CN1704426A - Cancer gene and its medical use - Google Patents
Cancer gene and its medical use Download PDFInfo
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- CN1704426A CN1704426A CN 200410012307 CN200410012307A CN1704426A CN 1704426 A CN1704426 A CN 1704426A CN 200410012307 CN200410012307 CN 200410012307 CN 200410012307 A CN200410012307 A CN 200410012307A CN 1704426 A CN1704426 A CN 1704426A
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Abstract
The invention discloses a cancer gene and its pharmaceutical use, wherein the gene has the name of PPO and cDNA nucleic acid sequence of SEQ ID NO.1, its DNA length is approx. 36-kb, the cDNA length is 6022 bases, the PPO amino acid sequence has the genes of the open-reading frame of 993 amino acid residues, the gene is expressed in multiple tissues of human cancer, including oophoron cancer, mammary cancer, pancreas adenocarcinoma, colon cancer, lung carcinoma and prostate cancer. The reactive composition can be prepared into diagnosis agent or reagent kit for cancer diagnosis.
Description
Technical field
The present invention relates to new gene and uses thereof, specifically the new gene and the medicinal use thereof in No. 1 the short arm of a chromosome 36 districts of human body (1p36).
Background technology
Modern biology studies show that, cancer be the rapid complicated event of a kind of multiplefactor multistep, be the result of multiple oncogene activation or cancer suppressor gene inactivation.At present, the oncogene medical research mainly comprises three aspects: (1) seeks the goal gene with treatment meaning.(2) set up effective carrier system to target cell transfer destination gene.(3) goal gene is expressed in target cell, the performance biological effect.Therefore, searching oncogene or cancer suppressor gene are the most important things in the present cancer research.
The heterozygosity disappearance of human body 1p36 and karyomit(e) obtain existing (reviewed by Schwab et al.1996 Kraggerud et al., 2000 reported; Loveday et al., 2000; Stock et al., 2000; Alvarez et al., 2001; Zielenska et al., 2001).These result of study promptings exist oncogene or cancer suppressor gene in this district.
Summary of the invention
Purpose of the present invention is exactly screening and isolates new gene and the transcript relevant with cancer that occurs in the 1p36.13 zone, and develops the medicinal use that it has.
The object of the present invention is achieved like this:
The inventor utilizes the colony screening technology, on human (homo sapiens) No. 1 karyomit(e), has found a new oncogene.This gene (ovarian cancer, mammary cancer, carcinoma of the pancreas, colorectal carcinoma, lung cancer, prostate cancer, the cancer of the brain, cancer of the stomach, liver cancer, uterus carcinoma etc.) in the multiple cancerous tissue of the mankind all had expression, PCR quantitatively to show, it is hundreds of to thousands of times that it exceeds healthy tissues.Studies confirm that further this gene is relevant with propagation and phosphorylation.It is subcutaneous that this gene transfered cell is inoculated in nude mice, through one month observation, can grow tumour.In addition, this gene is relevant with very famous MAPK pathway.It can stimulate RAS to cross expression and MEK, ERK2 phosphorylation.With the mode of RNA interfering (RNAi), block this expression of gene, can reduce the expression of phosphorylation and PCNA.Cell levels and live body all confirmed can anticancer growth.
It is PPO gene (Phosphorylation and ProliferationOncogene) that the inventor names this gene.
By the cDNA nucleotide sequence of PPO as described in the SEQ ID NO.1.The about 36-kb of its DNA length, cDNA length 6022 bases.
The aminoacid sequence that the cDNA nucleotide sequence of PPO (SEQ ID NO.1) is inferred is as described in the SEQ ID NO.2.It has the gene of the open reading-frame (ORF) of 993 amino-acid residues shown in the coding SEQ ID NO.2.
The amino acid molecular amount of PPO genes encoding of the present invention is 100kD.
The example of the combination of codon is represented in the coding region of this nucleotide sequence, and described codon is at the amino-acid residue separately in the aminoacid sequence shown in the SEQ ID NO:2.
The combination of codon is not limited to the example shown in the SEQ ID NO:1 in the gene of the present invention.The arbitrary combination of codon can be used for amino-acid residue separately.The selection of codon can adopt usual manner to carry out.
The production method of gene of the present invention is to prepare the cDNA library with conventional procedure or directly buy from the appropriate sources of wherein expressing gene of the present invention, and the antibody that uses suitable probe or be specific to gene of the present invention screens required clone from this library.
Being suitable for what do the cDNA source is for example to express the various cells of gene of the present invention and tissue and deutero-culturing cell therefrom.
From the separation of total RNA in this source, obtaining and cloning of the separation of mRNA and purifying and cDNA can be carried out in the usual way.
Commercially available cDNA library is as purchasing the production that can be used for gene of the present invention in the various cDNA library of Clontech (PaloAltocA) company.
The gene of producing from the cDNA library of the present invention, its screening method has no particular limits, and can adopt conventional method.
The example of screening method, comprise that use is at screened corresponding cDNA clone immunoscreening method by the proteic specific antibody that cDNA produced, use probe optionally with target DNA sequence bonded method, as plaque hybridization method or colony hybridization method, and the combination of these methods.
Act on the probe of aforesaid method, generally can use according to the information of the nucleotide sequence of gene of the present invention and the DNA of chemosynthesis.Acquired gene of the present invention or its fragment also can be used for probe.
According to the information of the nucleotide sequence of gene of the present invention and have adopted primer and the antisense primer that design can be used as the probe of screening.
In obtaining gene of the present invention, can be by the DNA/RNA amplification of PCR.Particularly can adopt the RNACE method to obtain the cDNA of total length.
The primer that is used for these PCR method can carry out appropriate design with reference to the nucleotide sequence information of gene of the present invention, also can be synthetic by conventional procedure.
Gene of the present invention can detect by conventional procedure, as PCR method.
When selecting PCR method to be used to detect, it can be any primer that only can cause gene Selection amplification of the present invention that primer to be used is arranged.
The screening of PPO gene of the present invention, separation, determine, clone, nucleotide sequence determine undertaken by following method.
