CN1712542A - Screen and use for labelled proto-protein 18 of protein molecule related to hepatocellular carcinoma - Google Patents
Screen and use for labelled proto-protein 18 of protein molecule related to hepatocellular carcinoma Download PDFInfo
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Abstract
This invention relates to oncoprotein 1 or stathmin 1 and its use in diagnosis of liver cell cancer. Compared with normal liver cells, expression of stathmin 1 is obviously higher, so that it can be an effective mark as diagnosis of liver cancer.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to relevant protein molecular marker-proto-protein 18 (being also referred to as Oncoprotein 1 or stathmin 1 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, liver cancer has become serious harm China people life property safety's dead enemy, and be 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 generation 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 methods of treatment 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 is gi|5031851 to proto-protein 18 (Stathmin 1), the accession number of NCBI is NP_005554, the accession number of SWISS-PROT is spP16949) be a kind of solubility endochylema phosphorprotein of high conservative, claim p19, prosolin, p18 or pp20-pp21-pp23 again.Its nucleotide sequence is shown in SEQ ID NO:1, and wherein ORF is positioned at the 177-626 position, and code length is 149 amino acid whose albumen (SEQ ID NO:2).It has at least 2 kinds of non-phosphorylating hypotypes and the phosphorylation hypotype more than 12 kinds, and (molecular weight is about 19,000Da; PI=5.5-6.2).(comprise 11 Serines and 2 Threonines, all be possible phosphorylation site to rat and people's proto-protein 18 (Stathmin 1).Stathmin 1 is very important in intracellular function, is numerous kinase whose substrates in the cell, for example: map kinase, cdc2 kinases, cAMP-dependant kinase and protein kinase C.Thereby Stathmin 1 is called rly. (intracellular relay) in the born of the same parents.The mistake of Stathmin 1 is expressed in all report (Mistry in acute leukemia, lymphoma, neuroblastoma, prostate cancer, ovarian cancer, mammary cancer and the lung cancer, S.J.and Atweh, G.F.Role of stathmin in the regulation of themitotic spindle:potential applications in cancer therapy.Mt.SinaiJ.Med.2002,69,299-304; Cassimeris, L.The oncoprotein 18/stathminfamily of microtubule destabilizers.Curr.Opin.Cell Biol.2002,14,18-24; Sobel, A.Stathmin:a relay phosphoprotein for multiple signaltransduction? Trends Biochem.Sci.1991,16,301-305; Gigant, B.; Martin-Barbey, C.; Curmi, P.A.; Sobel, A.; Knossow, M.[The stathmin-tubulin interaction and the regulation of the microtubule assembly] .Pathol.Biol. (Paris) 2003,51,33-38.).But the relevant report that does not also have up to now, proto-protein 18 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 proto-protein 18 (being called " stathmin 1 albumen " again) new, high expression level in liver cancer
Another object of the present invention provides the application of proto-protein 18 aspect the detection hepatocellular carcinoma.
In a first aspect of the present invention, the purposes of a kind of stathmin 1 albumen or its encoding sequence is provided, be used to prepare the reagent that detects liver cancer.
In a second aspect of the present invention, a kind of external method of determining that stathmin 1 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 stathmin 1 transcript, be template with the cDNA of step (a), obtain the amplified production of stathmin 1 by the quantitative PCR method amplification;
(c) the stathmin 1 amplified production quantity with hepatocellular stathmin 1 amplified production quantity to be measured and normal liver cell in the step (b) compares, and the stathmin 1 amplified production quantity that is higher than normal liver cell just represents that stathmin 1 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 a third aspect of the present invention, a kind of external method of determining that stathmin 1 expressing quantity in the liver cell to be measured is whether unusual is provided, it comprises step:
(a) with stathmin 1 proteic quantity in the anti-stathmin 1 proteic antibody test of the specificity liver cell to be measured;
(b) the stathmin 1 albumen quantity with hepatocellular stathmin 1 albumen quantity to be measured and normal liver cell in the step (a) compares, and the stathmin 1 albumen quantity that is higher than normal liver cell just represents that stathmin 1 expressing quantity exists unusual in the liver cell to be measured.
In another preference, described antibody is monoclonal antibody or polyclonal antibody.
In a fourth aspect of the present invention, a kind of test kit that detects liver cancer is provided, it comprises: the antibody of the primer of specific amplification stathmin 1 transcript and/or the anti-stathmin 1 of specificity, and specification sheets.
