CN1712541B - Screen and use for labelled heterogenic ribonucleoprotein K of protein molecule related to hepatocellular carcinoma - Google Patents
Screen and use for labelled heterogenic ribonucleoprotein K of protein molecule related to hepatocellular carcinoma Download PDFInfo
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Abstract
Heterogeneous nuclear ribonucleoprotein K relating to liver cell cancer and its use are disclosed. It has high expression and can be used for effective marker.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to relevant protein molecular marker-xenogenesis ribonucleoprotein K (hnRNP K albumen) of a kind of hepatocellular carcinoma and uses thereof.
Background technology
Liver cancer is a kind of serious harm Human diseases.The sickness rate of western developed country liver cancer is lower; Still comparatively weak to the fundamental research of liver cancer in the world, and China country occurred frequently that is liver cancer, M & M presents ascendant trend; And age of onset constitutes rejuvenation; The medical expense that is used for liver cancer treatment every year greatly increases, and liver cancer has become serious harm China people life property safety's dead enemy, and is an important factor that influences socio-economic development; The fundamental research of going into overdrive to carry out China's liver cancer has strategic importance, and separates and identify that new liver cancer related gene is the advanced subject in the present liver cancer fundamental research.
Up to the present; The gene unconventionality expression that does not have 20 kinds is determined relevant with the incidence and development of liver cancer; But the unconventionality expression rate of fixed liver cancer related gene in liver cancer is not high, and the pathogenesis of liver cancer is not illustrated so far yet, and the early diagnostic rate of liver cancer still remains to be improved.In addition; Traditional operation of liver cancer adds chemotherapy and the several genes treat-ment that is used does not in recent years still have obviously to improve the survival rate of liver cancer patient, thereby especially liver cance high-expression gene is significant for the pathogenesis of inquiring into liver cancer to seek new liver cancer related gene.
The Genebank accession number of xenogenesis ribonucleoprotein K (heterogeneous nuclear ribonucleoprotein K, hnRNP K) is gi|13384620, and the accession number of NCBI is NP_079555.Its nucleotide sequence is shown in SEQ ID NO:1, and wherein ORF is positioned at the 210-1604 position, and code length is 464 amino acid whose albumen (SEQ ID NO:2).HnRNP K is a kind of RNA, dna binding protein dna, and (for example, Ser 302 is action site (Schullery, D.S. of PKC δ to comprise several potential phosphorylation sites; Ostrowski, J.; Denisenko, O.N.; Stempka, L.; Shnyreva, M.; Suzuki, H.; Gschwendt, M.; Bomsztyk; K.Regulated interaction of protein kinase Cdeltawith the heterogeneous nuclear ribonucleoprotein K protein.J.Biol.Chem.1999; 274; 15101-15109.)) and three KH structural domains, participate in sequence specific RNA or DNA combination, transcribe, important biological procedureses such as translation, RNA processing, caryoplasm transportation, signal transduction, gene expression regulation.HnRNP K and epidermal growth factor family (Epidermal growth factor family, EGFfamily), nonreceptor tyrosine kinase pp60 (c-src) etc. all has interaction relationship (Ritchie, S.A.; Pasha, M.K.; Batten, D.J.; Sharma, R.K.; Olson, D.J.; Ross, A.R.; Bonham; K.Identification of the SRC pyrimidine-binding protein (SPy) as hnRNP K:implications in the regulation of SRC1A transcription.Nucleic Acids Res.2003; 31,1502-1513; Mandal, M.; Vadlamudi, R.; Nguyen, D.; Wang, R.A.; Costa, L.; Bagheri-Yarmand, R.; Mendelsohn, J.; Kumar, R.Growth factorsregulate heterogeneous nuclear ribonucleoprotein K expression andfunction.J.Biol.Chem.2001,276,9699-9704; Ostareck-Lederer, A.; Ostareck, D.H.; Cans, C.; Neubauer, G.; Bomsztyk, K.; Superti-Furga, G.; Hentze, M.W.c-Src-mediated phosphorylation of hnRNP K drivestranslational activation of specifically silenced mRNAs.Mol.Cell Biol.2002,22,4535-4543; Bomsztyk, K.; Van, S., I; Suzuki, H.; Denisenko, O.; Ostrowski, J.Diverse molecular interactions of the hnRNP K protein.FEBSLett.1997,403,113-115; Habelhah, H.; Shah, K.; Huang, L.; Ostareck-Lederer, A.; Burlingame, A.L.; Shokat, K.M.; Hentze, M.W.; Ronai, Z.ERKphosphorylation drives cytoplasmic accumulation of hnRNP-K and inhibitionof mRNA translation.Nat.Cell Biol.2001,3,325-330; Miau, L.H.; Chang, C.J.; Shen, B.J.; Tsai, W.H.; Lee; S.C.Identification ofheterogeneous nuclear ribonucleoprotein K (hnRNP K) as a repressor ofC/EBPbeta-mediated gene activation.J.Biol.Chem.1998; 273,10784-10791; Michael, W.M.; Eder, P.S.; Dreyfuss, G.The K nuclear shuttling domain:anovel signal for nuclear import and nuclear export in the hnRNP K protein.EMBOJ.1997,16,3587-3598.).
The mistake of xenogenesis ribonucleoprotein K is expressed in mammary cancer breast cancer and colorectal carcinoma colon cancer has report.But the relevant report that does not also have up to now, xenogenesis ribonucleoprotein K and hepatocellular carcinoma.
Therefore, to research and develop in liver cancer the gene and/or the albumen of high expression level significant for treatment and diagnostic purpose.This area press for new in liver cancer the gene and/or the albumen of high expression level.
Summary of the invention
The purpose of this invention is to provide a kind of people's xenogenesis ribonucleoprotein K (being called " hnRNP K albumen " again) new, high expression level in liver cancer
Another object of the present invention provides the application of xenogenesis ribonucleoprotein K aspect the detection hepatocellular carcinoma.
In first aspect of the present invention, the purposes of a kind of hnRNP K albumen or its encoding sequence is provided, it is used to prepare the reagent that detects liver cancer.
In second aspect of the present invention, a kind of external method of confirming that hnRNP K gene expression amount in the liver cell to be measured is whether unusual is provided, it comprises step:
(a) extracting hepatocellular mRNA to be measured, and reverse transcription is cDNA;
(b) with the primer of specific amplification hnRNP K transcript, be template with the cDNA of step (a), obtain the amplified production of hnRNP K through the quantitative PCR method amplification;
(c) the hnRNP K amplified production quantity with hepatocellular hnRNP K amplified production quantity to be measured and normal liver cell in the step (b) compares, and the hnRNP K amplified production quantity that is higher than normal liver cell just representes that hnRNP K gene expression amount exists unusual in the liver cell to be measured.