The cDNA library screening
The tire liver, the tire brain, pancreas and kidney cDNA library purchase in Clontech (Palo Alto, CA).Large intestine, lung, liver, the cDNA library of ovary and fibroblast purchase in Stratagene. (La Jolla, CA).Design 10 groups of primers, be used for screening the cDNA library,, finally determine EST Hs.154797 and clone full-length cDNA by the cDNA library screening.Accurate location PPO is in 1p36.13 and measure dna sequence dna
With the own 1p36 contig of being done (contig), the mode of hybridizing by southern blotting technique hybridization (SouthernBlotting) finds BAC clone b203I24, b140B13, and b81G11, and b57B11 accurately is positioned 1p36.13 and measures dna sequence dna.
Point out exon sequence
Utilize GenScan program (
Http:// genes.mit.edu/GENSCAN.html) (Burge andKarlin, 1997) point out exon and intron sequences.Utilize the RACE method to find the sequence of 5 ' end.(Clontech, Palo Alto CA), find the sequence of 5 ' end from renal cancer cell line ACHN to utilize 5 ' SMART RACE test kit.
Primer is as follows:
R1，5’-AACCAACTTCTTGGATCCAGGGGA-3’.
R2，5’-TTGGAATTCAATCAAACTTGCCCACCTG-3.
Cell cultures:
OVK18, OVK18#102 (Uehara et al., 1984), OVCAR3 (Hamilton et al., 1984), CoLoTC (Quinn et al., 1979), Lu65 (Hirohashi et al., 1989), HT1 (Tsuchiya et al., 1982), HCT15 (Dexter et al., 1979), HEC1A (Kuramotoet al., 1972), ACHN (Giard et al., 1973), U251N (Hirohashi et al., 1978), SUN5 (Brattain et al., 1981), LK-2 (Yoshioka et al., 1989) and NIH/3T3 (Aaronson SA et al) PK1, PK8 and PK9, (Kobari et al., 1986), used cell all northeast university add Institute for Medical Research in age.LNCaP (Gibas et al., 1983), HEK 293 (Graham et al., 1977) and U87 (Beckma.et al., 1971) purchase in U.S. ATCC (Manassas, VA). and cultivate in strict accordance with explanation.
Reverse transcription PCR:
RNA extracts in freezing tissue or cell strain.After becoming cDNA by upset record, following 94 ℃ of PCR conditions 5 minutes, 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 30 seconds, 40 circulations.72 ℃ 7 minutes.
Primer is as follows:
F，5’-TTTGATTGGAGACAGCAATATG-3’。
R，5’-CTGCGATGTACCTTACAGAC-3’。
Quantitative reverse transcription PCR:
Quantitative PCR machine ABI PRISM 7000 Sequence Detection System (AppliedBiosystems, Foster City, CA).
Primer is as follows:
F, 5 '-GCTTTTCGGAGAAGTCTAGTTCAAAG-3 '. (from 1117th nt to 1142nd nt).
R, 5 '-TCTCCACGAGGTATAGGTTAATGGT-3 '. (from 1197th nt to 1173rd nt).
Probe, 5 '-CTTGCTTCAATCAGACCTA-3 '. (from 1153rd nt to 1171st nt).
β 2-microglobulin gene is as controlling gene.
Primer is as follows:
F, 5 '-GTGACTTTGTCACAGCCCAAGA-3 '. (from 373rd nt to 394th nt).
R, 5 '-AACCTCCATGATGCTGCTTACA-3 '. (from 437th nt to 416th nt)
Probe, 5 '-AGTTAAGTGGGATCGAGAC-3 '. (from 396th nt to 414th nt)
The result measures with calibration curve method.
The structure of plasmid:
The structure of plasmid in two steps, at first from the KpnI site to the EcoRI site, then from the EcoRI site to the XbaI site, earlier handle carrier and PCR product respectively with KpnI and EcoRI enzyme, connect with ligase enzyme again.Cultivation was in a large number again handled carrier and PCR product respectively with EcoRI and XbaI enzyme then after the order-checking proof was suddenlyd change, and connected with ligase enzyme again, cultivated back transfection NIH/3T3 cell strains after the order-checking proof is suddenlyd change again in a large number and obtained stably express.3000 base pairs of total length.The expression vector of packing into be pcDNA3.1/V5His (Invitrogen, Carlsbad, CA).
Western blot hybridization (Western Blotting) is analyzed
After cell cultures reclaimed, method saw reference (Kondo et al., 1999) for details, antibody V5, PCNA, Phospho-MAPK, anti-beta actin purchase in (Sigma, St Louis, MO).ERK2 purchase in (BDBioscience, Palo Alto, CA), RAS (Oncogen, Boston, MA), MEK1/2 andPhospho-MEK1/2 purchase in (Cell Signaling Beverly, MA)
Cellular immunization dyeing
Plasmid transfection NIH/3T3 cell strain also obtains the stably express cell and reclaims the back smear, and through the processing of PBS damping fluid, V5 is anti-as one, and 1%FITC is as two anti-dyeing, and 4,6-diamidino-2-phenylindole (DAPI) dyes nuclear.Show the position that the protein of this gene exists in cell.
MTT analyzes
Cell cultures detected once totally 6 days in per 24 hours with 96 hole culture dish.With MTT dyeing, MTT purchase in (MO), VERSA max microplate reader machine detects the light transmission of solution for Sigma, St Louis, machine purchase in Japan (Molecular Devices, Tokyo, Japan).
Colony on the soft agar forms capability analysis
With 6 orifice plate culture dish, and 1% soft agar (DIFCO LABORATORIES, Detroit, MI), with 1 * 10
4Individual cell seeding after 4 weeks, is used the MTT chromoscopy in every hole, see the propagation situation of cell.
, can stablize and produce its expression product in large quantities or contain the albumen of same expression product by conventional gene engineering by gene of the present invention.
Albumen of the present invention and production method thereof:
The proteic aminoacid sequence of PPO of the present invention is shown in SEQ ID NO.2.
Albumen of the present invention also comprises its any homologue.Described homologue can be to have identical aminoacid sequence and keep the active albumen of PPO.
The proteic homologue of the present invention includes same derived from human, horse, sheep, dog, ape, cat and other mammalian species and rodent the albumen with identical activity or function with the aminoacid sequence shown in the SEQ IP NO:2 of having as rat, mouse and rabbit.
From table 2 also as can be seen, in other biological, there is the homotype gene of PPO gene, comprises mouse, fruit bat, mosquito, nematode and yeast.
Albumen of the present invention can prepare according to PPO gene nucleotide series information disclosed by the invention by conventional recombinant DNA technology.
As make up plasmid, allow coding target protein expression of gene in host cell, by to transduction vector transformed host cell wherein; Make the transforming factor growth of gained, from training liquid, collect albumen.
