CN103937871A - Application of SRRP35 gene and expression product thereof to cancer diagnosis and treatment - Google Patents

Application of SRRP35 gene and expression product thereof to cancer diagnosis and treatment Download PDF

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CN103937871A
CN103937871A CN201310025221.XA CN201310025221A CN103937871A CN 103937871 A CN103937871 A CN 103937871A CN 201310025221 A CN201310025221 A CN 201310025221A CN 103937871 A CN103937871 A CN 103937871A
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srrp35
cancer
cell
albumen
gene
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CN103937871B (en
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高勇
李砚东
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Shanghai East Hospital
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57438Specifically defined cancers of liver, pancreas or kidney
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/136Screening for pharmacological compounds
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value
    • G01N2500/10Screening for compounds of potential therapeutic value involving cells

Abstract

The invention discloses application of SRRP35 gene and an expression product thereof to prepare products for cancer diagnosis and treatment. SRRP35 gene and the expression product can be used a specific marker gene for diagnosing cancers especially liver cancer; and SRRP35 gene and the expression product also can be used as a target gene for preparing medicines for treating cancers especially liver cancer, and help to provide a new cancer treatment approach.

Description

The application in cancer diagnosis and treatment of SRRP35 gene and expression product
Technical field
The present invention relates to oncology.More specifically, the present invention relates to the application aspect cancer detection of SRRP35 gene and expression product, especially in the application of the detection of liver cancer.The invention still further relates to the application in cancer therapy of SRRP35 gene, albumen and agonist thereof.
Background technology
Hepatocellular carcinoma (hepatocellularcarcinoma, HCC) is the common malignant tumour of China, and its mortality ratio occupies second.The diagnosis of liver cancer, especially early diagnosis are the keys of clinic diagnosis and prognosis.Can be used as the biomolecules of primary liver cancer markers, it has been generally acknowledged that following four classes: cancer embryo and glycoprotein antigen; Enzyme and isozyme; Cytokine; Gene.In the world, to the etiologic diagnosis of liver cancer, to detect Serum AFP (alpha-fetoprotein) as main, but the susceptibility of AFP (40%~65%) and specificity (76%~96%) are all unsatisfactory.
In addition, the treatment of liver cancer makes great progress in 20 years in the past, has occurred some local non-operative treatment schemes, still, substantially continues to use the medicine of other tumours due to the current chemotherapeutics of liver cancer, and specific aim is poor, and therefore, general curative effect is not good.
Therefore, this area can be used for the associated protein of diagnosing cancer of liver in the urgent need to exploitation, and in order effectively to suppress the growth of liver cancer cell, this area can be used for suppressing the medicine of liver cancer cell growth in the urgent need to exploitation, to improve specificity and the validity of chemotherapy.
Summary of the invention
The invention discloses the application of a kind of people SRRP35 gene and expression product thereof, for the preparation of diagnosis and the treatment product of cancer especially liver cancer.
A first aspect of the present invention provides the purposes of a kind of SRRP35 gene or SRRP35 albumen, for the preparation of the reagent or the test kit that detect cancer;
In another preference, described cancer is liver cancer.
In another preference, described test kit comprises: the reagent and corresponding label or the specification sheets that SRRP35 albumen or mRNA are carried out to detection by quantitative.
In another preference, described reagent comprises SRRP35 Auele Specific Primer, specific antibody, probe and/or chip.
In another preference, above-mentioned reagent comprises detection chip, comprises nucleic acid chip and protein chip.
In another preference, described nucleic acid chip comprises substrate and the point sample specific oligonucleotide probe at on-chip cancer related gene, and the specific oligonucleotide probe of described cancer related gene comprises the probe with SRRP35 gene or mRNA specific binding.
In another preference, described protein chip comprises substrate and the point sample specific antibody in on-chip cancer associated protein, and the specific antibody of described cancer associated protein comprises the specific antibody of anti-SRRP35 albumen.
In another preference, described SRRP35 albumen comprises fusion rotein and non-fusion rotein.
A second aspect of the present invention, provides a kind of diagnostic kit for detection of cancer, and described test kit contains a container, contains the detection reagent that detects SRRP35 albumen or mRNA in described container; And label or specification sheets, described label or specification sheets indicate described test kit for detection of cancer.
In another preference, in described label or specification sheets, indicate following content:
When ratio≤1 of the mrna expression amount of the mrna expression amount of the relative beta-actin of the SRRP35 of detected object and the relative beta-actin of SRRP35 of cancer beside organism, point out the cancered probability of this detected object higher than general population.
In another preference, described detection reagent comprises: Auele Specific Primer, specific antibody, probe and/or chip;
In another preference, described test kit is for detection of people's neoplasmic tissue sample or blood sample;
In another preference, described neoplasmic tissue sample is liver cancer sample.
A third aspect of the present invention, provides the purposes of a kind of SRRP35 albumen, SRRP35 gene or its agonist, for the preparation of the medicine of anticancer growth or propagation, or for the preparation of the medicine for the treatment of cancer.