In another preference, described test kit also contains the probe that specificity is incorporated into stathmin 1.
In another preference, described test kit also contains the reagent that is selected from down group:
(a) positive control;
(b) negative control.
In a fifth aspect of the present invention, the purposes of the antibody of a kind of anti-stathmin 1 is provided, it is used to prepare the reagent that detects liver cancer or is used to prepare the medicine for the treatment of liver cancer.
In a sixth aspect of the present invention, a kind of method that detects liver cancer or liver cancer susceptibility is provided, it comprises step:
(a) with the primer of specific amplification stathmin 1 transcript, be template with the cDNA of individual hepatocyte to be detected, obtain the amplified production of stathmin 1 by 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 anti-stathmin 1 proteic antibody of specificity is contacted with the sample of individuality to be measured, and whether the antibody complex quantity that detection forms is higher than normal control;
(b) whether amplified production number of Xing Chenging or antibody complex quantity 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
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 shows proto-protein 18 (Stathmin 1) (some SSP 4010) obviously rise of expression in hepatocellular carcinoma patient's cancerous tissue.
Fig. 2 has shown the immunoblotting assay result of 1-DE glue.
Fig. 3 has shown the immunoblotting assay result of 2-DE glue.
Embodiment
The inventor is with non-enzymolysis sample preparation method (nonenzymatic sample preparation, NESP) cancerous tissue of Zhi Bei hepatocellular carcinoma and cancer beside organism's protein example are identified with the differential expression protein point and the mass spectrum of conventional two dimensional gel electrophoresis technology screening.Found that proto-protein 18 (stathmin 1) high expression level in the hepatocellular carcinoma cancerous tissue.Immunoblot experiment confirms that further proto-protein 18 there are differences expression really in the cancerous tissue of hepatocellular carcinoma and cancer beside organism.Finished 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 by the pairing of complementary base two single-chain nucleic acids be combined.
" be incorporated into substantially " 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 by the tight degree that reduces hybridization medium.This term also refers under stringent condition, a part in conjunction with, compound or hybridize in specific nucleotide sequence, when this sequence is present in the compound mixture (as total cell NDA or RNA).Term " stringent condition " refers to that probe hybridization decides subsequence in target and do not hybridize condition in other sequences.Stringent condition is sequence-dependent, and is different under different situations.Long sequence can be under higher temperature specific hybrid.Usually, Xuan Ding stringent condition is than low about 5 ℃ of the pyrolysis chain temperature (Tm) of inferring sequence under ionic strength that limits and pH.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 0.02Na ionic concn (or other salt), pH7.0-8.3, and temperature is at least about 60 ℃.Stringent condition also can be realized by 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 substantially with disclosed primer or probe " basic identical ".
Stathmin 1 albumen and gene
Based on disclosed stathmin 1 and the dependency of liver cancer and the sequence information of stathmin 1, those skilled in the art can utilize the ordinary skill in the art to produce stathmin 1 gene, albumen or its fragment.
In the present invention, term " proto-protein 18 ", " stathmin 1 albumen ", " stathmin 1 polypeptide ", " liver cance high-expression albumen stathmin 1 " or " albumen of the present invention " etc. are used interchangeably, (the Genebank accession number is gi|5031851 all to refer to have people or other mammiferous proto-proteins 18, the accession number of NCBI is NP_005554, the accession number of SWISS-PROT is spP16949), it is a kind of solubility endochylema phosphorprotein of high conservative.The stathmin 1 that a kind of particularly preferred proto-protein 18 is people, its aminoacid sequence are shown in SEQ ID NO:2, and nucleotide sequence is shown in SEQ ID NO:1, and ORF is positioned at the 177-626 position, and code length is 149 amino acid whose albumen.
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, " isolating stathmin 1 albumen or polypeptide " is meant that stathmin 1 polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use purified technology of protein purifying stathmin 1 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 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.
The present invention also comprises people stathmin 1 proteic fragment, derivative and analogue.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 stathmin 1 polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of people stathmin 1 protein-active.This term also comprises having and variant form people stathmin 1 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.This term also comprises people stathmin 1 proteic active fragments 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 stathmin 1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people stathmin 1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people stathmin 1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people stathmin 1 polypeptide.Usually, this fragment have people stathmin 1 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 stathmin 1 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 SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature 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 (as PCR) of nucleic acid to determine and/or separation coding stathmin 1 proteic polynucleotide.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People stathmin 1 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.