Host cell can be any prokaryotic cell prokaryocyte and eukaryotic cell.
When prokaryotic cell prokaryocyte was used as host cell, the construction process of expression plasmid was to use reproducible carrier in the host cell, and added promotor and SD (Shine and Dalgqrno) sequence in the upstream of gene of the present invention, so that this gene can be explained therein.
As expression carrier of the present invention, can utilize any conventional fusion protein expression vector (as pcDNA3.1).
Method and relevant method for transformation that described expression vector is imported in the host cell can adopt ordinary method to carry out.The transformant of gained can adopt usual manner to cultivate.
The recombinant protein of the present invention that obtains like this can separate by routine techniques, purifying.These technology comprise reconstruction process, albumen precipitation, centrifugal, ultrafiltration, adsorption chromatography, ion-exchange, affinity chromatography and high performance liquid chromatography or the like.
The antigen that albumen of the present invention or its fragment can be used as the preparation specific antibody uses this antigen can prepare required polyclonal antibody and monoclonal antibody.
The method for preparing this antibody can adopt preparation method conventional in the immunology.
This antibody can be used for the proteic purifying of the present invention and mensuration or evaluation.
Gene of the present invention all has in multiple cancerous tissue as previously mentioned and is higher than the hundreds of expression to thousands of times of healthy tissues, and therefore, this antibody can be used for the diagnosis of cancer.Its medicinal use is: with this antibody is that activeconstituents is used to prepare cancer diagnosis agent or test kit.
Cancer diagnosis agent of the present invention or test kit comprise the active ingredient of PPO gene or segmental active ingredient or its complementary nucleotide sequence at least.Can choose wantonly and contain other components, as mark medium and PCR reagent.
The mark medium can be radioactivity with element or chemical modification object such as gold-tinted material.
Test kit can contain suitable reaction diluent, standard antibody, damping fluid, reaction terminating liquid or the like.
Another medicinal use of PPO gene of the present invention is: the antibody with the expression product of the separated DNA of PPO gene is activeconstituents, is prepared into cancer treatment drugs preparation or thinner with suitable pharmaceutical carrier.
Medicine preparation of the present invention can be prepared into various formulations (as liquid preparation, solid preparation) according to different preparations carriers.
But the carrier of using always in the agent of preparations carrier drug of choice length of schooling is as thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, tensio-active agent, isotonic agent, stablizer (as albumen, L-amino acid, carbohydrate and the derivatived cellulose of human serum) or the like.
Be included in the content of the active ingredient of the present invention in the pharmaceutical preparation, there is no particular limitation, generally can select according to treatment needs and medication, the course of treatment and patient's age factor.
With regard to generalized case, common dose is: adult's per kilogram of body weight about 0.01 μ g～10mg/ days.Every day 1-3 time.
The medicinal use of another of PPO gene of the present invention is: be prepared into pharmaceutical preparation as activeconstituents with medicine effective quantity and suitable pharmaceutical carrier or thinner with the antisense nucleotide transfer vector of PPO gene or with its antisense nucleotide conversion/cells transfected system.
This pharmaceutical preparation has translation and the expression that suppresses PPO gene PPO gene in cell, thereby reaches the propagation of anticancer.
Introducing the initial vector of goal gene or play source carrier to goal gene is the known technology of this area.
With the antisense nucleotide transfer vector or be that the dosage of its activeconstituents is not subjected to special qualification as active ingredient drug prepared preparation with its antisense nucleotide/conversion transfection.
Generally, its dosage can be tired every kg body weight every day about 1 * 10 according to the sick record of reverse transcription
2Pfu.～1 * 10
10Pfu..
In this manual, gene of the present invention (DNA) molecule is called PPO or KIAA0090, become PPO albumen by the albumen of this genes encoding, and inferior proteic functionally active is called the PPO protein-active.
The invention provides following major topic based on what above-mentioned result of study was developed
1) comprises a kind of separated dna molecular in the following polynucleotide
A) polynucleotide of the polypeptide formed by the aminoacid sequence shown in the SEQ ID NO.2 of coding.
B) with the polynucleotide that relatively have at least 95% homology of nucleotide sequence shown in the SEQ ID NO.1.
C) under stringent condition can with the polynucleotide sequence of the nucleotide sequence hybridization shown in the SEQ ID NO.1.
D) with above-mentioned a or b complementary polynucleotide.
The polynucleotide sequence of the polypeptide that 2) as above 1) described separated dna molecular, its coding are made up of the aminoacid sequence shown in the SEQ ID NO.2.
3) as 2) affiliated separated dna molecular, it has the nucleotide sequence shown in the SEQ ID NO.1.
4) comprise the aminoacid sequence expression product shown in the SEQ ID NO.2.
5) comprise as above 1) or 3) under the recombinant expression vector of separated dna molecular.
6) including as above 5) host cell of described recombinant expression vector is NIH/3T39.
7) a kind of PPO detects and uses probe, and it has at least 15 continuous nucleotide sequences that comprise from nucleotide sequence shown in the SEQ ID NO.1.
8) as 7) described PPO detects and uses probe, and it has at least 30 continuous nucleotide sequences that comprise from nucleotide sequence shown in the SEQ ID NO.1.
9) cancer diagnosis agent, it comprises as above 7) or 8) under probe or primer.
10) cancer diagnosing kit, it comprises as above probe or primer under (7) or (8).
11) antibody or antibody fragment, it can be with as above 1) under the expression product of separated dna molecular combine.
12) method of diagnosing cancer: it comprises the following steps: to prepare biological sample goods, preparation as above 11) described antibody or its segment, and above-mentioned sample and above-mentioned antibody or segment carried out immune reaction product in immune response and the test sample.
13) treatment for cancer method.It comprises the following steps: to prepare biological sample goods, preparation as above 11) described antibody or its segment, and above-mentioned sample and above-mentioned antibody or segment carried out immune response and utilize the RNA perturbation technique or methods of treatment that the Antisense gene therapy means are carried out.
14) a kind of antisense nucleotide of sequence, this sequence comprise at least 15 continuous nucleotides from nucleotide sequence shown in the SEQ ID NO.1.
15) as above 14) described antisense nucleotide, its antisense and a kind of sequence, this sequence comprise at least 30 continuous nucleotides that come from the nucleotide sequence shown in the SEQ ID NO.1.