A fourth aspect of the present invention, provides a kind of anticancer growth of external non-therapeutic or the method for propagation, comprises step: under SRRP35 albumen or the existence of its agonist, cultivate cancer cells, thus anticancer growth or propagation.
In another preference, described method comprises in the culture system of cancer cells adds SRRP35 agonist, thus anticancer growth or propagation.
In another preference, described cancer cells is liver cancer cell.
A fifth aspect of the present invention, provides a kind of method of screening the candidate compound for the treatment of cancer, comprises step:
(a) in test group, in the culture system of cell, add test compounds, and observe expression amount and/or the activity of SRRP35 in the cell of described test group; In control group, in isocellular culture system, do not add test compounds, and observe expression amount and/or the activity of SRRP35 in the described cell of control group;
Wherein, if the expression amount of the SRRP35 of cell and/or activity are greater than control group in test group, just show that this test compounds is expression and/or the active candidate compound that has the treatment cancer of promoter action to SRRP35.
In another preference, described cell comprises: cancer cells or normal cell;
In another preference, described cell is liver cancer cell or liver cell.
In another preference, described method also comprises step:
(b) for the candidate compound obtaining in step (a), further test its restraining effect to growth of cancer cells or propagation.
In another preference, described step (b) comprises step: in test group, in the culture system of cancer cells, add test compounds, and observe quantity and/or the growing state of cancer cells; In control group, in the culture system of cancer cells, do not add test compounds, and observe quantity and/or the growing state of cancer cells; Wherein, if the quantity of cancer cells or the speed of growth are less than control group in test group, just show that this test compounds is the candidate compound that growth to cancer cells or propagation have inhibiting treatment cancer.
A sixth aspect of the present invention, also provides a kind of method that suppresses or treat cancer, comprises step: the purposes of using the SRRP35 agonist of safe and effective amount to the object (Mammals) of needs treatment.
In another preference, described cancer comprises liver cancer.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tire out and state no longer one by one at this.
Brief description of the drawings
Figure 1A is that in embodiment 1, real-time quantitative PCR detects the expression schematic diagram of SRRP35 gene in 32 routine hepatocarcinoma patient cancerous tissues and cancer beside organism, and wherein, " N " refers to cancer beside organism, and " C " refers to liver cancer tissue.Result shows in 20 examples (62.5%) patient's cancerous tissue that SRRP35 gene expression amount is lower than corresponding cancer beside organism.
Figure 1B has shown that real-time quantitative PCR detects the expression pattern of SRRP35 gene in human hepatoma cell strain.
Fig. 2 A has shown the excessively expression of SRRP35 in hepatoma cell strain Sk-Hep-1 and WRL-68, and SRRP35 successful expression in carrier for expression of eukaryon is described.
Fig. 2 B has shown that the SRRP35 albumen of excessively expressing has weakened the clonality of hepatoma cell strain Sk-Hep-1 and WRL-68.
Fig. 2 C has shown that the SRRP35 albumen of excessively expressing suppresses the increment of Sk-Hep-1 and WRL-68 cell.
Fig. 3 A has shown that the siRNA of synthetic has effectively disturbed the expression of SRRP35 gene.
Fig. 3 B has shown that the expression of the reticent SRRP35 of method disturbing by RNA promotes the increment of liver cancer cell Huh-7 and Hep3B.
Fig. 4 has shown that the expression of the reticent SRRP35 of method disturbing by RNA has improved liver cancer cell Huh-7 and the clonality of Hep3B in soft agar, and then explanation SRRP35 suppresses the pernicious sign of liver cancer cell.
Fig. 5 A has shown that the SRRP35 gene inhibition liver cancer cell WRL-68 that crosses expression is in the ability of nude mice by subcutaneous formation tumour.On volume and tumor weight, to cross expression group all little and light than control group for SRRP35, and two groups have statistical significance simultaneously.
Fig. 5 B has shown that reticent SRRP35 genetic expression promotes liver cancer cell Huh-7 to form the ability of tumour at nude mice by subcutaneous.On volume and tumor weight, the reticent expression group of SRRP35 is all large than control group and heavy, and two groups have statistical significance simultaneously.
Embodiment
The inventor, through extensive and deep research, is surprised to find that first, and SRRP35 is low expression in cancerous tissue, and in cancer beside organism and healthy tissues high expression level, the mark that therefore SRRP35 can be used as cancer detection for detection of or complementary detection cancer.In addition the growth that, the agonist of SRRP35 can anticancer (especially liver cancer cell).Complete on this basis the present invention.
The inventor, also taking carrier for expression of eukaryon system as mediation system, crosses expression SRRP35 gene (through identifying that expression multiple was for being greater than 20 times) in liver cancer cell Sk-hep-1 and WRL-68.Test discovery by Growth of Cells: the growth that obviously suppresses liver cancer cell Sk-hep-1 and WRL-68 is expressed in crossing of SRRP35 gene.Therefore, can the gene therapy for liver cancer by SRRP35 gene.