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 stathmin 1 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people stathmin 1 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.
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 isolation technique etc.
Anti-stathmin 1 proteic antibody
On the other hand, the present invention also comprises people stathmin 1 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 stathmin 1 gene product or fragment.Preferably, refer to that those can combine with people stathmin 1 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 people stathmin 1 proteic molecule, comprise that also those do not influence the antibody of people stathmin 1 protein function.The present invention also comprise those can with modify or without the people stathmin 1 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, people stathmin 1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human stathmin 1 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 be utilized hybridoma technology to prepare (to see people such as Kohler, N
Ature256; 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 stathmin 1 protein function and the antibody that does not influence people stathmin 1 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people stathmin 1 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 stathmin 1 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).
Anti-people stathmin 1 proteic antibody can be used in the immunohistochemistry technology, detects people stathmin 1 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of people stathmin 1 protein bound monoclonal antibody, 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 stathmin 1 albumen, as liver cancer.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 stathmin 1 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, by 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 stathmin1 protein positive.Because stathmin 1 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 stathmin 1 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.
Pharmaceutical composition
The proteic antagonist of the present invention (as antibody) when using (administration) in treatment, 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 by 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 stathmin 1 albumen of the present invention, also can use the other treatment agent simultaneously, as 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 (as 0.01-99wt%) stathmin 1 polypeptide of the present invention 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.
When making pharmaceutical composition, be that stathmin 1 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 also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
People stathmin 1 proteic polynucleotide also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or metabolic disturbance (as liver cancer) due to the stathmin 1 proteic expression of stathmin 1 proteic abnormal expression or representative.
Suppress the oligonucleotide (comprising sense-rna and DNA) of people stathmin 1 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.
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.
Detection method
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people stathmin 1 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.Whether people stathmin 1 protein level that is detected in the test can be higher than one of index of normal population as the probability that definite detected individuality suffers from liver cancer or liver cancer susceptibility.
Whether having stathmin 1 proteic method in a kind of detection test sample is to utilize stathmin 1 proteic specific antibody to detect, and it comprises: sample is contacted with stathmin 1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample stathmin 1 albumen.
Stathmin 1 proteic polynucleotide can be used for the diagnosis of stathmin 1 gene-correlation disease.Aspect diagnosis, stathmin 1 proteic polynucleotide can be used for detecting stathmin 1 expression of gene and whether is higher than normal population.Correlation technique comprises quantitative PCR method (as quantitative fluorescent PCR) etc., and 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 stathmin 1 albumen and also can detect stathmin 1 proteic transcription product.
Because the very high expression level rate of stathmin 1 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 stathmin 1 polynucleotide, stathmin 1 albumen and antibody thereof, and the relevant antagonist of stathmin 1 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 stathmin 1 to express diagnostic kit.In preference, a kind of test kit comprises the primer of specific amplification stathmin 1 transcript and/or the antibody of the anti-stathmin 1 of specificity.Test kit also can contain description and how use test kit to detect the illustrative material of stathmin 1.Test kit also can contain one or more in the following group: be used to assist the various markers 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 understand, after the present invention has disclosed the dependency of the unusual and liver cancer of stathmin 1 expression of gene first, but those skilled in the art can design the primer that specific amplification goes out stathmin 1 easily, determine its expression amount by 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 method that contains the test kit of these primers and use these primers is 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 stathmin 1.A kind of preferred primer is to having the sequence of SEQ ID N0:3 and 4.
Though the length of amplified production is not particularly limited, 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, superiority of the present invention mainly shows: in view of the closely related property of stathmin 1 and liver cancer (has high expression level in the liver cancer case 70% or more (in the liver cancer cases of 60 example detections 43 examples being arranged), 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 better than existing hepatocarcinoma gene diagnostic method, 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 and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Material
1. hepatocellular carcinoma sample: 10 routine hepatocellular carcinoma samples clearly are hepatocellular carcinoma from east hospital of liver and gall surgical department by 2 doctors of Pathology Deparment.Be the male sex, 47.3 years old mean age (31～56 years old), serum detects the hepatitis B virus infection positive, and 10 examples (100%) belong to TNM classification III level.Wherein, AFP is higher than 9 examples (90%) of 25 μ g/L; 9 routine tumours are greater than 5cm.The pathological data of 10 routine hepatocellular carcinoma samples sees table 1 for details.