16) gene therapy medicament preparation, it comprises as above 14) or 15) described antisense nucleotide is as activeconstituents.
17) screen the method for one or more materials (agonist and/or antagonist or agent interfering), described one or more materials as above 1) expression product of described separated dna molecular interacts, described method comprises the following step: in the substratum that contains test substances to be selected to containing as above 1) host cell of described separated dna molecular cultivates, then quantitatively as above 1) expression product of described separated dna molecular.
18) be used for the novel remedies for cancer agent, its comprise significant quantity as above 11) rnai agent of described antibody or its segment and pharmaceutically useful carrier or gene itself.
Amino acid in the specification sheets, peptide, nucleotide sequence, nucleic acid etc. are write by the rule of " Nucleotide and or aminoacid sequence table electronic standard file " (the intellecture property industry standard ZC003-2001 of the People's Republic of China (PRC)) with the abbreviation expression.
Description of drawings:
The cDNA nucleotide sequence of SEQ ID NO.1:PPO.
The aminoacid sequence of SEQ ID NO.2:PPO.
Fig. 1: the expression of EST Hs.154797 in the human body different tissues wherein of nucleotide sequence shown in the SEQ ID NO.1.
Fig. 2: reverse transcription PCR shows.
Beta2-MG among the figure is inherent controlling gene.
Fig. 3: the nucleotide sequence of clone's part PPO gene.
Fig. 4: the structural representation of PPO gene.
Fig. 5 A, Fig. 5 B: be RNA blot hybridization result.
The as can be seen from the figure expression of this gene in different tissues.
Among Fig. 5 A: 1 heart, 2 brains, 3 placentas, 4 lungs, 6 skeletal muscle, 7 kidneys, 8 pancreases
Among Fig. 5 B: blood leukocytes around 1 spleen, 2 Tiroidina, 3 prostate glands, 4 testis, 5 ovaries, 6 small intestines, 7 large intestines, 8
Fig. 6: the functional domain analysis of people and mouse relatively.
Its Smalt letter is represented B.cAMP-and cGMP-dependent kinases phosphorylation point, red cassettes filial generation table C. protein phosphorylation point, green cassettes filial generation table casein kinase i I phosphorylation point.The mazarine box is represented Tyrosylprotein kinase phosphorylation point.
Fig. 7, Fig. 8: people's gene and other species are relatively.
Fig. 9 A-B: quantitative PCR shows.
Fig. 9 A: quantitative PCR shows that this gene had expression in multiple cancerous tissue and cell strain.Wherein 1 and 5 is normal ovary tissues, the 2nd, and gonad cell strain VK18, the 3rd, gonad cell strain VK18#102, the 4th, gonad cell strain OVCAR-3.From 6-14 is ovarian cancer tissue's sample.The 15th, normal body of uterus sample, 16-19 is a carcinoma of uterine body, the 20th, carcinoma of uterine body cell strain HEC1A.The 21st, normal renal tissue, the 22nd, normal cell strain 293, the 23rd, renal cancer cell strain ACHN, 24-27 is the kidney cancer tissue sample, and 28 and 29 is normal liver organizations, and 30-34 is the liver cancer tissue sample, the 35th, normal galactophore tissue, 36-39 is breast cancer tissue's sample, the 40th, and normal colonic tissue, 41-44 is the cell strain of colorectal carcinoma.The 45th, normal gastric mucosa, 46-49 is the stomach organization sample, 50-51 is a normal cerebral tissue, and 52 and 53 is cell strains of cerebral glioma, the 54th, and normal pancreas tissue, 55-57 is the cell strain of pancreas, the 58th, cancer of pancreas tissue sample, 59-60 are normal lungs tissues, and 62-63 is the cell strain of lungs, the 64th, normal prostatic sample, the 65th, prostate cancer cell strain.
Fig. 9 B: express quantitative analysis.
Figure 10: cellular immunization dyeing display result.
A represents tenuigenin, and B represents nucleus, and C represents the image after overlapping.
Figure 11: MTT analyzes.
Wherein: A represents the plasmid transfection NIH/3T3 after gene imports.
B represents blank carrier transfection NIH/3T3.
The performance relevant of Figure 12 PPO gene with phosphorylation.
Figure 12 A: expression ERK2 phosphorylated protein obviously increases the stably express clone.
Figure 12 B: expression MEK1/2 phosphorylated protein obviously increases the stably express clone.
By Figure 12 A: comparing with Figure 12 B, the PPO gene is relevant with phosphorylation as can be seen.
Figure 12 C: expression cell nuclear proliferating antigen PCNA obviously increases.
Figure 12 D: expression BRAF does not change, and RAS had expression.
Figure 12 E: immunoprecipitation represents that this gene may be relevant with RAS.
The proteinic length of Figure 12 F:PPO.
Colony on Figure 13 soft agar forms the ability experimental analysis
A among Figure 13 and B: be the normal performance of 3T3 cell on soft agar.
C among Figure 13 and D: the performance of cell on soft agar that imports carrier for the PPO gene.This gene transfered cell can form big colony group as shown in the figure.
E among Figure 13 and F: the comparison that is normal cell and blank carrier transfered cell colony group size.As can be seen from the figure, colony group size is different.
The G of Figure 13: be the comparison of normal cell and blank carrier transfered cell colony group quantity, as can be seen from the figure, colony group's quantity also has tangible difference.
The H of Figure 13: blank carrier transfered cell can not form the cell superposition phenomenon.
The I of Figure 13: this gene transfered cell can form the cell superposition phenomenon.
These results represent that the PPO gene imports the carrier cells transfected and has the characteristics of cancer cells.
Figure 14 in-vivo analysis shows figure.
Figure 14 A: show that the 3T3 cell after this gene imports can be at stimulate cell growth on one's body the mouse.
Figure 14 B: after showing that this gene imports, the tumour under mouse is dissected on one's body relatively.
Figure 14 C: growth curve chart, zero line are the growth curve that gene imports the back transfectional cell, and the * line is the growth curve of control group.
As can be seen from the figure, can the growth of obvious stimulation cell after this gene imports and have tumorigenicity.
Figure 15 A.: the result of quantitative PCR.
As seen from the figure, after the RNA interference, PPO is in 2 ovarian cancer cell strains, and this expression of gene amount reduces.
Figure 15 .B-C.D. Western blot hybridization display result.Western blot hybridization shows that phosphorylation and propagation index reduce as seen from the figure.Growth at the cell levels anticancer.