SRRP35 albumen and polynucleotide
In the present invention, " albumen of the present invention ", " polypeptide of the present invention ", " SRRP35 albumen " are used interchangeably, and refer to referred to as SRRP35).Should be understood that described term also comprises active fragments and the derivative of SRRP35.
In the present invention, " gene of the present invention ", " polynucleotide of the present invention " refer to the to encode nucleotide sequence of SRRP35 albumen or its active fragments and derivative, comprises justice and antisense nucleic acid.The SRRP35 assignment of genes gene mapping is in cell chromosome 6q15, and cDNA total length is 786bp, 261 amino acid whose albumen of coding total length.
In the present invention, term " SRRP35 albumen ", " SRRP35 polypeptide " or " cancer markers SRRP35 " are used interchangeably, and all refer to have albumen or the polypeptide of people's Protein S RRP35 aminoacid sequence.
SRRP35, has another name called SRSF12(serine/arginine-rich splicing factor12, is rich in Serine and arginic shear factor 12), be that 35kD gains the name because finding at first as supressor and the molecular weight of SR.SR albumen is the shear factor that a class is rich in Serine and the arginic RRM of having RNA binding domains, has the function that regulation and control eukaryotic cell pre-mRNA transcript alternative splicing falls intron and splices exon.Research is found in body, and SRRP35 can other SR albumen of antagonism and activated the alternative splicing (Alison E, et al.2001.THE JOURNALOF BIOLOGICAL CHEMISTRY) of the pre-mRNA5 ' least significant end of adenovirus E 1 A.
The cDNA sequence of SRRP35 gene is as shown in SEQ IDNO.:1; Genbank accession number 135295, the cDNA sequence C CDS47459.1 of SRRP35 with and coding aminoacid sequence NP542781.3.
The aminoacid sequence of SRRP35 coded by said gene albumen is as shown in SEQ ID NO.:2.
As used herein, " separation " refers to that material separates (if natural substance, primal environment is natural surroundings) from its primal environment.As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, for separation and purification.
As used herein, " SRRP35 albumen or the polypeptide of separation " refers to that SRRP35 albumen does not basically contain natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can be purified SRRP35 albumen with the purified technology of protein of standard.Substantially pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.In the present invention, SRRP35 albumen comprises fusion rotein and non-fusion rotein.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferably recombinant polypeptide.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
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 polynucleotide of the mature polypeptide of coding SRRP35 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence (with optional additional code sequence) and the non-coding sequence of mature polypeptide.Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and can be also the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or the fragment of polypeptide, analogue and derivative with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing in fact the function of its coded polypeptide.
The invention still further relates to and the nucleic acid fragment of above-mentioned sequence hybridization, comprise the nucleic acid fragment of justice and antisense.As used herein, the length of " nucleic acid fragment ", containing 15 Nucleotide, is at least better 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 to separate the polynucleotide of coding SRRP35 albumen.
People SRRP35 Nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be according to published relevant 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.In the time that sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment amplifying for each time is stitched together by proper order.
Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated and obtains relevant sequence from the host cell propagation by ordinary method.
In addition, also can synthesize relevant sequence by the method for synthetic, especially fragment length more in short-term.Conventionally, by first synthetic multiple small segments, and then connect and can obtain the fragment that sequence is very long.
The method of application round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.Primer for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment increasing by gel electrophoresis separation and purifying.
The present invention also relates to the carrier that comprises polynucleotide of the present invention, and the host cell producing through genetically engineered with carrier of the present invention or SRRP35 albumen coded sequence, and produce the method for polypeptide of the present invention through recombinant technology.
By conventional recombinant DNA technology, can utilize polynucleotide sequence of the present invention to can be used to the SRRP35 albumen of expression or Restruction.In general there are following steps:
(1). with the polynucleotide (or varient) of encoding human SRRP35 albumen of the present invention, or transform or the suitable host cell of transduceing with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of cultivating in suitable substratum;
(3). separation, protein purification from substratum or cell.
Method well-known to those having ordinary skill in the art can be used for building containing people SRRP35 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, to instruct mRNA synthetic.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of the host cell transforming, as eukaryotic cell is cultivated Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of use, or for colibacillary tsiklomitsin or amicillin resistance.
Comprise above-mentioned suitable DNA sequence dna and the suitable carrier of promotor or control sequence, can be for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, the bacterial cell of streptomyces; Fungal cell is as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS or 293 cells etc.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host is prokaryotic organism during as intestinal bacteria, the competent cell that can absorb DNA can, in exponential growth after date results, be used CaCl 2method processing, step used is well-known in this area.Another kind method is to use MgCl 2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is as microinjection, electroporation, liposome packaging etc.
The transformant obtaining can be cultivated by ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional mediums.Under the condition that is suitable for host cell growth, cultivate.When host cell grows into after suitable cell density, the promotor of selecting with suitable method (as temperature transition or chemical induction) induction, cultivates cell for some time again.