In another preference, described quantitative PCR method is the quantitative fluorescence PCR method.
In second aspect of the present invention, a kind of external method of confirming that hnRNP K expressing quantity in the liver cell to be measured is whether unusual is provided, it comprises step:
(a) with the proteic quantity of hnRNP K in the proteic antibody test of the specific anti hnRNP K liver cell to be measured;
(b) the hnRNP K albumen quantity with hepatocellular hnRNP K albumen quantity to be measured and normal liver cell in the step (a) compares, and the hnRNP K albumen quantity that is higher than normal liver cell just representes that hnRNP K expressing quantity exists unusual in the liver cell to be measured.
In another preference, described antibody is monoclonal antibody or polyclonal antibody.
In the third aspect of the invention, a kind of test kit that detects liver cancer is provided, it comprises: the primer of specific amplification hnRNP K transcript and/or the antibody of specific anti hnRNP K, and specification sheets.
In another preference, described test kit also contains the agent that is selected from down group:
(a) positive control;
(b) negative control;
(c) specificity is incorporated into the probe of hnRNP K.
In fourth aspect of the present invention, the purposes of a kind of hnRNP K albumen or its transcript is provided, it is used as the affinity tag that detects liver cancer.
Aspect the of the present invention the 5th, the purposes of the antibody of a kind of anti-hnRNP K is provided, it is used to prepare the reagent that detects liver cancer or is used to prepare the medicine of treating liver cancer.
Aspect the of the present invention the 6th, a kind of method that detects liver cancer or liver cancer susceptibility is provided, comprise step:
(a) with the primer of specific amplification hnRNP K transcript, be template with the cDNA of individual hepatocyte to be detected, obtain the amplified production of hnRNP K through the quantitative PCR method amplification, detect the amplified production number that forms and whether be higher than normal control; Perhaps under the condition that is fit to the formation antibody complex, the proteic antibody of specific anti hnRNPK is contacted with the sample of individuality to be measured, and whether the antibody complex quantity that detection forms is higher than normal control;
Whether the amplified production number or the antibody complex quantity that (b) form are higher than normal control, just represent that this individuality suffers from liver cancer or liver cancer susceptibility is higher than normal population.
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
Attached drawings is used to explain specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 shows that xenogenesis ribonucleoprotein K (hnRNP K) (some SSP 3709,3729) expresses obviously rise in hepatocellular carcinoma patient's cancerous tissue.
Fig. 2 has shown the immunoblotting assay result to hnRNP K.
Embodiment
The inventor is with non-enzymolysis sample preparation method (nonenzymatic sample preparation; NESP) cancerous tissue and cancer beside organism's protein example of the hepatocellular carcinoma of preparation are identified with the differential expression protein point and the mass spectrum of conventional two dimensional gel electrophoresis technology screening.The result finds xenogenesis ribonucleoprotein K (hnRNP K) high expression level in the hepatocellular carcinoma cancerous tissue.Immunoblot experiment confirms that further xenogenesis ribonucleoprotein K there are differences expression really in the cancerous tissue of hepatocellular carcinoma and cancer beside organism.With meticulous laser capture microdissection technology (laser-capturemicrodissection; LCM) the hepatocellular carcinoma cancerous tissue of preparation and the hepatic parenchymal cells protein example of cancer beside organism; (isotope-coded affinity tagging ICAT) has also further confirmed the differential expression of hnRNP K with isotropic substance code affinity tag technology.Accomplished the present invention on this basis.
Definition
As used herein, " nucleic acid " refers to the deoxyribonucleotide or the ribonucleoside acid polymer of strand or double chain form.This term has comprised the analogue of the natural nucleotide that can play a role with the natural nucleotide similar fashion unless otherwise indicated.
" hybridization " refers to through the pairing of complementary base two single-chain nucleic acids combined.
" be incorporated into basically " or " specific combination " or " selective binding " or " specific hybrid in "; The complementary hybridization of finger between oligonucleotide and target sequence; And can comprise small mispairing, these mispairing can realize detecting required target polynucleotide sequence through the tight degree that reduces hybridization medium.This term also refers under stringent condition, and a part combines, compound or hybridize in specific nucleotide sequence, when this sequence is present in the compound mixture (like total cell NDA or RNA).Term " stringent condition " refers to that probe hybridization decides subsequence and do not hybridize the condition in other sequences in target.Stringent condition is sequence-dependent, and under different situations, is different.Long sequence can be under higher temperature specific hybrid.Usually, selected stringent condition is than low about 5 ℃ of the pyrolysis chain temperature (Tm) of under ionic strength that limits and pH, inferring sequence.This Tm is when reaching balance, have an appointment 50% with the probe of target complement sequence the temperature (under ionic strength, pH and the nucleic acid concentration of qualification) during with target sequence hybridization.Usually, for short probe, stringent condition is such condition, and wherein salt concn is at least about 0.02 Na ionic concn (or other salt), pH7.0-8.3, and temperature is at least about 60 ℃.Stringent condition also can be realized through adding destabilizing agent such as methane amide.
Person of skill in the art will appreciate that the concrete primer described herein and the definite sequence of probe can be revised (modifications) to a certain extent, to produce but reservation is incorporated into the ability of target sequence basically with disclosed primer or probe " basic identical ".
HnRNP K albumen and gene
Based on the dependency of disclosed hnRNP K and liver cancer and the sequence information of hnRNP K, those skilled in the art's ordinary skill in the art capable of using produces hnRNP K gene, albumen or its fragment.
In the present invention; Term " xenogenesis ribonucleoprotein K ", " hnRNP K albumen ", " hnRNP K polypeptide ", " liver cance high-expression albumen hnRNP K " or interchangeable uses such as " albumen of the present invention "; All refer to have people or other mammiferous xenogenesis ribonucleoprotein K (the Genebank accession number is gi|13384620, and the accession number of NCBI is NP_079555), it is a kind of solubility endochylema phosphorprotein of high conservative; Mainly be present in the karyon, can between caryoplasm, shuttle back and forth.A kind of preferred especially xenogenesis ribonucleoprotein K is people's hnRNPK, and its aminoacid sequence is shown in SEQ ID NO:2, and nucleotide sequence is shown in SEQ ID NO:1, and ORF is positioned at the 210-1604 position.
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 like 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, " isolating hnRNP K albumen or polypeptide " is meant that hnRNP K 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 hnRNP K albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
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 from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.
The present invention also comprises the proteic fragment of people hnRNP K, verivate and analogue.Polypeptide fragment of the present invention, verivate 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 so substituted amino-acid residue 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 (like 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, verivate and analogue belong to the known scope of those skilled in the art.