Figure 15 .B is ovarian cancer cell strain OVK18.
Figure 15 C is ovarian cancer cell strain OVCAR-3.
Figure 15 D is the 3T3 cell strain after the PPO transfection.Western blot hybridization shows that phosphorylation and propagation index all reduce.
Figure 16. the effect synoptic diagram of this gene in the MAPK path.Can infer that thus this gene may be in the effect in the MAPK path.Stimulate RAS to cross the phosphorylation of expression and MEK1/2 and MAPK.
Figure 17-19. is for being injected in RNAi reagent the experimental result of mice with tumor.
Figure 17 A injects sub-mice with tumor with RNAi reagent and injects performance after 21 days.
The comparison of Figure 17 B both sides tumour.
Figure 17 C growth curve, green is represented crt gene Lamin, left side, the red PPO gene of representing.
As seen from the figure, RNAi reagent is injected in the growth that mice with tumor can obviously suppress cancer.
Figure 18 is that the revision test condition is the same with Figure 17 with the result.
Figure 19 is a growth curve.
RNA disturbs the growth that can suppress cancer in the experiment made on the living proof.
Figure 20 A used carrier and cloning process synoptic diagram.
Figure 20 B is concrete sequence, begins from preceding 3 Nucleotide of initial son.
Figure 20 C is a first half to EcoRI enzyme point of contact.
It is latter half that Figure 20 D begins to terminator from EcoRI enzyme point of contact, and terminator TAA is substituted by XbaI enzyme cutting point and continues coding V5 albumen.
Figure 21 is the normal NIH/3T3 of PPO gene transfection.
Figure 22 A is used for the RNA sequence of RNA perturbation technique.
Figure 22 B is used for the primer and the probe of diagnostic kit.
The connecting portion of table 1 exon and intron meets the length of ag-gt rule and exon and intron.
Table 2: be illustrated in other biology, the homotype gene of PPO gene comprises mouse, fruit bat, mosquito, nematode and yeast.
Table 3: be illustrated in amino acid the most conservative in the organic evolution.And these amino acid are relevant with phosphorylation.
Embodiment
Embodiment
The cDNA library screening
The tire liver, the tire brain, pancreas and kidney cDNA library purchase in Clontech (Palo Alto, CA).Large intestine, lung, liver, the cDNA library of ovary and fibroblast purchase in Stratagene. (La Jolla, CA).The many groups of design primer is used for screening the cDNA library, by the cDNA library screening, finally determines EST Hs.154797 and clones full-length cDNA, 6022 base pairs, double-stranded spirane structure.
As shown in Figure 1,1 EST Hs.154797 can have clear and definite product in the cDNA library.It is at the tire brain, and nephridial tissue has expression in hepatic tissue and the pancreatic tissue.At ovary tissue, do not express in big intestinal tissue and the lungs.
Accurate location KIAA0090 is in 1p36.13 and measure dna sequence dna
With the own 1p36 contig of being done (contig), the mode of hybridizing by southern blotting technique hybridization (Southern Blotting) finds BAC clone b203I24, b140B13, and b81G11, and b57B11 accurately is positioned 1p36.13 and measures dna sequence dna.
As scheme shown in the SEQ ID NO.1: the about 36-kb of its DNA length of PPO gene of the present invention, cDNA length 6022 bases.
The nucleotide sequence of PPO gene is shown in SEQ ID NO.1.
Point out exon sequence
Utilize GenScan program (
Http:// genes.mit.edu/GENSCAN.html) (Burge andKarlin, 1997) point out exon and intron sequences.
Utilize RACE (Rapid Amplification of cDNA Ends) method to find the sequence of 5 ' end.
(Clontech, Palo Alto CA), at first extract RNA from renal cancer cell line ACHN and press SuperScript IIRT Kit (GIBCO, BRL) (Rockville, MD) the synthetic first chain cDNA of schedule of operation to utilize 5 ' SMART RACE test kit.Synthetic afterreaction volume dilution to 120 μ l, 1 μ l contains the total RNA of 8ng approximately, the first chain cDNA that obtains after the reverse transcription.The reaction conditions of RACE is as follows:
cDNA 2μl
Adaptor?Pimer 0.25μl
10mMdNTP 0.25μl
10XPCR?Buffer 1.25μl
50XPolymerase?Mix 0.25μl
Gene-specific?Primer(10pmol/μl 0.25μl
H2O 7.75μl
Total 12μl
The primer of gene specific is as follows
R1，5’-AACCAACTTCTTGGATCCAGGGGA-3’.
R2,5 '-TTGGAATTCAATCAAACTTGCCCACCTG-3 ' RACE product directly checks order, and condition requires operation in strict accordance with test kit.Find the sequence of 5 ' end.
As shown in Figure 3: utilize SMART RACE test kit, cloned initial son and the non-coding region part of totally 28 bases and EST Hs.154797.Beginning from tilted letter is the part of KIAA0090cDNA.
As shown in Figure 4: the PPO gene contains 25 exons, and initiation codon is positioned at first exon, and termination codon is positioned at the 23rd exon.
The connecting portion of exon and intron meets the length (seeing Table 1) of ag-gt rule and exon and intron.
Cell cultures:
OVK18, OVK18#102 (Uehara et al., 1984), OVCAR3 (Hamilton et al., 1984), CoLoTC (Quinn et al., 1979), Lu65 (Hirohashi et al., 1989), HT1 (Tsuchiya et al., 1982), HCT15 (Dexter et al., 1979), HEC1A (Kuramotoet al., 1972), ACHN (Giard et al., 1973), U251N (Hirohashi et al., 1978), SUN5 (Brattain et al., 1981), LK-2 (Yoshioka et al., 1989) and NIH/3T3 (Aaronson SA et al) PK1, PK8 and PK9, (Kobari et al., 1986), used cell all northeast university add Institute for Medical Research in age.LNCaP (Gibas et al., 1983), HEK 293 (Graham et al., 1977) and U87 (Beckma.et al., 1971) purchase in U.S. ATCC (Manassas, VA). and cultivate in strict accordance with explanation.
Reverse transcription PCR:
Reverse transcription: the RNA with in Trizol extraction cell and the freezing tissue, get total RNAl μ g, total reaction volume 20 μ l press SuperScript IIRT Kit (GIBCO, BRL) (Rockville, MD) the synthetic first chain cDNA of schedule of operation.Synthetic afterreaction volume dilution to 120 μ l, 1 μ l contains the total RNA of 8ng approximately, the first chain cDNA that obtains after the reverse transcription.