Extracellular can be expressed or be secreted into recombinant polypeptide in the above methods in cell or on cytolemma.If needed, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processing, with the combination of protein precipitant processing (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Antibody
The present invention also comprises that people SRRP35 albumen is had to specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " refers to that antibody capable is incorporated into people SRRP35 gene product or fragment.Preferably, refer to that those can be combined with people SRRP35 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.
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 scFv molecule; Or chimeric antibody.
The antibody of anti-human SRRP35 albumen can be used in immunohistochemistry technology, detects the people SRRP35 albumen in biopsy specimen.
Agonist and pharmaceutical composition
Utilize albumen of the present invention, by various conventional screening methods, can filter out with SRRP35 albumen interactional material occurs, especially agonist etc.
The agonist of SRRP35 albumen of the present invention, when use (administration) in treatment time, can promote expression and/or the activity of SRRP35 albumen, and then growth or the propagation of anticancer (comprising liver cancer).Conventionally, these agonists can be formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, and preferably pH is about 6-8, although pH value can change to some extent with being formulated the character of material and illness to be treated.The pharmaceutical composition preparing can carry out administration by conventional route, comprising (but being not limited to): in knurl, intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
The present invention also provides a kind of pharmaceutical composition, the SRRP35 albumen of the present invention that it contains safe and effective amount or its agonist and pharmaceutically acceptable carrier or vehicle.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the present invention can be made into injection form, for example, be prepared by ordinary method with physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as Tablet and Capsula, can be prepared by ordinary method.Pharmaceutical composition should be manufactured as injection, solution, Tablet and Capsula under aseptic condition.The dosage of activeconstituents be treatment significant quantity, for example every day approximately 1 microgram-10 mg/kg body weight.
Detection method and test kit
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people SRRP35 protein level or mRNA level.These tests are known in the art.The people SRRP35 protein level detecting in test, can be for diagnosing liver cancer.
A kind ofly detect that in sample, whether to have the method for SRRP35 albumen be to utilize the specific antibody of SRRP35 albumen to detect, it comprises: sample is contacted with SRRP35 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in sample SRRP35 albumen.
SRRP35 albumen or its polynucleotide can be used for diagnosis and the treatment of SRRP35 protein related diseases.Part or all of polynucleotide of the present invention can be used as probe and is fixed on microarray or DNA chip, for analyzing Differential expression analysis and the gene diagnosis of tissue gene.The antibody of anti-SRRP35 can be fixed on protein chip, for detection of the SRRP35 albumen in sample.
The present invention also provides a kind of test kit that detects liver cancer, the primer pair that it contains specific amplification SRRP35 and/or SRRP35 specific antibody.
Screening method
The present invention also provides the method for carrying out drug screening based on SRRP35.One method is first to screen impact (promotion) SRRP35 expression or active compound, then the compound filtering out is further tested to it to cancer cells.A kind of screening method can be based on SRRP35 the expression level of mRNA.
Wherein, representational cancer cells comprises (but being not limited to): liver cancer cell.
Universal method:
(1) obtaining of clinical tissue sample
Liver cancer and cancer beside organism take from the liver cancer patient of operative treatment, have all signed Informed Consent Form with patient obtaining before sample.The liver of excision is once in vitro, cuts rapidly primary tumors and the cancer beside organism beyond 5cm around, drops in liquid nitrogen quick-frozen and moves to-80 DEG C of Refrigerator stores, when transport, is stored in liquid nitrogen.Cancer and cancer beside organism all make last diagnostic by pathology expert.Sample is divided into I-II I level according to Edmondson grade scale.
(2) tissue and cell RNA extracting
Adopt TRIzol Reagent(Invitrogen) reagent extracting RNA, concrete operations are as follows:
1) mortar, grind the vessel such as pestle and homogenizer and clean, use again respectively ddH 2o and DEPC H 2o rinses, and then in 180 DEG C of baking ovens, dries approximately 4 hours, to remove RNA enzyme;
2) in mortar, add appropriate liquid nitrogen to make it precooling, will organize from liquid nitrogen and take out rapidly, cut approximately 50 – 100mg sizes, grind into powder in mortar;
3) with curet, the ground powder of organizing is moved in the EP pipe without RNA enzyme as far as possible completely, EP pipe is added appropriate volume (1ml) TRIzol reagent in advance, fully homogenate;
4) room temperature is placed 5 minutes, adds chloroform (200 μ l/1ml TRIzol) in proportion in centrifuge tube, rapidly thermal agitation 15 seconds, and room temperature leaves standstill 2 – 3 minutes, and 4 DEG C, under 12000 × g condition centrifugal 15 minutes;
5) upper strata water is transferred to as much as possible in the new EP pipe without RNA enzyme, adds isopyknic Virahol, put upside down and mix 5 times, room temperature leaves standstill 10 minutes, and 4 DEG C, under 12000 × g condition centrifugal 10 minutes, now visible RNA precipitation;
6) supernatant is outwelled, added 75% ethanol (1ml/1ml TRIzol), mix, washing RNA, centrifugal 4 DEG C, under 7500 × g condition centrifugal 5 minutes;
7) abandon supernatant, eliminate as far as possible residual ethanol, precipitation seasoning 5 – 10min(note being sure not complete drying); Add 30 – 50 μ l DEPC H 2o, pressure-vaccum several times, dissolves RNA precipitation;
8) microplate reader is measured RNA concentration and purity OD260/280(1.8 – 2.0); Gel electrophoresis is observed and is had or not degraded ,-80 DEG C of preservations.