In the present invention, term " people hnRNP K polypeptide " refers to have the SEQ IDNO.2 polypeptide of sequence of people hnRNP K protein-active.This term also comprises having and variant form people hnRNP K 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; 1-20 more preferably, 1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20; Preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people hnRNP K 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 hnRNP K DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people hnRNP K polypeptide to obtain.The present invention also provides other polypeptide, as comprises people hnRNP K polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people hnRNP K polypeptide.Usually; This fragment have people hnRNP K 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.
In the present invention; " people hnRNP K 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.
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 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 (like PCR) of nucleic acid to confirm and/or the proteic polynucleotide of separation coding hnRNP K.
People hnRNP K Nucleotide full length sequence of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain 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 need carries 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 come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, through first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
Recombinant DNA technology (Science, 1984 through routine; 224:1431), polymerized nucleoside acid sequence of the present invention capable of using can be used to express or produce the hnRNP K polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding human hnRNP K polypeptide of the present invention, or with recombinant expression vector conversion that contains these polynucleotide or transduction proper host cell;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
It all is well known to those skilled in the art being used for the proteic carrier of recombinant expressed the present invention, host cell and cultivation and stripping technique etc.
The proteic antibody of anti-hnRNP K
On the other hand, the present invention also comprises people hnRNP K DNA or the polypeptide of its segment encoding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people hnRNP K gene product or fragment.Preferably, refer to that those can combine with people hnRNP K gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody comprises that those can combine and suppress the proteic molecule of people hnRNP K among the present invention, comprises that also those do not influence the antibody of people hnRNP K protein function.The present invention also comprise those can with modify or without the people hnRNP K 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, like Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, United States Patent(USP) No. 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 prepare through the known various technology of those skilled in that art.For example, the people hnRNP K gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human hnRNP K 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 hnRNP K protein function and the antibody that does not influence people hnRNP K protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people hnRNP K gene product, obtains through the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can use the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) to come immune animal and produce with the unmodified form bonded antibody of people hnRNP K gene product; With posttranslational modification form bonded antibody (like the albumen or the polypeptide of glycosylation or phosphorylation), can use the gene product that produces in the eukaryotic cell (for example yeast or insect cell) to come immune animal and obtain.
The proteic antibody of anti-people hnRNP K can be used in the immunohistochemistry technology, detects the people hnRNP K albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people hnRNP K, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.
Antibody among the present invention can be used for detecting and the relevant disease of people hnRNP K albumen, like liver cancer.Antibody also can be used for being designed to the immunotoxin to a certain privileged sites in the body.As the monoclonal antibody of people hnRNP K albumen high-affinity can with bacterium or plant poison (like 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, through the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell (for example liver cancer cell etc.) of people hnRNP K protein positive.Because hnRNP K albumen of the present invention is specificity overexpression in liver cancer cell, this hybrid antibody can be used for directionally killing liver cancer cell.
The production of polyclonal antibody can choose hnRNP K albumen or polypeptide immune animal, like rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Pharmaceutical composition
The proteic antagonist of the present invention (like antibody) when in treatment, using (administration), can provide the effect of treatment.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 through conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
The antagonist of polypeptide of the present invention can be used for the especially treatment of liver cancer of tumour.When using hnRNP K albumen of the present invention, also can use the other treatment agent simultaneously, like IFN-α, IFN-β, TNF-α, TNF-β etc.
The present invention also provides a kind of pharmaceutical composition, and it contains antagonist and the pharmaceutically acceptable carrier or the vehicle of (like 0.01-99wt%) hnRNP K of the present invention polypeptide of safe and effective amount.This type carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical prepn should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example prepares through ordinary method with the saline water or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can prepare through 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.
When making pharmaceutical composition; Be that the hnRNP K albumen of safe and effective amount or its antagonist, agonist are 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 is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.
The proteic polynucleotide of people hnRNP K also can be used for various therapeutic purposes.Gene therapy technology can be used for treating because cell proliferation, growth or metabolic disturbance (like liver cancer) due to the proteic expression of hnRNP K of the proteic abnormal expression of hnRNP K or representative.
Suppress the oligonucleotide (comprising sense-rna and DNA) of people hnRNP K mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme appearance RNA molecule that a kind of ability 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 use existing any RNA or DNA synthetic technology to obtain, like the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external through carrier (like virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Detection method
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people hnRNP K protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.Whether the people hnRNPK protein level that is detected in the test can be higher than one of index of normal population as the probability that definite individuality to be detected suffers from liver cancer or liver cancer susceptibility.
Whether having the proteic method of hnRNP K in a kind of detection test sample is to utilize the proteic specific antibody of hnRNP K to detect, and it comprises: sample is contacted with hnRNP K protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample hnRNP K albumen.
The proteic polynucleotide of hnRNP K can be used for the diagnosis of hnRNP K gene-correlation disease.Aspect diagnosis, the proteic polynucleotide of hnRNP K can be used for detecting hnRNP K expression of gene and whether is higher than normal population.Correlation technique comprises quantitative PCR method (like quantitative fluorescent PCR) etc., and these technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial sources.
Polynucleotide of the present invention a part or all can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), be 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 hnRNP K albumen and also can detect the proteic transcription product of hnRNP K.
Because the very high expression level rate of hnRNP K in liver cancer; Apparently higher than the AFP that is used for diagnosing cancer of liver at present (the about 50-60% of its recall rate); So hnRNP K polynucleotide, hnRNP K albumen and antibody thereof; And the relevant antagonist of hnRNP K albumen, agonist etc. can be treatment and comprise that multiple disease such as liver cancer provides new treatment approach, thereby have great application prospect.
Test kit
Whether unusual the present invention also provide diagnosis hnRNP K to express diagnostic kit.In preference, a kind of test kit comprises the primer of specific amplification hnRNP K transcript and/or the antibody of specific anti hnRNP K.Test kit also can contain description and how use test kit to detect the illustrative material of hnRNP K.Test kit also can contain one or more in the following group: be used to assist the various affinity tags or the labelled reagent that detect; The reagent (comprising damping fluid) that is used for PCR; The reagent that is used for immuning hybridization; And positive and negative control etc.
Should be understood that after the present invention has disclosed the dependency of the unusual and liver cancer of hnRNP K expression of gene first, but those skilled in the art can design the primer that specific amplification goes out hnRNP K easily, confirm its expression amount through methods such as detection by quantitative then.Usually, the length of primer is 15-50bp, preferably is 20-30bp.Though the complete complementation of primer and template sequence is preferred, one skilled in the art will appreciate that at primer and template to have under the situation of certain not complementary (especially 5 of primer ' end) also can increase specifically (promptly only amplifying required fragment).The test kit that contains these primers and the method for using these primers are all within the scope of the invention, as long as the amplified production that this primer amplification goes out contains total length or the partial sequence of hnRNP K.A kind of preferred primer is to having the sequence of SEQ ID NO:3 and 4.