PCR reaction: add following reagent successively
Reverse transcription strand cDNA 1 μ l (dilution back)
10XPCR damping fluid 1.5 μ l
2nMdNTP 1.5μl
Upstream primer F 1.5 μ L
Downstream primer R 1.5 μ l
Taq enzyme (promaga 0.5u/ μ l) 1.5 μ l
Water 6.5
The reverse transcription PCR condition is as follows; 94 ℃ 5 minutes, 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 30 seconds, 40 circulations.72 ℃ 7 minutes.
Primer is as follows
F，5’-TTTGATTGGAGACAGCAATATG-3’.
R，5’-CTGCGATGTACCTTACAGAC-3’.
As shown in Figure 2, reverse transcription shows that the PPO gene had expression in ovarian cancer.
Quantitative reverse transcription PCR:
On the basis of reverse transcription PCR, utilize same cDNA as masterplate, do the quantitative PCR machine with ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). primer is as follows
Forward?primer，5’-GCTTTTCGGAGAAGTCTAGTTCAAAG-3’.(from?1117th?nt?to1142nd?nt).
Reverse?primer，5’-TCTCCACGAGGTATAGGTTAATGGT-3’.(from?1197th?nt?to1173rd?nt).
Probe，5’-CTTGCTTCAATCAGACCTA-3’.(from?1153rd?nt?to?1171st?nt).
β 2-microglobulin gene is as controlling gene, and primer is as follows
Forward?primer，5’-GTGACTTTGTCACAGCCCAAGA-3’.(from?373rd?nt?to?394thnt).
Reverse?primer，5’-AACCTCCATGATGCTGCTTACA-3’.(from?437th?nt?to?416thnt)
Probe，5’-AGTTAAGTGGGATCGAGAC-3’.(from?396th?nt?to?414th?nt)
The result measures with calibration curve method.
All reagent are all purchased in Applied Biosystems company and in strict accordance with description operation.Particularly, we are according to explanation ready reaction solution, the standard curve making of two genes, and the cDNA of PPO and β 2-microglobulin presses 5 times of doubling dilutions of cDNA, (8 * 10
-2Ng, 1.6 * 10
-2Ng, 3.2 * 10
-3Ng, 6.4 * 10
-4Ng, 1.28 * 10
-4Ng, 2.56 * 10
-5Ng and negative control). every part of sample is prepared two fens samples, and machine calculates its mean value automatically, and human genomic dna is as negative control.The relation conefficient of typical curve is between 0.98 and 0.99.For guaranteeing the result, more than the identical test triplicate, obtain mean value and typical curve.
Shown in Fig. 9 A and 9B: this gene had expression in multiple cancerous tissue (as ovarian cancer, mammary cancer, carcinoma of the pancreas, colorectal carcinoma, lung cancer, prostate cancer, the cancer of the brain, cancer of the stomach, liver cancer, uterus carcinoma etc.) and cell strain.
The structure of plasmid
The structure of plasmid in two steps, at first cDNA library oneself preparation, in renal cancer cell line ACHN, extract RNA after, reverse transcription is cDNA, as mentioned above, utilizes this cDNA to be masterplate, the primer of design comprises KpnI site and EcoRI site respectively,
Forward?primer，5’-CATGGTACCGCATCATGGCGGCTGAGTG-3’.
Reverse?primer，5’-CAGCTCAGTGGTGAGATCCT-3’.
The PCR product is handled respectively through KpnI and EcoRI enzyme, and simultaneously, (Invitrogen, Carlsbad CA) also handle respectively through KpnI and EcoRI enzyme expression vector pcDNA3.1/V5His, connect with ligase enzyme again.Shown in Figure 20 A, B, C, D.Like this, the cDNA of about 1.5kb is loaded into carrier, confirm whether successfully import with southern blotting technique method (Southern blotting), and order-checking is confirmed.And as new carrier, same, utilize own synthetic cDNA to be masterplate, the primer of design comprises EcoRI site and XbaI site respectively,
Forward?primer，5’-ATGAGATCAACATTGACACC-3’.
Reverse?primer，5’-CCCTCTAGATCGCCAGGCCC-3’.
The PCR product is handled respectively through EcoRI and XbaI enzyme, and new carrier connects with ligase enzyme also after EcoRI and XbaI enzyme are handled respectively again.The cDNA of about like this 1.5kb is loaded into new carrier.The about 3.0kb of cDNA of pcDNA3.1/V5His carrier altogether packs into.The sequence that comprises all proteins encoded.And the characteristics that have the pcDNA3.1/V5His carrier, to the Xin Meisu resistance, and the albumen that produces can be discerned by the V5 monoclonal antibody.The order-checking proof is sudden change back plasmid transfection intestinal bacteria not, and after LB cultivated, the cesium chloride ultracentrifugation was isolated plasmid, alcohol and acetic acid precipitation and purification plasmid, detailed method is pressed molecular cloning (beautiful J.Sambrook, E.F.Fritsch, T.Maniatis. the gold winter wild goose, Li Mengfeng etc. translate.Second edition in 1992, Science Press) extracts the method preparation of plasmid in the book.Order-checking proves and suddenlys change back transfection NIH/3T3 cell strain and obtain stably express.Confirm the monoclonal cell of stably express with the Western blot hybrid method.At last, having two strain cells to be determined obtains stably express and does further research.
Stably express clone's screening
Blank carrier and gene import new plasmid 3 pairs of NIH/3T3 cell strains of the transfection culture dish respectively that makes up, and transfection method is in strict accordance with LIPOFECTAMINE PLUS Reagent (Invitrogen company) test kit explanation.First pair of culture dish is used for verifying whether transfection is successful.Other two pairs of culture dish add the Xin Meisu screening, and persister can be survived, and are last, obtain 30 clones, go checking whether can express V5 antibody with the Western blot hybridizing method, have only the V5 of expression antibody, could prove the transfection success.At last, obtain 2 strain stably express clone.