Cell strain RNA extracting, the cell in the vegetative period of taking the logarithm, draws nutrient solution, adds TRIzol reagent (the 1ml TRIzol/10cm of respective amount according to the area of culture dish 2) lysing cell, several times, the cell harvesting that cracking is got off is in the EP pipe without RNA enzyme in piping and druming, all the other are according to above-mentioned steps 4) – 8) complete Lv Fang – isopropanol method separation and purification RNA.
(3) reverse transcription of RNA
With M-MLV Reverse Transcriptase(Promega) reverse transcription, operate as follows:
1) in the EP of nuclease free pipe, add following component:
Be placed in PCR instrument, 70 DEG C, 5 minutes, then immediately at cooled on ice 5min.
2) in above-mentioned system, add again following component:
After mixing gently, be placed in PCR instrument, 37 DEG C, 60min.
The cDNA that reverse obtains is placed in 4 DEG C of preservations.
(4) real-time quantitative PCR
Real-time quantitative PCR reaction is used premix ExTaq tMthe reaction system of (Perfect RealTime) test kit (TaKaRaBiotechnology Co., Ltd.Dalian, China), utilizes Thermal CyclerDice tMrealTime System(TP800 real-time fluorescence quantitative PCR instrument, TaKaRa) operate.The amplified production length of quantitative PCR is with 80bp – 150bp the most suitable (can extend to 300bp).
Reaction system is as follows:
Reaction conditions:
Solubility curve analytical procedure:
95℃ 15sec
60℃ 30sec
95℃ 15sec
Dissociation time is 4sec.
The default value that fluorescence background signal and threshold value adopt instrument to arrange, can generate after each PCR reaction finishes automatically, the cycle number that the fluorescent signal in the each reaction tubes of Ct value representation experiences while reaching setting threshold (baseline fluorescence intensity 10 times); The each template of goal gene SRRP35 is done 3 multiple pipes, and the Ct value obtaining is averaged; The Ct mean value of SRRP35 gene deducts the Ct mean value of the reference gene (β-actin) of corresponding template, obtains Δ Ct.The Δ Ct of liver cancer group deducts the Δ Ct of corresponding adjacent tissues, obtains Δ Δ Ct value, and the multiple relation of the SRRP35 gene in the other group of liver cancer group and cancer is with 2 -Δ Δ Ctrepresent.
(5) construction of eukaryotic expression vector
1) template: the cDNA library of people liver immortalized cells L02.
2) selection of carrier for expression of eukaryon: pcDNA tM3.1/myc-His (-) A, 5522nucleotides.
3) according to SRRP35mRNA( nM080743.4) sequence, in conjunction with expression vector pcDNA tMthe restriction enzyme site design primer of 3.1/myc-His (-) A, primer sequence is as SEQ ID NO.:3(forward) and SEQIDNO.:4(reverse) as shown in.Wherein in reverse primer, the terminator codon of SRRP35 is removed, and makes c-myc and 6xHi s label on the C-terminal band of SRRP35.Utilize hi-fi archaeal dna polymerase PrimeSTAR tMhS DNA Polymerase(TaKaRa), taking L02cDNA as template amplification gene SRRP35 total length opening code-reading frame, 50 μ l total reaction system compositions are as follows:
Employing two-step PCR (98 DEG C, 10sec; 60 DEG C, 90sec), 35 circulations of increasing.The about 0.8kb of PCR product size, 1% agarose gel electrophoresis qualification size, the PCR product that (glue purification test kit: MACHEREY-NAGEL) meets clip size is reclaimed in rubber tapping.
EcoRV, Hind III (TaKaRaBiotechnology Inc.Dalian, China) double digestion reclaims PCR product and vector plasmid pcDNA tM3.1/myc-His (-) A, endonuclease reaction system is as follows:
37 DEG C of endonuclease reactions 1 hour; Rubber tapping is reclaimed enzyme and is cut product.
4) connect: the PCR product that enzyme cuts back to close mixes according to the ratio of mole ratio (4:1) with carrier, the link of DNA ligase system, also comprises 2.5 μ l4 × Solution I (TaKaRa Code:D102A), ddH in system 2o polishing to 10 μ l, 16 DEG C connect 2h and even spend the night;
5) transform: get 10 μ l connection products and mix with 100 μ l competence bacteriums (TOP10 or DH5 α), place 30min on ice, 42 DEG C of heat shock 90sec, be placed in immediately 5min on ice, add 800 μ l not containing antibiotic LB nutrient solution, 37 DEG C, 200rpm shaking culture 30min, make thalline recovery and an amplification generation, the centrifugal 2min of 3000rpm, remove most of supernatant, stay 50 – 100 μ l bacterium liquid, piping and druming precipitation gently mixes, and has then evenly been applied to amicillin resistance (Amp +) LB flat board on, cultivate 12 – 16 hours for 37 DEG C.