Though the not special restriction of the length of amplified production, the length of amplified production is 100-3000bp usually, preferably is 150-2000bp, more preferably is 200-1000bp.These amplified productions all should contain the sequence corresponding to SEQ ID NO:1.
Compare with the existing additive method that is used for liver cancer clinical diagnosis and prognosis; Meliority of the present invention mainly shows: seeing that the closely related property of hnRNP K and liver cancer (has high expression level in the liver cancer case that (in the liver cancer cases that 60 examples detect, 55 examples is arranged) more than 90%; Expression amount in the liver cancer cell: the expression amount in the normal liver cell＞2.5: 1); The sensitivity that the inventive method is detecting; Accuracy all significantly is superior to existing hepatocarcinoma gene diagnostic method, has not only filled up the blind area of detecting, and easy and simple to handle.
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 the restriction scope of the present 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.
Embodiment 1
Material
1. hepatocellular carcinoma sample:
11 routine hepatocellular carcinoma samples (10 examples are used for non-enzymolysis sample preparation, and 1 example is used for the laser capture micro-dissections) from east hospital of liver and gall surgical department, clearly are hepatocellular carcinoma by 2 doctors of Pathology Deparment.Be the male sex, 47.5 years old mean age (31-56 year), it is positive that serum detects hepatitis B virus infection, and 11 examples (100%) belong to TNM classification III level.Wherein, AFP is higher than 9 examples (81.8%) of 25 μ g/L; 10 routine tumours are greater than 5cm.The pathological data of 11 routine hepatocellular carcinoma samples sees table 1 for details.
The pathological data of the routine hepatocellular carcinoma sample of table 1.11.
| No. | Sex | Age | HBV | HCV | Grade (TNM classification) | AFP | The tumour size |
| f31 | The male sex | 56 | + | - | III | ＞1000 | 7×6 |
| f32 | The male sex | 51 | + | III | ＞1000 | 14×12×12 | |
| f33 | The male sex | 50 | + | - | III | ＞1000 | 5×6 |
| f39 | The male sex | 55 | + | - | III | ＞1000 | 5×5.5 |
| 327 | The male sex | 44 | + | - | III | ＞1000 | 8×8×7 |
| 328 | The male sex | 45 | + | - | III | ＞1000 | 7.5×6 |
| 415 | The male sex | 40 | + | - | III | ＞1000 | 10×8×6 |
| 418 | The male sex | 31 | + | - | III | 3.7 | 8×5×8 |
| 422 | The male sex | 57 | + | - | III | ＞1000 | 3.5×4 |
| 429 | The male sex | 44 | + | - | III | ＞1000 | 7.2×6 |
| H38 | The male sex | 50 | + | - | III | 7.3 | 15×13×10.5 |
2. main agents and instrument:
Urea, 3-[(3-courage amido propyl)-diethyl ammonium]-1-propanesulfonic acid (CHAPS), sodium laurylsulfonate (SDS), WR 34678 (DTT) are available from Sigma company; Iodo-acid amide (IAA), acrylic amide, N, N-methylene diacrylamide etc. are available from Fluka company.
Ammonium Persulfate 98.5, TEMED, Tri-n-butylphosphat (TBP), Bio-Rad Protean II electrophoresis equipment (180 * 3 * 0.5mm strips), PDQuest software etc. are the Bio-Rad product.
LCQ
TMDeca XP system and ProteomeX
TMWorkstation is available from Thermo Finnigan company.
The prefabricated adhesive tape of non-linear solid phase pH gradient (IPG dry strips; PH3-10NL, 130 * 3 * 0.5mm or 180 * 3 * 0.5mm), IPGphore isoelectrofocusing system (Amersham Pharmacia Biotech), Amersham Pharmacia Ettan Dalt II systems etc. are Amersham Bioscience Company products.
Reflex
TMIII MALDI-TOF mass spectrum is the Bruker product.
Pixcell
TMLaser capture microdissection system purchases the company in Arcturus Engineering.
Cleavable ICAT light chain and heavy chain reagent, avidin post etc. are Applied Biosystems Company products.
Method
1. non-enzymolysis sample preparation method (nonenzymatic sample preparation, NESP):
The flesh tissue piece of excision places rapidly on ice, is cut into fast that several naked eyes are visible, the fritter of no necrotic zone.After organizing fritter for several times with the washing of the RPMI1640 damping fluid of precooling; In liquid nitrogen, grind to form cell precipitation fast; Cell precipitation is dissolved in respectively in an amount of lysate (8mol/L urea, 4%CHAPS, 40mmol/LTris and 65mmmol/L DTT), and (Soniprep 150, Britain for the ultrasonic cell disintegration appearance; MSE) ice bath ultrasonic 2min at intermittence, 15000r/min, 4 ℃ of centrifugal 1h.Get supernatant, it is quantitative that the Bradford method of improvement is carried out gross protein, the hepatocellular carcinoma cancerous tissue for preparing and the protein example packing of corresponding adjacent tissues ,-80 ℃ of preservations.The NESP method has prepared 10 pairs of hepatocellular carcinoma cancerous tissues and cancer beside organism's protein example.
2. two dimensional gel electrophoresis:
Two-dimensional electrophoresis is mainly by people's such as Sanchez (Sanchez, the J.C. of improving one's methods; Rouge, V.; Pisteur, M.; Ravier, F.; Tonella, L.; Moosmayer, M.; Wilkins, M.R.; Hochstrasser, D.F.Improved and simplified in-gel sample application usingreswelling of dry immobilized pH gradients.Electrophoresis 1997,18 324-327.) carries out.Method is following:
60 μ g protein examples and the Chong Pao liquid (8mol/L urea, 2%CHAPS, 0.5%IPG damping fluid, 18mmol/L DTTh and trace tetrabromophenol sulfonphthalein) that rises mixes; TV 250 μ l or 350 μ l; Use 130 * 3 * 0.5mm or 180 * 3 * 0.5mm pH3-10NL adhesive tape; In IPGphore isoelectrofocusing system, carry out one to separation, total voltage hour is about 80000Vhrs.Adhesive tape is successively in balance liquid I (6M Urea, 30%Glycerol, 2%SDS, 1%DTT) and balance liquid II (DTT replaces with 2.5%IAA among the balance liquid I) balance after the isoelectrofocusing, each 15min.Adhesive tape is transferred to SDS-PAGE system (12%), and deposition condition is 15mA/ glue 30min, and 30mA/ glue is retained to tetrabromophenol sulfonphthalein from glue lower edge 0.5cm then.Preparative electrophoresis, applied sample amount are 1.5mg, and total Vhrs is about 90000, and employing can detect with the compatible Kao Masi light blue method of mass spectrum, and other is identical with the analysis mode electrophoresis method.