The pathological data of table 1. 10 routine hepatocellular carcinoma samples.
| ??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 |
| ??3?27 | The male sex | ??44 | ??+ | ??- | ?III | ??1000 | ??8×8×7 |
| ??3?28 | The male sex | ??45 | ??+ | ??- | ?III | ??1000 | ??7.5×6 |
| ??4?15 | The male sex | ??40 | ??+ | ??- | ?III | ??1000 | ??10×8×6 |
| ??4?18 | The male sex | ??31 | ??+ | ??- | ?III | ??3.7 | ??8×5×8 |
| ??4?22 | The male sex | ??57 | ??+ | ??- | ?III | ??1000 | ??3.5×4 |
| ??4?29 | The male sex | ??44 | ??+ | ??- | ?III | ??1000 | ??7.2×6 |
2. main agents and instrument:
Urea, 3-[(3-courage amido propyl)-the diethyl ammonium]-1-propanesulfonic acid (CHAPS), sodium laurylsulfonate (SDS), dithiothreitol (DTT) (DTT) be available from Sigma company; Iodo-acid amide (IAA), acrylamide, N, N-methylene diacrylamide etc. are available from Fluka company.Ammonium Persulfate 98.5, TEMED, Bio-Rad Protean II electrophoresis equipment (180 * 3 * 0.5mm strips), PDQuest software etc. are the Bio-Rad product.LCQ
TMDecaXP system is available from Thermo Finnigan company.The prefabricated adhesive tape of non-linear solid phase pH gradient (IPG drystrips, pH3-10NL, 130 * 3 * 0.5mm or 180 * 3 * 0.5mm), IPGphore isoelectrofocusing system (Amersham Pharmacia Biotech), Amersham Pharmacia Ettan Dalt II system etc. are Amersham Bioscience company product.
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/L Tris and 65mmmol/L DTT), (Soniprep 150 for the ultrasonic cell disintegration instrument, Britain, 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 applicationusing reswelling of dry immobilized pH gradients.Electrophoresis 1997,18 324-327.) carries out.Method is as follows:
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, cumulative volume 250 μ l or 350 μ l, use 130 * 3 * 0.5mm or 180 * 3 * 0.5mm pH3-10NL adhesive tape, carry out one to separation in IPGphore isoelectrofocusing system, 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 Kao Masi light blue method of mass spectrum compatibility, 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 examining of mass spectrum compatibility,, decolours vacuum lyophilization, 5 μ l 50mmol/L NH among the 30%ACN at 100mM NH4HCO3
4HCO
3(pH8.3, protein: trypsinase=1: 5, W/W) in 4 ℃ place 2hr, add 20 μ l 50mmol/L NH
4HCO
3(pH8.3), 37 ℃ of enzymolysis spend the night.Extracting albumen (60%ACN, 0.1%TFA), vacuum lyophilization.LCQ
TMDeca XP system (ThermoFinnigan) identifies the good sample of enzymolysis, and Bioworks software carries out database search.
5. immunoblotting:
Respectively getting 20 μ g protein examples separates with 12% SDS-PAGE, be transferred on the pvdf membrane (available from Amersham Biosciences company), the one anti-anti-people stathmin more than 1 of rabbit that uses resists (available from Calbiochem company, 1: 20000), two anti-for goat anti-rabbit antibody (available from Calbiochem company, 1: 10000), ECL plus (Amersham Biosciences company) reagent detects.
The result
1. the foundation of the 2DE-PAGE difference protein expression profile of cancerous tissue and corresponding adjacent tissues and proto-protein 18 (Stathmin 1) variance analysis:
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 sees Table two) that 3 PDQuest softwares have carried out cancerous tissue and cancer beside organism.