6) clone identification: the bacterium colony that picking is grown after ammonia benzyl resistance screening enlarged culturing in the liquid nutrient medium that adds penbritin, extraction plasmid carries out enzyme and cuts qualification: get the little plasmid EcoRV that takes out of 1 – 2 μ g, Hind III double digestion, agarose gel electrophoresis qualification endonuclease bamhi size, carrier pcDNA tMthe about 5.5kb of 3.1/myc-His (-) A clip size, the about 800bp of SRRP35 reading frame clip size, meets big or small clone and send order-checking to confirm the exactness of Insert Fragment sequence.
(6) mensuration of cell growth curve
1) different types of HCC cell is pressed to 3-5 × 10 according to its growth characteristics 3cell total amount is calculated in/100 μ l/ holes, fully, after peptic cell, is diluted to desired concn, is inoculated in 96 orifice plates.Every group three of every day multiple hole, by 7 days inoculating cells of 5 –;
2) observation of cell state and number after cell is substantially adherent.Carry out color reaction with CCK-8 developer (Cell CountingKit-8, DOJINDO, Japan), every 100 μ l nutrient solutions add 10 μ l CCK-8,37 DEG C, 5%CO 2incubator is placed and is hatched 1h, and microplate reader is measured the absorbancy at 450nm place, and record is determined the initial density of cell reality, as the relative zero of growing.
3) every day or every other day half amount change liquid, specifically depending on requirement of experiment;
4) observation of cell form under microscope, Fixed Time Interval is measured, and records cell growth condition;
5) generally survey 5 to 7 days.After end to be determined, collect data and process, draw chart with Excel.
(7) cell clonal formation experiment
1) transfection: adopt Lipofectamine tM2000(Invitrogen) transfectional cell, the expression of crossing SRRP35 gene in expression or reticent cell;
2) cell after transfection, after normal nutrient solution cultivation 24h for 6 orifice plates or 35mm culture dish, digestion is counted, be seeded to 100mm culture dish (different cell strain number difference) by certain number, continue to cultivate 24h, then add the G418(600 – 1000 μ g/ml of proper concn according to cell category), to screen the cell clone of the transfection positive;
3) cultivate 2-3 week, changed fresh medium therebetween every 3-5 days and add G418 screening, until there is macroscopic cell clonal formation;
4) suck the training liquid in culture dish, 1 × PBS washes twice, coomassie brilliant blue R_250 dyeing 2h, and after water rinses gently, then with the coomassie brilliant blue staining destainer 30 – 60min that decolour;
5) clone's formation coloration result is taken pictures, and according to identical standard (cell clone size), the cell clone on each culture dish is counted.
(8) western blotting (Western Blot)
1) protein sample preparation: cultured cells sucks after culture supernatant, wash twice with the 1XPBS of precooling, add 2 × SDS lysate (100mM Tri s-Cl, pH=6.8,4%SDS, 20% glycerine), fully, after cracking, boiling water bath heats 10min, the centrifugal 10min of 12000 × g, supernatant is transferred in new pipe bCA ProteinAssayKit carries out quantitatively the albumen obtaining ,-80 DEG C of preservations;
2) protein electrophoresis separates: add the sample-loading buffer (loading buffer) that contains in right amount 200mM DTT at protein sample, boiling water bath heating 10min, slightly do centrifugal, SDS-PAGE proteins gel electrophoresis sample separation;
3) transferring film: running gel, nitrocellulose membrane, thick (thin) filter paper backing plate are dipped in to (24mMTris, 192mM glycine, 20% methyl alcohol) balance 15-20min in transferring film damping fluid.Put the wet instrument (XCellSureLock that turns well by the order of 2 layers of thin filter paper backing plate – negative pole of the fine dimension of positive utmost point – 1 bed thickness filter paper backing plate – nitric acid film – electricity swimming glue – tM, invitrogen) and 30 volts of transferring film 30-40min;
4) sealing: 5% skim-milk/0.1%PBST is as confining liquid, horizontal shaking table, room temperature sealing 30min-2h;
5) primary antibodie: confining liquid dilution (reference antibody specification sheets recommended density) for primary antibodie, incubated at room 2h or 4 DEG C of overnight incubation, 0.1%PBST washes three times, each 5min;
6) two is anti-: fluorescence two is anti-with confining liquid dilution (1:1000), incubated at room 30min, and 0.1%PBST washes three times, each 5min;
7) sweep film: ODYSSEY infrared imaging system scanning nitrocellulose filter, preserve image.