3. atlas analysis:
Silver dyes back glue with the scanning of GS-710 image reading apparatus (Bio-Rad) transmission mode, resolving power 84.7 μ m/pixel.(Version 7.0, Bio-Rad) analyze with PDQuest software for the detection of point and coupling.The iso-electric point pI of protein spots and molecular weight M
rWith 2DE standard protein (Bio-Rad) as Marker, Input Software is used to analyze other proteic pI and M
r
4. enzymolysis and mass spectrum are identified in the glue:
Protein spots dyes manual cutting on the glue by compatible the examining of mass spectrum, at 100mM NH
4HCO
3, decolour vacuum lyophilization, 5 μ l 50mmol/L NH among the 30%ACN
4HCO
3(pH 8.3, protein: trypsinase=1: 5, W/W) in 4 ℃ place 2hr, add 20 μ l 50mmol/L NH
4HCO
3(pH 8.3), 37 ℃ of enzymolysis spend the night.Extracting albumen (60%ACN, 0.1%TFA), vacuum lyophilization.LCQ
TMDecaXP system (Thermo Finnigan) identifies the good sample of enzymolysis, and Bioworks software carries out database search.
Perhaps, get sample and HCCA matrix (sigma, the St.Louis that 0.5 μ L enzymolysis is good and redissolve; MO) mix Bruker REFLEX III MALDI-TOF mass spectrum (Bruker-Franzen Analytik, Bremen; German) analyze; (UK) software analysis is confirmed protein sequence for http://www.matrixscience.com, MatrixSicence Ltd with Mascot.
5. immunoblotting:
Respectively getting 20 μ g protein examples separates with 12%SDS-PAGE; Be transferred on the pvdf membrane (available from AmershamBiosciences company); The one anti-goat-anti people hnRNP K how anti-(available from Santa Cruz company, 1: 1000) that uses, two anti-for the anti-goat-anti body of monkey (available from Santa Cruz company; 1: 10000), ECLplus (Amersham Biosciences) reagent detects.
Laser capture microdissection technology (Laser Capture Microdissection, LCM):
Adopt Emmert-Buck, M.R.; Bonner, R.F.; Smith, P.D.; Chuaqui, R.F.; Zhuang, Z.; Goldstein, S.R.; Weiss, R.A.; Liotta, L.A.Laser capturemicrodissection.Science 1996,274,998-1001; And Bonner, R.F.; Emmert-Buck, M.; Cole, K.; Pohida, T.; Chuaqui, R.; Goldstein, S.; Liotta, L.A.Lasercapture microdissection:molecular analysis of tissue.Science 1997,278, the method described in the 1481-1483.Detailed process is following:
The flesh tissue piece of excision is processed 8 μ m frozen tissue sections, and is air-dry through toluidine blue dyeing, uses PixCell
TMLaser Capture Microdissection (the Arcturus Engineering of system; Mountain View CA) carries out micro-dissections, uses diameter 7.5 μ m, 70mW, continues the 2-3mS laser pulse parameters; Cut about 50; 000 hepatocellular carcinoma cancerous tissue cell or corresponding adjacent tissues cell are collected in little cutting cap and are stored in-80 ℃, are dissolved in respectively in an amount of lysate (8mol/L urea, 4%CHAPS, 40mmol/L Tris and 65mmmol/L DTT); Ultrasonic cell disintegration appearance ice bath is ultrasonic 2min intermittently, 15000r/min, 4 ℃ of centrifugal 1h.Get supernatant, it is quantitative to carry out gross protein with the Bradford method of improvement.The hepatic parenchymal cells protein example packing of hepatocellular carcinoma cancerous tissue for preparing and corresponding adjacent tissues ,-80 ℃ of preservations.The LCM technology has prepared 1 pair of hepatocellular carcinoma cancerous tissue and cancer beside organism's protein example (case numbering H38).
Isotropic substance code affinity tag technology (isotope-coded affinity tagging, ICAT) identify with mass spectrum:
Adopt Li, J.; Steen, H.; Gygi, S.P.Protein profiling with cleavableisotope coded affinity tag (cICAT) reagents:The yeast salinity stressresponse.Mol.Cell Proteomics.2003 and Gygi, S.P.; Rist, B.; Gerber, S.A.; Turecek, F.; Gelb, M.H.; Aebersold, R.Quantitative analysis of complexprotein mixtures using isotope-coded affinity tags.Nat.Biotechnol.1999,17, the method described in the 994-999.Detailed process is following:
Hepatocellular carcinoma cancerous tissue and the corresponding adjacent tissues protein example of laser capture microdissection technology preparation are got 100 μ g respectively, and be first with TBP crude protein also, then uses cleavable ICAT reagent (C respectively
12And C
13) mark, after the mixing, trypsin digestion 16-20 hour.Peptide section mixture behind the enzymolysis is used LCQ again after the Avidin affinity column is purified into mark peptide section
TMProteomeX
TMWorkstation carries out the 2D-LC-MS/MS mass spectrum and identifies that Bioworks software carries out database search and x-press numerical evaluation.
The result
1. the foundation of the 2DE-PAGE difference protein expression profile of cancerous tissue and corresponding adjacent tissues and the variance analysis of hnRNP K:
Present embodiment prepares 10 couples of hepatocellular carcinoma patients' the cancerous tissue and the protein example of corresponding adjacent tissues altogether with non-enzymolysis sample preparation method; Obtain the width of cloth surplus the 2-DE collection of illustrative plates 40 altogether, wherein the 5 pairs of samples repeat the differentially expressed spectrum analysis of protein (analytical results is seen table 2) that 3 PDQuest softwares have carried out cancerous tissue and cancer beside organism.
Table 2.PDQuest software analysis result.
More in T=protein spots of up-regulated in liver cancer tissue
More in N=protein spots of down-regulated expression in liver cancer tissue
Only in T=detected protein spots in liver cancer tissue only
Only in N=detected protein spots in the non-liver cancer tissue only
Relation conefficient between the T-T=tumour.
Relation conefficient between the paired non-tumour of N-N=.