Table 2.PDQuest software analysis result.
| The sample title | Matching point | Statistical study (t test, p＜0.05) | Quantitative analysis | Qualitative analysis | Relation conefficient | |||
| More in T | More in N | Only in T | Only in N | T-T or N-N | ??T-N | |||
| 3?27 | ??1283 | ??230 | ??35 | ?54 | ??16 | ??22 | ????r[0.82718- ????0.86666] | ??r[0.69826- ??0.76055] |
| 3?28 | ??1316 | ??97 | ??24 | ?9 | ??9 | ??6 | ????r[0.75556- ????0.84765] | ??r[0.63577- ??0.73870] |
| 4?15 | ??1121 | ??393 | ??51 | ?56 | ??48 | ??75 | ????r[0.82165- ????0.85401] | ??r[0.66092- ??0.72361] |
| 4?18 | ??1125 | ??362 | ??41 | ?74 | ??46 | ??57 | ????r[0.827149- ????0.863555] | ??r[0.673221- ??0.729310] |
| 4?29 | ??1213 | ??264 | ??67 | ?46 | ??44 | ??95 | ????r[0.79004- ????0.86468] | ??r[0.60064- ??0.68952] |
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 4010 (Fig. 1) expresses in cancerous tissue obviously and raises, and enzymolysis, 1D-LC-MS/MS mass spectrum identify that getting 5 peptide sections with database search conforms to proto-protein 18 (Stathmin 1) in point of contact, glue, and the amino acid fraction of coverage is 34.9%.And the up-regulated expression of proto-protein 18 (Stathmin 1) in cancerous tissue all obtains repetition in the 2-DE of all 10 pairs of samples collection of illustrative plates.
2. the immunoblotting of proto-protein 18 (Stathmin 1) differential expression checking:
For confirming the differential expression of proto-protein 18 (Stathmin 1) in two dimensional gel electrophoresis, cancerous tissue and corresponding adjacent tissues protein example (preparation of NESP method) to the hepatocellular carcinoma patient, carry out immunoblotting assay, result consistent with the two-dimensional electrophoresis result (Fig. 2) with anti-Stathmin 1 antibody of buying.Wherein, 429 samples have carried out the immunoblotting assay of 2-DE glue, two kinds of different phosphorylation forms finding proto-protein 18 (Stathmin1) also have up-regulated expression (Fig. 3) in cancerous tissue, the relative position of three kinds of multi-form proto-proteins 18 (Stathmin 1) conform to bibliographical information (Schweppe, R.E.; Haydon, C.E.; Lewis, T.S.; Resing, K.A.; Ahn, N.G.The characterization ofprotein post-translational modifications by mass spectrometry.Acc.Chem.Res.2003,36,453-461.).
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 10 routine hepatocellular carcinoma cases have the case diagnosis index of fairly similar: be the male sex, 47.3 years old mean age (31～56 years old), serum detects the hepatitis B virus infection positive, and 10 examples (100%) belong to TNM classification III level.Wherein, AFP is higher than 9 examples (90%) of 25 μ g/L; 9 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 in conjunction with mass spectrum identification and detection hepatocellular carcinoma patient's cancerous tissue and cancer beside organism in the whole difference aspect the protein expression.
On the pH3-10 adhesive tape, silver dyes that two class tissue samples show that all about 1200 silver dye a little under the condition.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 class 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 proto-protein 18 (Stathmin 1) all obtains repetition at the up-regulated expression of cancerous tissue in the 2DE collection of illustrative plates of 10 routine hepatocellular carcinoma patients' cancerous tissue and cancer beside organism.The mistake of proto-protein 18 (Stathmin 1) is expressed in all report in acute leukemia, lymphoma, neuroblastoma, prostate cancer, ovarian cancer, mammary cancer and the lung cancer, but the relevant report that does not also have up to now, proto-protein 18 (Stathmin 1) and hepatocellular carcinoma.Present embodiment prompting proto-protein 18 (Stathmin 1) is a tumor correlated albumen matter important in the hepatocellular carcinoma.
In the present embodiment behind two dimensional gel electrophoresis, use conventional immunoblotting 1DE and 2DE level detection the expression of proto-protein 18 (Stathmin 1) in the two class tissue samples, result and two dimensional gel electrophoresis are in full accord, and differential expression has obtained checking.In addition, the immunoblotting of 2DE level has found two kinds of different phosphorylation forms in addition of former cancer egg 18 (Stathmin 1) that up-regulated expression (Fig. 3) is also arranged in cancerous tissue, and the relative position of three kinds of multi-form proto-proteins 18 (Stathmin 1) conforms to bibliographical information.The multi-form phosphorylation modification of prompting proto-protein 18 (Stathmin 1) also is important change indicator.Though the detailed problem of the multi-form phosphorylation modification of relevant proto-protein 18 (Stathmin 1), dynamic biological function and tumour related mechanism are still waiting further research, but are sure with it as the marker that detects liver cancer or susceptibility.