(9) soft-agar cloning forms experiment
1) prepare respectively 1% and 2% low melting point Agarose(TaKaRa company), autoclave sterilization;
2) preparation 2 × DMEM nutrient solution (2.5 × DMEM, containing 20%FBS);
3) 2%Agarose of 37 DEG C of insulations and 2 × DMEM nutrient solution are mixed by same volume, be added in 24 orifice plates with every hole 0.5ml, be placed in 4 DEG C of refrigerators, after solidifying, use;
4) fully digest cultured cells and become individual cells, counting, is diluted to same concentrations (3000-5000/0.5ml);
5) cell suspension is mixed with same volume with the 1%Agarose of 37 DEG C of insulations, be added in 24 orifice plates of completing lower floor's glue, every hole 0.5ml, is placed in 4 DEG C of refrigerator 10min;
6) on the soft agar solidifying, add 0.2ml nutrient solution, be placed in 37 DEG C, 5%CO 2in incubator, continue to cultivate 2-3 week;
7) growing state of the inside, the each hole of micro-Microscopic observation cell clone, counting, analyzes.
(10) nude mice becomes knurl experiment
1) mouse used is the male BLAB/c nu nude mice of 5-6 week size, is provided by Shanghai Slac Experimental Animal Co., Ltd., raises in southern model animal Culture Center;
2) getting cell after treatment, be inoculated in mouse subcutaneous (different cell category inoculation number difference) with equal amts, is the error of avoiding individual difference to cause, and allogenic cell different treatment can symmetrically be inoculated in same mouse;
3) after there is visible tumour, every 3 days monitoring tumor sizes, vernier callipers reads tumour major diameter and minor axis,
Calculate as follows gross tumor volume: volume=major diameter x minor axis 2;
4) continue to monitor after about 7-8 time disposal data, statistics.
(11) antibody obtains and immunodetection
1) antigen protein obtains
The cDNA sequence that obtains people SRRP35 gene from Genebank database, obtains encoder block by pcr amplification, inserts in prokaryotic organism or eukaryote expression vector, expresses SRRP35 albumen, and presses the purification system purifying protein of gene engineering expression product.
2) antibody preparation
Can adopt following several method Dispersal risk:
A cytogamy method: with the SRRP35 protein immune animal (comprising rabbit, goat etc.) of above-mentioned preparation, obtain spleen cell, then merge with myeloma cell, and monoclonal antibody technology of preparing is prepared monoclonal antibody routinely.
B utilizes phage display storehouse, and the spleen IgG variable region of clone immune animal is also expressed as gene engineering monoclonal antibody.
C utilizes the protein immune animal of purifying, prepares polyvalent antibody.
3) detect
The antibody (how anti-or monoclonal antibody) of preparation for a, carries out the pathology detection of liver cancer with histochemical method, negative signal is liver cancer.
B gets patients serum, and with the detection of ELISA method, negative reaction is the suspicious patient of liver cancer.
One of c probe using SRRP35 antibody as protein chip, diagnoses for kinds of tumors.
Embodiment 1: real-time quantitative PCR detects the expression of SRRP35 in clinical sample
The total mRNA that adopts universal method 1~2 to obtain, and by the expression of universal method 4SRRP35 in liver cancer tissue.The primer sequence of the real-time quantitative PCR that in experiment, detection SRRP35 used expresses is as shown in SEQIDNO.:5 and SEQ ID NO.:6.As the primer sequence of the β-actin of internal reference as shown in SEQ ID NO.:7 and SEQID NO.:8.
Result is as Figure 1A: in 32 pairs of clinical samples, detect by real-time quantitative PCR the expression that finds that there is SRRP35 in 20 pairs of sample liver cancer tissues and be starkly lower than corresponding cancer beside organism, downward rate reaches 62.5%, and statistical analysis shows p<0.01, and the reliability of result is described.We have also detected the expression pattern of SRRP35 in 12 kinds of hepatoma cell strains simultaneously, and as shown in Figure 1B, SRRP35 presents extremely low expression in 10 kinds of cells, also further illustrates the expression level of SRRP35 in cancer lower.
Embodiment 2: cross the propagation of expressing SRRP35 gene inhibition liver cancer cell
Build respectively carrier for expression of eukaryon with universal method 5,8, and the cDNA of SRRP35 is cloned into pcDNA tM3.1/myc-His (-) A, the plasmid that transfection builds enters liver cancer cell successful expression, as shown in Figure 2 A.For the primer sequence that builds SRRP35 carrier for expression of eukaryon as SEQ ID NO.:9(forward) and SEQ ID NO.:10(reverse) as shown in.
Then, then carry out functional experiment growth curve and clone's formation test with universal method 6~7, result is as shown in Fig. 2 B, C: the overexpression of SRRP35 suppresses propagation and the clonality of liver cancer cell Sk-hep-1 and WRL-68.