Relation conefficient between T-N=tumor tissues and the paired nonneoplastic tissue
Use preparative electrophoresis, the interior zymolysis technique of glue, MALDI-TOF-MS and 1D-LC-MS/MS technology and identified 116 differential points, corresponding 102 kinds of differentially expressed protein.Wherein, hepatocellular carcinoma cancerous tissue high expression level or what only express therein is 61 differential points, corresponding 54 kinds of protein; Hepatocellular carcinoma cancer beside organism high expression level or what only express therein is 55 differential points, corresponding 48 kinds of protein.Wherein, some SSP 3709 and SSP 3729 (Fig. 1) up-regulated in cancerous tissue, enzymolysis in point of contact, glue, some SSP 3729 usefulness 1D-LC-MS/MS mass spectrums identify that getting 9 peptide sections with database search conforms to hnRNP K, the amino acid fraction of coverage is 24.62%.Point SSP 3709 usefulness MALDI-TOF mass spectrums identify that 6 peptide sections of database search conform to hnRNP K, and getting score value is 64, and the albumen fraction of coverage is 18%; Identify that with the 1D-LC-MS/MS mass spectrum getting 20 peptide sections with database search conforms to hnRNP K, the amino acid fraction of coverage is 44.49%.And the up-regulated expression of hnRNP K in cancerous tissue obtains good repetition (SSP 3709 is 50%, and SSP 3709 is 80%) in the 2-DE collection of illustrative plates of different hepatocellular carcinoma patients' cancerous tissue and cancer beside organism.
2.hnRNP the immunoblotting of K differential expression checking:
For confirming the differential expression of hnRNP K in two dimensional gel electrophoresis; Cancerous tissue and corresponding adjacent tissues protein example (preparation of NESP method) to the hepatocellular carcinoma patient; Anti-hnRNP K antibody (available from SantaCruz company) with buying carries out immunoblotting assay, result consistent with the two dimensional gel electrophoresis result (Fig. 2).
3.LCM the differential expression of hnRNP K is further verified in the coupling of technology and ICAT technology:
The hepatocellular carcinoma cancerous tissue of laser capture microdissection technology preparation and corresponding adjacent tissues protein example (case numbering H38) are got 100 μ g respectively, use cleavable ICAT reagent (C respectively
12And C
13) mark, trypsin digestion, avidin affinity column purifying, mass spectrum evaluation, Bioworks software carry out database search and x-press numerical evaluation after mixing.Obtain 283 kinds of protein that quantitative information is arranged through once testing (test 903), comprising the quantitative information of hnRNP K (cancerous tissue:, consistent cancer beside organism=1.80: 1) with the result of up-regulated in cancerous tissue of hnRNP K in the two dimensional gel electrophoresis.
Discuss
The used cancerous tissue of present embodiment and cancer beside organism's sample are the paired samples of taking from same hepatocellular carcinoma patient; All 11 routine hepatocellular carcinoma cases have the case diagnosis index of fairly similar: be the male sex; 47.5 years old mean age (31-56 year); It is positive that serum detects hepatitis B virus infection, and 11 examples (100%) belong to TNM classification III level.Wherein, AFP is higher than 9 examples (81.8%) of 25 μ g/L; 10 routine tumours are greater than 5cm.This sampling method helps reducing between individuality difference to the influence of experimental analysis work.Present embodiment utilization two dimensional gel electrophoresis technology combines mass spectrum identification and detection hepatocellular carcinoma patient's cancerous tissue and the cancer beside organism whole difference aspect protein expression.
On pH 3-10 adhesive tape, silver dyes the following two types of tissue samples of condition and shows that all about 1200 silver dye a little.To dye a matching rate be 0.75-0.86 to silver between cancerous tissue, and to dye a matching rate be 0.60-0.76 to silver between cancerous tissue and cancer beside organism, and the science of two types of tissue sample sampling methods is described to a certain extent.PDQuest software analysis and mass spectrum are identified and have been obtained a plurality of differential expression proteins.
Identifying in the differentially expressed protein that hnRNP K obtains good repetition (SSP 3729 is 50%, and SSP 3709 is 80%) at the up-regulated expression of cancerous tissue in the 2-DE collection of illustrative plates of different hepatocellular carcinoma patients' cancerous tissue and cancer beside organism.The mistake of hnRNP K is expressed in all has report in mammary cancer and the colorectal carcinoma, but up to now, does not also have the relevant report of hnRNP K and hepatocellular carcinoma.Present embodiment prompting hnRNP K is a tumor correlated albumen matter important in the hepatocellular carcinoma.In addition, detected on the 2-DE glue is two parallel points (SSP 3709 and SSP3729), is hnRNP K, and the multi-form phosphorylation modification of prompting hnRNP K also is important change indicator.
Behind two dimensional gel electrophoresis, use the expression that conventional immunoblotting has detected hnRNP K in two types of tissue samples of cancerous tissue and cancer beside organism in the present embodiment, the result is consistent with two dimensional gel electrophoresis, and differential expression has obtained checking.
The LCM technology can well solve heterogeneous and different contaminative two hang-ups in the tissue sample preparation; Can implement accurate micro-catching to single type cells such as cancerous tissue cell or cancer beside organism's cells, thereby realize accurate preparation cancerous tissue and cancer beside organism's protein example.The ICAT technology can provide the quantitative relationship of protein on different sample room integral levels, in conjunction with LCM technology with the 2D-LC-MS/MS mass spectrum detection just can realize becoming more meticulous simultaneously, mass-producing and quantification.And 2-DE glue divides a kind of protein in different positions sometimes, and for example, detected on the 2-DE glue in the present embodiment is exactly two parallel hnRNP K protein spots-SSP 3709 and SSP 3729.Further confirmed the differential expression of hnRNPK among the 2DE result in the present embodiment with the special separation hepatic parenchymal cells of LCM technology; The differential expression that is illustrated in the hnRNPK that obtains in full tissue sample part at least is that differential expression by hepatic parenchymal cells causes that this meaning and value of inquiring into liver cell differential expression hnRNPK from cell levels for the later stage is laid a good foundation.
Existing research shows that the phosphorylation of 302 Ser of hnRNP K receives the adjusting of PKC δ, and important biological function is arranged; HnRNP K also comprises some potential phosphorylation sites; And hnRNP K participates in multiple important biological procedures, the regulation and control of wide participation genetic transcription and translation, and its differential expression in hepatocellular carcinoma possibly played a role to the generation and the development of canceration; Because liver cancer is the same with other tumour, be the rapid process of polygene multistep.In addition, a kind of plasmosin matter-p62 albumen that contains RNA combination Motif contains four hnRNP K homeodomains (KH structural domain), and bibliographical information is arranged, and it is a kind of autoantigen (Autoantigen) (Zhang, the J.Y. of hepatocellular carcinoma; Chan, E.K.; Peng, X.X.; Tan, E.M.A novelcytoplasmic protein with RNA-binding motifs is an autoantigen in humanhepatocellular carcinoma.J.Exp.Med.1999,189,1101-1110.).Though the detailed problem of the multi-form phosphorylation modification of relevant xenogenesis ribonucleoprotein K (hnRNP K), dynamic biological function and tumour related mechanism are still waiting further research, but are sure with it as the affinity tag that detects liver cancer or liver cancer susceptibility.
Present embodiment shows that hnRNP K can be used as the potential sign of hepatocellular carcinoma, and its biological function prompting hnRNP K in born of the same parents maybe be as the prognosis molecule mark of liver cancer and the target molecule of clinical treatment.