There are some researches show that people's proto-protein 18 (Stathmin 1) comprises 11 Serines and 2 Threonines, all is possible phosphorylation site.Proto-protein 18 (Stathmin 1) is very important in intracellular function, is numerous kinase whose substrates in the cell, for example: for example: map kinase, cdc2 kinases, cAMP-dependant kinase and protein kinase C.Thereby proto-protein 18 (Stathmin 1) is called rly. (intracellular relay) in the born of the same parents.In addition, the different phosphorylation form of proto-protein 18 (Stathmin 1) passes through closely related to the adjusting and the regulation and control of cell cycle of tubulin stability.Present embodiment shows that proto-protein 18 (Stathmin 1) can be used as the potential sign of hepatocellular carcinoma, and its biological function prompting proto-protein 18 (Stathmin 1) in born of the same parents may be as the prognosis molecule mark of liver cancer and the target molecule of clinical treatment.
Embodiment 2
The preparation of detection kit
As described in embodiment 1, Stathmin 1 proteic abnormal expression 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:
| Title | Sequence | Concentration |
| Adopted primer is arranged | SEQ ID NO:3 (corresponding to 71-90 position among the SEQ ID NO:1) | 100pmol |
| Antisense primer | SEQ ID NO:4 (corresponding to 1408-1427 position among the SEQ ID NO:1) | 100pmol |
| The PCR reaction solution | Contain Taq enzyme dNTP magnesium ion PCR reaction buffer | |
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 and compares with normal control, thereby determine Stathmin 1 proteic expression amount height.
The liver cancer susceptibility that detected result, Stathmin 1 proteic expression amount are higher than the detected object of normal control (expression amount is high 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-stathmin:
As described in embodiment 1, proteic abnormal expression of stathmin and liver cancer diseases are closely related.Therefore, the stathmin 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 |
| Anti-people stathmin more than 1 anti-(available from Calbiochem company) (the placing a container) of rabbit | 50 microlitres, concentration are 200ug/ml, use previous crops dilution in 1: 1000 |
During use, the conventional preparation of clinical fresh tumor specimen paraffin section, through dewaxing, antigen retrieval is handled, stathmin 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.
The result has high expression level, the expression amount in the liver cancer cell in the liver cancer case that (in the liver cancer case that 60 examples detect 43 examples is arranged) more than 70%: the expression amount in the normal liver cell＞2.5.
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
Dongfang Inst of Hepatobiliary Surgery, Military Medical Univ No.2
＜120〉screening and the application thereof of the protein molecular marker proto-protein 18 that hepatocellular carcinoma is relevant
<130>041287
<160>4
<170>PatentIn?version?3.1
<210>1
<211>1542
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>1
gctctcggcc?aatgcggagc?cccgcgcgga?ggtcacgtgc?ctctgtttgg?cgcttttgtg?????60
cgcgcccggg?tctgttggtg?ctcagagtgt?ggtcaggcgg?ctcggactga?gcaggacttt????120
ccttatccca?gttgattgtg?cagaatacac?tgcctgtcgc?ttgtcttcta?ttcaccatgg????180
cttcttctga?tatccaggtg?aaagaactgg?agaagcgtgc?ctcaggccag?gcttttgagc????240
tgattctcag?ccctcggtca?aaagaatctg?ttccagaatt?ccccctttcc?cctccaaaga????300
agaaggatct?ttccctggag?gaaattcaga?agaaattaga?agctgcagaa?gaaagacgca????360
agtcccatga?agctgaggtc?ttgaagcagc?tggctgagaa?acgagagcac?gagaaagaag????420
tgcttcagaa?ggcaatagaa?gagaacaaca?acttcagtaa?aatggcagaa?gagaaactga????480