Embodiment 3: the expression Promote cell's growth of reticent SRRP35
Synthetic by Shanghai Ji Ma pharmaceutical Co. Ltd for the siRNA that disturbs SRRP35 to express, sequence is respectively as SEQIDNO.:11(positive-sense strand) and SEQ IDNO.:12(antisense strand) as shown in.Classify as the non-specific nucleotides sequence of NC that disturbs contrast: SEQIDNO.:13(positive-sense strand) and SEQIDNO.:14(antisense strand)
Through real-time quantitative PCR detected result as shown in Fig. 3 A, 3B: the interference siRNA of synthetic can effectively lower the expression level of SRRP35, and growth curve experiment has also proved that the downward of SRRP35 can promote the growth of liver cancer cell Huh-7 and liver cancer cell Hep3B.
As can be seen here, SRRP35 can suppress liver cancer cell growth.
Embodiment 4:SRRP35 suppresses the pernicious sign of liver cancer cell
Adopt WRL-68 to cross the research object of expression as SRRP35, chosen Huh7 cell as SRRP35 reticent express research object.By universal method 9, the number of cell clones being grown in soft agar is analyzed.
Result is as shown in Figure 4: cross and express the grade malignancy that SRRP35 can effectively suppress WRL-68 cell and the reticent SRRP35 of expression and can effectively promote Huh-7 cell, show that SRRP35 affects the pernicious of liver cancer cell.
Embodiment 5:SRRP35 affects the one-tenth knurl ability of liver cancer cell at nude mice by subcutaneous
By universal method 10, by 1 × 10 6individual mistake is expressed the WRL-68 cell, 3 × 10 of SRRP35 6the Huh-7 cell of individual reticent expression SRRP35 and control cells respectively symmetry are injected into nude mice subcutaneous abdomen, observe the growing state of knurl body.
Result is as shown in Fig. 5 A, 5B: SRRP35 excessively expresses gross tumor volume is obviously dwindled compared with control group, illustrates: SRRP35 excessively expresses and can effectively suppress the one-tenth knurl ability of WRL-68 cell at nude mice by subcutaneous; And the silence of SRRP35 obviously increases gross tumor volume, illustrate that the downward of SRRP35 has effectively promoted Huh-7 cell nude mice by subcutaneous to become the ability of knurl.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that 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 limited range equally.

Claims (10)

1. a purposes for SRRP35 gene or SRRP35 albumen, is characterized in that, for the preparation of the reagent or the test kit that detect cancer.
2. purposes as claimed in claim 1, is characterized in that, described test kit comprises: the reagent and corresponding label or the specification sheets that SRRP35 albumen or mRNA are carried out to detection by quantitative.
3. purposes as claimed in claim 1, is characterized in that, described reagent comprises SRRP35 Auele Specific Primer, specific antibody, probe and/or chip; And/or
Described cancer comprises liver cancer.
4. for detection of a diagnostic kit for cancer, it is characterized in that, described test kit contains a container, contains the detection reagent that detects SRRP35 albumen or mRNA in described container; And label or specification sheets, described label or specification sheets indicate described test kit for detection of cancer.
5. test kit as claimed in claim 4, is characterized in that, indicates following content in described label or specification sheets:
When ratio≤1 of the mrna expression amount of the mrna expression amount of the relative beta-actin of the SRRP35 of detected object and the relative beta-actin of SRRP35 of cancer beside organism, point out the cancered probability of this detected object higher than general population.
6. a purposes for SRRP35 albumen, SRRP35 gene or its agonist, is characterized in that, is used to prepare the medicine of anticancer growth or propagation, or for the preparation of the medicine for the treatment of cancer.
7. a method for the growth of the anticancer of external non-therapeutic or propagation, is characterized in that, comprises step: under SRRP35 albumen or the existence of its agonist, cultivate cancer cells, thus anticancer growth or propagation.
8. a method of screening the candidate compound for the treatment of cancer, is characterized in that, described method comprises step:
(a) in test group, in the culture system of cell, add test compounds, and observe expression amount and/or the activity of SRRP35 in the cell of described test group; In control group, in isocellular culture system, do not add test compounds, and observe expression amount and/or the activity of SRRP35 in the described cell of control group;
Wherein, if the expression amount of the SRRP35 of cell and/or activity are greater than control group in test group, just show that this test compounds is expression and/or the active candidate compound that has the treatment cancer of promoter action to SRRP35.
9. method as claimed in claim 8, is characterized in that, described method also comprises step:
(b) for the candidate compound obtaining in step (a), further test its restraining effect to growth of cancer cells or propagation.
10. method as claimed in claim 9, is characterized in that, described step (b) comprises step: in test group, in the culture system of cancer cells, add test compounds, and observe quantity and/or the growing state of cancer cells; In control group, in the culture system of cancer cells, do not add test compounds, and observe quantity and/or the growing state of cancer cells; Wherein, if the quantity of cancer cells or the speed of growth are less than control group in test group, just show that this test compounds is the candidate compound that growth to cancer cells or propagation have inhibiting treatment cancer.
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CN105624275A (en) * 2014-11-06 2016-06-01 上海市东方医院 Applications of EIF4G1 in diagnosis and treatment of squamous cell carcinomas
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