Western blot hybridization (Western Blotting)
The antibody V5 of Western blot hybridization, PCNA, Phospho-MAPK, anti-beta actin purchase in (Sigma, St Louis, MO).ERK2 purchase in (BD Bioscience, Palo Alto, CA), RAS (Oncogen, Boston, MA), MEK1/2 and Phospho-MEK1/2 purchase in (Cell SignalingBeverly, MA).The cell strain of stably express adds 10% calf serum and 500 μ g/ml G418 simultaneously through the DMEM substratum, cultivates after 48 hours and collects, and cell is dissolved among the 3%SDS after the PBS flushing.Proteic concentration BSA test kit (Bio-Rad, Hercules, CA). measure.Concrete grammar is referring to reference (Kondo et al., 1999).Preparation 10-20% spacer gel and SDS-polyacrylamide separation gel, get and respectively organize suppressor proteins 40 μ g, with equal-volume sample-loading buffer (0.05mol Tris-HClpH6.8,2%SDS, the 10%beta-mercaptoethanol, 20% glycerine, 0.1% bromjophenol blue) mixes, boiling water bath 5 minutes, be splined on behind the naturally cooling in the gel well, condition by spacer gel 8V/cm and separation gel 15V/cm is carried out the constant voltage electrophoresis, and electrophoretic buffer is Tris-glycine-SDS damping fluid, and bromjophenol blue moves to the glue bottom and ends electrophoresis.With albumen go to Clear blot membrane-P film (ATTO, Tokyo, Japan).The sealing condition of film places 2.5% skim-milk sealing 1 hour according to the explanation of producer with film.Add and spend the night 4 ℃, washed film 3 * 10 minutes with PBST (PBS that contains 0.05%Tween20), adding two resists, hatched 1 hour for 37 ℃, washed film 3 * 10 minutes with PBST, signal is observed the (AmershamBiosciences with ECL Detection Reagent, Buckinghamshire, U.K.) analyze with LAS 1000 Plus with a ScienceLab 99 Image Gauge (Fuji Photo Film, Minamiashigara, Japan).
Cellular immunization dyeing
Plasmid transfection NIH/3T3 cell strain also obtains the stably express cell and reclaims the back smear, and through the processing of PBS damping fluid, V5 is anti-as one, and 1%FITC is as two anti-dyeing, and 4,6-diamidino-2-phenylindole (DAPI) dyes nuclear.Show that as Figure 10 the protein of this gene is located in the tenuigenin.
MTT reduction method (MTT assay) detects cell proliferation
The cell strain of stably express is seeded in 96 well culture plates (1 * 10
4Individual cell seeding is in every hole), detected once totally 6 days in per 24 hours.With MTT dyeing, cytolysis is shaken and was made it mixing in 10 minutes in 100% alcohol, selects the 490nm wavelength, calculates per-cent and is compared.MTT purchase in (MO), VERSA max microplate reader machine detects the light transmission of solution for Sigma, St Louis, machine purchase in Japan (Molecular Devices, Tokyo, Japan)
As can be seen from Figure 11, empty carrier and this gene transfecting cell strain do not have tangible difference on the speed of growth.
Immunoprecipitation test Immunoprecipitation (IP)
This method sees (Kondo, E., 1999) for details, in brief, the cell strain of stably express through the PBS flushing, is dissolved in Brij97 cell extract damping fluid (the 20mM Tris-HClpH7.4 of 500 μ l through after cultivating collection, 75mM NaCl, 1mM EDTA, 1mM Na
3VO
4, 1% Brij 97,1mM phenylmethylsulfonyl fluoride, 20 μ g/ml aprotinin) and (Higuchi, M., 1996). the protein expression amount detects identical with Western blot hybridization (Western Blotting).The result is shown in Figure 12 E, and this gene may be relevant with RAS.
For confirming that further the PPO gene has tumorigenicity, the inventor has carried out the colony on the soft agar and has formed capability analysis.
Method is:
The cell strain of blank expression vector and PPO newly make up the cell strain of plasmid expression vector and plant respectively in 6 orifice plate culture dish, 1% soft agar (DIFCO LABORATORIES, Detroit, MI) putting into culture dish is bottom, 0.5%agar/DMEM is the 2nd layer, 0.3%agar/DMEM is the 3rd layer, with 1 * 10
4Individual cell seeding is changed nutrient solution weekly one time, after 4 weeks in every hole, with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (SigmaChemical Co., St.Louis, the propagation situation of cell is seen in MO) chromoscopy.More than test is in strict accordance with producer's description operation.
Experimental result as Figure 13 A, B, C, D,, shown in E, the F.
Shown in Figure 13 A, B, its normal 3T3 cell can not form big colony group at soft agar.
The cell of this gene importing carrier can form big colony group shown in the C among Figure 13, D.
As can be seen, normal cell is different with blank carrier transfered cell colony group size among E from Figure 13 and the F.
From the G of Figure 13: normal cell and blank carrier transfered cell colony group's quantity has tangible difference as can be seen.
From the H of Figure 13: blank as can be seen carrier transfered cell can not form the cell superposition phenomenon.
From the I of Figure 13: this gene transfered cell can form the cell superposition phenomenon as can be seen.
Above result represents that the PPO gene imports the carrier cells transfected and has the characteristics of cancer cells.
Relevant in order to prove the PPO gene with the MAPK path, according to ordinary method, carried out the experiment of gene phosphorylation.As Fig. 6; Figure 12 A, B, D, E; Figure 15 B, Figure 15 C, Figure 15 D; Shown in Figure 16: gene of the present invention is positioned at tenuigenin and a plurality of phosphorylation points is arranged, and studies show that further it has the phosphorylation function, can make MEK and ERK2 phosphorylation, is arranged in the MAPK path.
As seen from Figure 6, this gene has a lot of phosphorylation points, may be relevant with phosphorylation.
By Figure 12 A as can be seen, the ERK2 phosphorylated protein obviously increases the stably express clone.
By Figure 12 B as can be seen, the MEK1/2 phosphorylated protein obviously increases the stably express clone.
Compared as can be seen with Figure 12 B by Figure 12 A., the PPO gene is relevant with phosphorylation.
By Figure 12 D as can be seen, BRAF does not change, and RAS had expression.
By Figure 12 E as can be seen, immunoprecipitation represents that this gene may be relevant with RAS.
Gene of the present invention can be regulated various cells or propagation therein, differentiation, and tumour generation and transcriptional activation etc. are (as Figure 12 C; Figure 13 A, B, C, D, E, F, G, H, I; Shown in Figure 14 A, B, the C), this gene can the obvious stimulation cell growth and have tumorigenicity.According to these activity, it can be used for explaining diagnosis and treatment with these the active relevant diseases such as the pathology of malignant tumour.