cccacaaaat?ggaagctaat?aaagagaacc?gagaggcaca?aatggctgcc?aaactggaac????540
gtttgcgaga?gaaggataag?cacattgaag?aagtgcggaa?gaacaaagaa?tccaaagacc????600
ctgctgacga?gactgaagct?gactaatttg?ttctgagaac?tgactttctc?cccatcccct????660
tcctaaatat?ccaaagactg?tactggccag?tgtcatttta?ttttttccct?cctgacaaat????720
attttagaag?ctaatgtagg?actgtatagg?tagatccaga?tccagactgt?aagatgttgt????780
tttaggggct?aaaggggaga?aactgaaagt?gttttactct?ttttctaaag?tgttggtctt????840
tctaatgtag?ctatttttct?tgttgcatct?tttctacttc?agtacacttg?gtgtactggg????900
ttaatggcta?gtactgtatt?ggctctgtga?aaacatattt?gtgaaaagag?tatgtagtgg????960
cttcttttga?actgttagat?gctgaatatc?tgttcacttt?tcaatcccaa?ttctgtccca???1020
atcttaccag?atgctactgg?acttgaatgg?ttaataaaac?tgcacagtgc?tgttggtggc???1080
agtgacttct?tttgagttag?gttaataaat?caagccatag?agcccctcct?ggttgatact???1140
tgttccagat?ggggcctttg?gggctggtag?aaatacccaa?cgcacaaatg?accgcacgtt???1200
ctctgccccg?tttcttgccc?cagtgtggtt?tgcattgtct?ccttccacaa?tgactgcttt???1260
gtttggatgc?ctcagcccag?gtcagctgtt?actttctttc?agatgtttat?ttgcaaacaa???1320
ccattttttg?ttctgtgtcc?cttttaaaag?gcagattaaa?agcacaagcg?tgtttctaga???1380
gaacagttga?gagagaatct?caagattcta?cttggtggtt?tgcttgctct?acgttacagg???1440
tggggcatgt?cctcatcctt?tcctgccata?aaagctatga?cacgagaatc?agaatattaa???1500
taaaacttta?tgtactgctg?tagcaaaaaa?aaaaaaaaaa?aa??????????????????????1542
<210>2
<211>149
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>2
Met?Ala?Ser?Ser?Asp?Ile?Gln?Val?Lys?Glu?Leu?Glu?Lys?Arg?Ala?Ser
1???????????????5????????????????????10??????????????????15
Gly?Gln?Ala?Phe?Glu?Leu?Ile?Leu?Ser?Pro?Arg?Ser?Lys?Glu?Ser?Val
20???????????????????25??????????????????30
Pro?Glu?Phe?Pro?Leu?Ser?Pro?Pro?Lys?Lys?Lys?Asp?Leu?Ser?Leu?Glu
35???????????????????40??????????????????45
Glu?Ile?Gln?Lys?Lys?Leu?Glu?Ala?Ala?Glu?Glu?Arg?Arg?Lys?Ser?His
50???????????????????55??????????????????60
Glu?Ala?Glu?Val?Leu?Lys?Gln?Leu?Ala?Glu?Lys?Arg?Glu?His?Glu?Lys
65???????????????????70??????????????????75??????????????????80
Glu?Val?Leu?Gln?Lys?Ala?Ile?Glu?Glu?Asn?Asn?Asn?Phe?Ser?Lys?Met
85??????????????????90??????????????????95
Ala?Glu?Glu?Lys?Leu?Thr?His?Lys?Met?Glu?Ala?Asn?Lys?Glu?Ash?Arg
100?????????????????105?????????????????110
Glu?Ala?Gln?Met?Ala?Ala?Lys?Leu?Glu?Arg?Leu?Arg?Glu?Lys?Asp?Lys
115?????????????????120?????????????????125
His?Ile?Glu?Glu?Val?Arg?Lys?Ash?Lys?Glu?Ser?Lys?Asp?Pro?Ala?Asp
130?????????????????135?????????????????140
Glu?Thr?Glu?Ala?Asp
145
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>3
tctgttggtg?ctcagagtgt?????????????????????????????????????????????20
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>4
gtaacgtaga?gcaagcaaac??????????????????????????????????????????????20
Claims (10)
1. the purposes of stathmin 1 albumen or its encoding sequence is characterized in that, is used to prepare the reagent that detects liver cancer.
2. external method of determining that stathmin 1 gene expression amount in the liver cell to be measured is whether unusual is characterized in that it comprises step:
(a) extracting hepatocellular mRNA to be measured, and reverse transcription is cDNA;
(b) with the primer of specific amplification stathmin 1 transcript, be template with the cDNA of step (a), obtain the amplified production of stathmin 1 by the quantitative PCR method amplification;
(c) the stathmin 1 amplified production quantity with hepatocellular stathmin 1 amplified production quantity to be measured and normal liver cell in the step (b) compares, and the stathmin 1 amplified production quantity that is higher than normal liver cell just represents that stathmin 1 gene expression amount exists unusual in the liver cell to be measured.