Embodiment 2
The preparation of detection kit
Of embodiment 1, proteic abnormal expression of hnRNP K and liver cancer diseases are closely related.Therefore, can design the HP gene-specific primer in view of the above is that template increases and detects at the DNA with patient.
Prepare a test kit (100 person-times), it contains:
Extract the patient's male sex to be detected hepatic tissue 1ml, use ordinary method (or using specific test kit) therefrom to extract mRNA, and be transcribed into cDNA.PCR primer in the liver cancer detection kit is diluted to 1 μ mol/ μ l, is that template is carried out the quantitative PCR reaction with the primer that is provided with prepared cDNA, and compares, thereby confirm the proteic expression amount height of hnRNP K with normal control.
The liver cancer susceptibility that detected result, the proteic expression amount of hnRNP K are higher than the detected object of normal control (expression amount is high 2.5 times) is higher than normal population.
Embodiment 3
The preparation of detection kit
In the present embodiment, with the following detection kit of the proteic Antibody Preparation of anti-hnRNP K:
Of embodiment 1, proteic abnormal expression of hnRNP K and liver cancer diseases are closely related.Therefore, the HnRNP K antibody that can use commercialization in view of the above or provide for oneself, the HnRNP K that histogenic immunity group method detects cancerous tissue and cancer beside organism expresses.
Prepare a test kit (100 person-times), it contains:
| Composition | Quantity |
| Goat-anti people hnRNP K resists (available from Santa Cruz company) (placing a container) more | 50 microlitres, concentration are 200ug/ml, use previous crops dilution in 1: 1000 |
During use, get the conventional preparation of clinical fresh tumor specimen paraffin section, through dewaxing; Antigen retrieval is handled; HnRNP K antibody (1: 1000), 4 ℃ of deposited educating are spent the night, and the Envision method detects positive signal; Mirror calculates positive cell ratio and intensity and scoring down, and the cancerous tissue score value is that there were significant differences for other 2.5 times greater than cancer.
In the liver cancer case that (in the liver cancer case that 60 examples detect, 55 examples is arranged) more than 90%, high expression level is arranged, the expression amount in the liver cancer cell: the expression amount in the normal liver cell＞2.5.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to 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
Dongfang Inst of Hepatobiliary Surgery, Military Medical Univ No.2
< 120>screening and the application thereof of the protein molecular marker xenogenesis ribonucleoprotein K that hepatocellular carcinoma is relevant
<130>041286
<160>4
<170>PatentIn version 3.1
<210>1
<211>2830
<212>DNA
< 213>homo sapiens (Homo sapiens)
<400>1
agggcgctcc aggcgacagc attgcagacg ccattatcct ctgtttctct gctgcaccga 60
cctcgacgtc ttgcctgtgt cccacttgtt cgcggcctat aggctactgc agcactgggg 120
tgtcagttgt tggtccgacc cagaacgctt cagttctgct ctgcaaggat atataataac 180
tgattggtgt gcccgtttaa taaaagaata tggaaactga acagccagaa gaaaccttcc 240
ctaacactga aaccaatggt gaatttggta aacgccctgc agaagatatg gaagaggaac 300
aagcatttaa aagatctaga aacactgatg agatggttga attacgcatt ctgcttcaga 360
gcaagaatgc tggggcagtg attggaaaag gaggcaagaa tattaaggct ctccgtacag 420
actacaatgc cagtgtttca gtcccagaca gcagtggccc cgagcgcata ttgagtatca 480
gtgctgatat tgaaacaatt ggagaaattc tgaagaaaat catccctacc ttggaagagg 540
gcctgcagtt gccatcaccc actgcaacca gccagctccc gctcgaatct gatgctgtgg 600
aatgcttaaa ttaccaacac tataaaggaa gtgactttga ctgcgagttg aggctgttga 660
ttcatcagag tctagcagga ggaattattg gggtcaaagg tgctaaaatt aaagaacttc 720
gagagaacac tcaaaccacc atcaagcttt tccaggaatg ctgtcctcat tccactgaca 780
gagttgttct tattggagga aaacccgata gggttgtaga gtgcataaag atcatccttg 840
atcttatatc tgagtctccc atcaaaggac gtgcacagcc ttatgatccc aatttttacg 900
atgaaaccta tgattatggt ggttttacaa tgatgtttga tgaccgtcgc ggacgcccag 960
tgggatttcc catgcgggga agaggtggtt ttgacagaat gcctcctggt cggggtgggc 1020
gtcccatgcc tccatctaga agagattatg atgatatgag ccctcgtcga ggaccacctc 1080
cccctcctcc cggacgaggc ggccggggtg gtagcagagc tcggaatctt cctcttcctc 1140
caccaccacc acctagaggg ggagacctca tggcctatga cagaagaggg agacctggag 1200
accgttacga cggcatggtt ggtttcagtg ctgatgaaac ttgggactct gcaatagata 1260
catggagccc atcagaatgg cagatggctt atgaaccaca gggtggctcc ggatatgatt 1320
attcctatgc agggggtcgt ggctcatatg gtgatcttgg tggacctatt attactacac 1380
aagtaactat tcccaaagat ttggctggat ctattattgg caaaggtggt cagcggatta 1440
aacaaatccg tcatgagtcg ggagcttcga tcaaaattga tgagccttta gaaggatccg 1500
aagatcggat cattaccatt acaggaacac aggaccagat acagaatgca cagtatttgc 1560
tgcagaacag tgtgaagcag tatgcagatg ttgaaggatt ctaatgcaag atattttttc 1620
ttttttatag tgtgaagcag tattctggaa agtttttcta agactagtga agaactgaag 1680
gagtcctgca tctttttttt tttatctgct tctgtttaaa aagccaacat tcctctgctt 1740
cataggtgtt ctgcatttga ggtgtagtga aatctttgct gttcaccaga tgtaatgttt 1800
tagttcttac aaacagggtt ggggggggga agggcgtgca aaaactaaca ttgaaatttt 1860
gaaacagcag cagagtgagt ggattttatt tttcgttatt gtggtggttt aaaaaattcc 1920
ccccatgtaa ttattgtgaa caccttgctt tgtggtcact gtaacatttg gggggtgggc 1980
cagggaggaa aagtaacaat agtccacatg tccctggcat ctgttcagag cagtgtgcag 2040
aatgtaatgc tcttttgtaa gaaacgtttt atgattttta aaataaattt agtgaaccta 2100
tttttggtgg tcattttttt tttaagacag tcattttaaa atggtggctg aatttcccaa 2160
cccaccccca aactaaacac taagtttaat tttcagctcc tctgttggac atataagtgc 2220
atctcttgtt ggacataggc aaaataactt ggcaaactta gttctggtga tttcttgatg 2280
gtttggaagt ctattgctgg gaagaaattc catcatacat attcatgctt ataataagct 2340
ggggattttt tgtttgtttt tgcaaatgct tgcccctact tttcaacaat tttctatgtt 2400
agttgtgaag aactaaggtg gggagcagta ctacaagttg agtaatggta tgagtatata 2460
ccagaattct gattggcagc aagtttatta atcagaataa cacttggtta tggaagtgac 2520
taatgctgaa aaaattgatt atttttatta gataatttct cacctataga cttaaactgt 2580
