gln Asp Leu Leu His Arg His Pro Asp Ala Phe Tyr
245 250 255
Leu Ala Arg Thr Ala Lys Ala Leu Gln Ala His Trp Gln Leu Met Lys
260 265 270
Gln Tyr Tyr Leu Leu Glu Asp Gln Thr Val Gln Pro Leu Pro Lys Gly
275 280 285
Asp Gln Val Leu Asn Phe Ser Asp Ala Glu Asp Leu Ile Asp Asp Ser
290 295 300
Lys Leu Lys Asp Met Arg Asp Glu Val Leu Glu His Glu Leu Met Val
305 310 315 320
Ala Asp Arg Arg Gln Lys Arg Glu Ile Arg Gln Leu Glu Gln Glu Leu
325 330 335
His Lys Trp Gln Val Leu Val Asp Ser Ile Thr Gly Met Ser Ser Pro
340 345 350
Asp Phe Asp Asn Gln Thr Leu Ala Val Leu Arg Gly Arg Met Val Arg
355 360 365
Tyr Leu Met Arg Ser Arg Glu Ile Thr Leu Gly Arg Ala Thr Lys Asp
370 375 380
Asn Gln Ile Asp Val Asp Leu Ser Leu Glu Gly Pro Ala Trp Lys Ile
385 390 395 400
Ser Arg Lys Gln Gly Val Ile Lys Leu Lys Asn Asn Gly Asp Phe Phe
405 410 415
Ile Ala Asn Glu Gly Arg Arg Pro Ile Tyr Ile Asp Gly Arg Pro Val
420 425 430
Leu Cys Gly Ser Lys Trp Arg Leu Ser Asn Asn Ser Val Val Glu Ile
435 440 445
Ala Ser Leu Arg Phe Val Phe Leu Ile Asn Gln Asp Leu Ile Ala Leu
450 455 460
Ile Arg Ala Glu Ala Ala Lys Ile Thr Pro Gln
465 470 475
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>3
atgacacgtg gcaccggggg aa 22
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>4
tcactgtggt gtgatcttgg ca 22
<210>5
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>5
atccgtcgag cagagtt 17
<210>6
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>6
cccttaccct tacccttacc ctaa 24
Claims (10)
1. isolating people MCRS2 polypeptide is characterized in that this polypeptide is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the function that suppresses telomerase activation by (a) polypeptides derived.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is to have 1-475 position or 1-162 amino acids polypeptide of sequence among the SEQ ID NO:2.
3. isolating polynucleotide is characterized in that, these polynucleotide are selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with the complete complementary polynucleotide of polynucleotide (a).
4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in 1-475 position among the SEQ ID NO:2 or the 1-162 position.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:
(a) has the sequence of 173-1597 position among the SEQ ID NO:1;
(b) has the sequence of 1-1953 position among the SEQ ID NO:1;
(c) has the sequence of 173-658 position among the SEQ ID NO:1.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. the preparation method of a peptide species is characterized in that, this method contains:
(a) under conditions suitable for the expression, cultivate the described host cell of claim 7;
(b) from culture, isolate people MCRS2 protein polypeptide.
9. energy and the described people MCRS2 of claim 1 protein-specific bonded antibody.
10. a pharmaceutical composition that is used to suppress telomerase activation or treatment tumour is characterized in that it contains the described MCRS2 polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410017051 CN1289524C (en) | 2004-03-19 | 2004-03-19 | Human telomerase active inhibitor protein and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410017051 CN1289524C (en) | 2004-03-19 | 2004-03-19 | Human telomerase active inhibitor protein and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1670032A CN1670032A (en) | 2005-09-21 |
| CN1289524C true CN1289524C (en) | 2006-12-13 |
Family
ID=35041495
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410017051 Expired - Fee Related CN1289524C (en) | 2004-03-19 | 2004-03-19 | Human telomerase active inhibitor protein and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1289524C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1948339B (en) * | 2005-10-14 | 2010-09-29 | 中国科学院上海生命科学研究院 | Preparation and purification of telomerase activity inhibition protein |
| CN112899251A (en) * | 2019-12-04 | 2021-06-04 | 陕西光子动力航天科技有限公司 | Preparation method of fixed-method telomerase |
-
2004
- 2004-03-19 CN CN 200410017051 patent/CN1289524C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1670032A (en) | 2005-09-21 |
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