3. method as claimed in claim 2 is characterized in that, described quantitative PCR method is the quantitative fluorescence PCR method.
4. external method of determining that stathmin 1 expressing quantity in the liver cell to be measured is whether unusual is characterized in that it comprises step:
(a) with stathmin 1 proteic quantity in the anti-stathmin 1 proteic antibody test of the specificity liver cell to be measured;
(b) the stathmin 1 albumen quantity with hepatocellular stathmin 1 albumen quantity to be measured and normal liver cell in the step (a) compares, and the stathmin 1 albumen quantity that is higher than normal liver cell just represents that stathmin 1 expressing quantity exists unusual in the liver cell to be measured.
5. method as claimed in claim 4 is characterized in that described antibody is monoclonal antibody.
6. a test kit that detects liver cancer is characterized in that it comprises: the antibody of the primer of specific amplification stathmin 1 transcript and/or the anti-stathmin 1 of specificity, and specification sheets.
7. test kit as claimed in claim 6 is characterized in that it also contains the probe that specificity is incorporated into stathmin 1.
8. test kit as claimed in claim 6 is characterized in that, it also contains the reagent that is selected from down group:
(a) positive control;
(b) negative control.
9. the purposes of the antibody of an anti-stathmin 1 is characterized in that, is used to prepare the reagent that detects liver cancer or is used to prepare the medicine for the treatment of liver cancer.
10. a method that detects liver cancer or liver cancer susceptibility is characterized in that, comprises step:
(a) with the primer of specific amplification stathmin 1 transcript, be template with the cDNA of individual hepatocyte to be detected, obtain the amplified production of stathmin 1 by 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 anti-stathmin 1 proteic antibody of specificity is contacted with the sample of individuality to be measured, and whether the antibody complex quantity that detection forms is higher than normal control;
(b) whether amplified production number of Xing Chenging or antibody complex quantity are higher than normal control, just represent that this individuality suffers from liver cancer or liver cancer susceptibility is higher than normal population.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101303352A (en) * | 2008-07-03 | 2008-11-12 | 中国人民解放军第二军医大学 | Method for testing and sorting free liver cancer cell |
| CN103328978A (en) * | 2011-01-21 | 2013-09-25 | 巴斯利尔药物股份公司 | Use of stathmin as a biomarker of drug response to furazanobenzimidazoles |
| CN101629958B (en) * | 2008-07-17 | 2014-04-09 | 中国医学科学院肿瘤研究所 | Detecting method by TGM2, detecting kit and application thereof |
| CN106198996A (en) * | 2016-06-29 | 2016-12-07 | 大连医科大学 | Early diagnosis biomarker, test kit and the application thereof of minimal change nephropathy |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002220131A1 (en) * | 2000-12-01 | 2002-06-11 | Board Of Trustees Of The University Of Arkansas | Compositions, methods, apparatus and products comprising a stathmin/oncoprotein 18 sequence for detecting and treating cancer |
| PL363009A1 (en) * | 2001-04-03 | 2004-11-15 | Merck Patent Gmbh | Renal cell carcinoma tumor markers |
-
2004
- 2004-06-25 CN CN 200410025465 patent/CN1712542B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101303352A (en) * | 2008-07-03 | 2008-11-12 | 中国人民解放军第二军医大学 | Method for testing and sorting free liver cancer cell |
| CN101629958B (en) * | 2008-07-17 | 2014-04-09 | 中国医学科学院肿瘤研究所 | Detecting method by TGM2, detecting kit and application thereof |
| CN103328978A (en) * | 2011-01-21 | 2013-09-25 | 巴斯利尔药物股份公司 | Use of stathmin as a biomarker of drug response to furazanobenzimidazoles |
| CN103328978B (en) * | 2011-01-21 | 2016-01-20 | 巴斯利尔药物股份公司 | The purposes of the biomarker that stathmin replys as furazano benzimidazoles residues |
| CN106198996A (en) * | 2016-06-29 | 2016-12-07 | 大连医科大学 | Early diagnosis biomarker, test kit and the application thereof of minimal change nephropathy |
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| CN1712542B (en) | 2012-07-04 |
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