caatttgctc tagtgtctta ttagttaaac tttgtaaaat atatatatac ttgtttttcc 2640
attgtatgca aattgaaaga aaaagatgta ccatttctct gttgtatgtt ggattatgta 2700
ggaatgtttg tgtacaattc aaaaaaaaaa aagatgaaaa aagttcctgt ggatgttttg 2760
tgtagtatct tggcatttgt attgatagtt aaaattcact tccaaataaa taaaacaccc 2820
atgatgctag 2830
<210> 2
<211> 464
<212> PRT
< 213>homo sapiens (Homo sapiens)
<400> 2
Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn
1 5 10 15
Gly Glu Phe Gly Lys Arg Pro Ala Glu Asp Met Glu Glu Glu Gln Ala
20 25 30
Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu
35 40 45
Leu Gln Ser Lys Asn Ala Gly Ala Val Ile Gly Lys Gly Gly Lys Asn
50 55 60
Ile Lys Ala Leu Arg Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp
65 70 75 80
Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr
85 90 95
Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu
100 105 110
Gln Leu Pro Ser ProThrAla Thr Ser Gln Leu Pro Leu Glu Ser Asp
115 120 125
Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp Phe Asp
130 135 140
Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala Gly Gly Ile Ile
145 150 155 160
Gly Val Lys Gly Ala Lys Ile Lys Glu Leu Arg Glu Asn Thr Gln Thr
165 170 175
Thr Ile Lys Leu Phe Gln Glu Cys Cys Pro His Ser Thr Asp Arg Val
180 185 190
Val Leu Ile Gly Gly Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile
195 200 205
Ile Leu Asp Leu Ile Ser Glu Ser Pro Ile Lys Gly Arg Ala Gln Pro
210 215 220
Tyr Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr
225 230 235 240
Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met Arg
245 250 255
Gly Arg Gly Gly Phe Asp Arg Met Pro Pro Gly Arg Gly Gly Arg Pro
260 265 270
Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met Ser Pro Arg Arg Gly
275 280 285
Pro Pro Pro Pro Pro Pro Gly Arg Gly Gly Arg Gly Gly Ser Arg Ala
290 295 300
Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly Asp Leu
305 310 315 320
Met Ala Tyr Asp Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met
325 330 335
Val Gly Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp
340 345 350
Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly
355 360 365
Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp Leu Gly
370 375 380
Gly Pro Ile Ile Thr Thr Gln Val Thr Ile Pro Lys Asp Leu Ala Gly
385 390 395 400
Ser Ile Ile Gly Lys Gly Gly Gln Arg Ile Lys Gln Ile Arg His Glu
405 410 415
Ser Gly Ala Ser Ile Lys Ile Asp Glu Pro Leu Glu Gly Ser Glu Asp
420 425 430
Arg Ile Ile Thr Ile Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln
435 440 445
Tyr Leu Leu Gln Asn Ser Val Lys Gln Tyr Ala Asp Val Glu Gly Phe
450 455 460
<210> 3
<211> 20
<212> DNA
< 213>artificial sequence
<220>
<221> misc feature
< 223>primer
<400> 3
taactgattg gtgtgcccgt 20
<210> 4
<211> 20
<212> DNA
< 213>artificial sequence
<220>
<221> misc feature
< 223>primer
<400> 4
gaactaagtt tgccaagtta 20
Claims (5)
1. xenogenesis ribonucleoprotein K is hnRNP K albumen or the purposes of its encoding sequence in the reagent that preparation detection liver cancer is used.
2. specific amplification xenogenesis ribonucleoprotein K is the purposes of primer in the test kit of preparation detection liver cancer of hnRNP K transcript.
3. test kit that detects liver cancer, it is characterized in that it comprises: specific amplification xenogenesis ribonucleoprotein K is the primer of hnRNP K transcript, and specification sheets, said primer is shown in SEQ ID NO:3 and SEQ ID NO:4.
4. test kit as claimed in claim 3 is characterized in that, it also contains the reagent that is selected from down group:
(a) positive control; With
(b) negative control.
5. the purposes of the antibody that anti-xenogenesis ribonucleoprotein K is hnRNP K in the reagent that preparation detection liver cancer is used.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410025464 CN1712541B (en) | 2004-06-25 | 2004-06-25 | Screen and use for labelled heterogenic ribonucleoprotein K of protein molecule related to hepatocellular carcinoma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410025464 CN1712541B (en) | 2004-06-25 | 2004-06-25 | Screen and use for labelled heterogenic ribonucleoprotein K of protein molecule related to hepatocellular carcinoma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1712541A CN1712541A (en) | 2005-12-28 |
| CN1712541B true CN1712541B (en) | 2012-07-25 |
Family
ID=35718335
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410025464 Expired - Fee Related CN1712541B (en) | 2004-06-25 | 2004-06-25 | Screen and use for labelled heterogenic ribonucleoprotein K of protein molecule related to hepatocellular carcinoma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1712541B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1060871A (en) * | 1990-10-01 | 1992-05-06 | 研究发展基金会 | A kind of new cancer tire gene and gene product and purposes |
| CN1272540A (en) * | 2000-04-11 | 2000-11-08 | 国家人类基因组南方研究中心 | Human immune factor related protein and its coded sequence |
| WO2002086096A2 (en) * | 2001-01-23 | 2002-10-31 | University Of Rochester Medical Center | Methods of producing or identifying intrabodies in eukaryotic cells |
-
2004
- 2004-06-25 CN CN 200410025464 patent/CN1712541B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1060871A (en) * | 1990-10-01 | 1992-05-06 | 研究发展基金会 | A kind of new cancer tire gene and gene product and purposes |
| CN1272540A (en) * | 2000-04-11 | 2000-11-08 | 国家人类基因组南方研究中心 | Human immune factor related protein and its coded sequence |
| WO2002086096A2 (en) * | 2001-01-23 | 2002-10-31 | University Of Rochester Medical Center | Methods of producing or identifying intrabodies in eukaryotic cells |
Non-Patent Citations (1)
| Title |
|---|
| Cao R.Role of histone H3 lysine 27 methylation in Polycombgroup silencing.《Science》.2002,第298卷1039-1043. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1712541A (en) | 2005-12-28 |
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