The amino acid molecular amount of PPO genes encoding of the present invention is 100kDa (shown in Figure 12 F).
Gene of the present invention all or part of can be used for producing can with gene expression product (albumen) bonded antibody or its segment.The antibody of gained or its segment can be used for the above-mentioned disease of diagnosing gene of the present invention to participate.Primer shown in Figure 22 B and probe can be used for preparing diagnosis box or test kit.
The antisense segment of gene of the present invention or RNA perturbation technique or its expression product can be used for controlling the outbreak of above-mentioned disease (as growth of tumor).
In this specification sheets, term " PPO gene (dna molecular) " not only comprises double-stranded DNA but also comprises the single stranded DNA of its composition, no matter that justice arranged or nonsense with and segment.Therefore, except as otherwise noted, term " PPO gene (dna molecular) " comprises the double-stranded DNA that contains the human gene group DNA, comprises the single stranded DNA (sense strand) of cDNA, has single stranded DNA (antisense strand) and their segment with sense strand complementary sequence.
Gene of the present invention can contain leader sequence, coding region, exon and intron.It contains 25 exons shown in Fig. 4 and table 1, and initiation codon is positioned at first exon, and termination codon is positioned at the 23rd exon.
Gene polynucleotide of the present invention comprise RNA and DNA.Described DNA comprises cDNA, genomic dna and synthetic DNA.Polypeptide comprises its segment, homologue and mutant.
People's gene is compared with other species, guards very much (as Fig. 7, shown in Figure 8) from some aminoacid sequence of angle of evolving.
In-vivo analysis is specific as follows:
Nude mice purchase in (Japan Clea, Tokyo, Japan) company, 5 ages in week, every nude mice, the cell strain of left side plantation blank expression vector, right side plantation PPO newly makes up the cell strain of plasmid expression vector, every side repopulating cell 5 * 10
6Individual, measured the tumour size once in per 4 days, totally 4 weeks.This test carries out twice altogether, comes to the same thing.Mouse is fed in the attached experimentation on animals of Northeastern University's medical board center.Condition meets international standard and can not bring pathogenic.
Experimental result can the growth of obvious stimulation cell after this gene imports and have tumorigenicity shown in Figure 14 A, B, C.
Experiment made on the living shows that this gene is relevant with propagation and phosphorylation.
Use the RNA interference method, obtain same result from the negative shown in Figure 15 A, after disturbing with RNA, the PPO gene is in 2 ovarian cancer cell strains, and this expression of gene amount reduces.Western blot hybridization shows that phosphorylation and propagation index reduce (shown in Figure 15 B, C, D).RNAi reagent is injected in the obviously growth of anticancer (as shown in figure 17) of mice with tumor.Revision test shows that it comes to the same thing.Experiment shows uses the single stranded RNA agent interfering, also can reach the effect (as shown in figure 17) that part suppresses the ovarian cancer cell growth.
RNA disturbs (RNAi) cell analysis, method:
RNAi reagent purchase in Japan Bioservice (Asaka, Japan) sequence is as follows:
Justice RNA, 5 '-UAUGUUGGGAAGGUCAAGUdTdT-3 ';
Sense-rna, 5 '-ACUUGACCUUCCCAACAUAdTdT-3 '.
Lamin is a controlling gene, and sequence is as follows
Justice RNA, 5 '-CUGGACUUCCAGAAGAACAdTdT-3 ';
Sense-rna, 5 '-UGUUCUUCUGGAAGUCCAGdTdT-3 '.
The method that the RNA interference method provides according to manufacturer, in brief, the just RNA of equimolecular quantity and sense-rna be transfection OVK18 after forming bifilar RNA after the annealing sex change, OVCAR-3 and KIAA0090 transfection 3T3 cell strain, transfection is purchased in (Invitrogen with oligofectamine reagent, Carlsbad is CA) and according to producer's description operation.Cultivate and reclaim cell after 72 hours and carry out Western blot hybridization and quantitative PCR inspection.
From Figure 15 A, B, C, D as can be seen,,, block this expression of gene, can reduce the expression of phosphorylation and PCNA with the mode of RNA interfering (RNAi) at cell levels.
RNA disturbs (RNAi) in-vivo analysis
Ovarian cancer cell strain OVCAR-3, every side repopulating cell 5 * 10 of nude mice
6Individual, measured the tumour size once in per 3 days, in the time of the 9th day, the tumour size is 9mm3, then, and bilateral injection rnai agent, the left side is controlling gene Lamin, the right side is the PPO gene, measures to be 3000pmol, measures once in per then 3 days, the 12nd day, again bilateral injection rnai agent 1000pmol put to death mouse after 21 days, measured the size of tumour.
Experimental result such as Figure 17; Figure 18; Shown in Figure 19.
Figure 17 A is injected in mice with tumor for RNAi reagent and injects performance after 21 days.
The comparison of Figure 17 B both sides tumour.
Figure 17 C growth curve, green is represented crt gene Lamin, left side, the red PPO gene of representing.
As seen from the figure, RNAi reagent is injected in the growth that mice with tumor can obviously suppress cancer.
Figure 18 is that the revision test condition is the same with Figure 17 with the result.
Figure 19 is the same with Figure 17 C, is growth curve.
More than experiment shows, with the mode of RNA interfering (RNAi), blocks this expression of gene, the growth that the live body level can anticancer.
The exon and the intron of table 1.PPO gene
exon?size intron?sequences exon?sequences intron?sequencesintron size
1 148 gtggtcccgcggaggcggagCCCGCGGCGG AGTTTGATTGgtgcgtacgtgtggaacata 1 6384
2 125 ctgtactctttttttctcagGAGACAGCAA GGGGAGATCTgtgagtgaactgagaactgt 2 890
3 66 ctaactttcatgtctctcagTGTGGCGCCA CACGGACAGGgtaagcagagtcctcccgtg 3 242
4 94 cacgatcttttcttttccagATGTGATCAC ACAGTGGCAGgtaggggagaaactggattt 4 1140
5 129 caacctgtacttgtgttcagTTTCCAGGCA TCCCAGAAAGgtaaggctgcctgctcacag 5 1202
6 127 tttcttcttccctctcttagTGACAGCATC TGTTCAGCAGgtacaggagggtgacaggag 6 596
7 150 taacgatgacttttctgtagGTTAGGGTTT CCCACTGCAGgtgagaggctgccactgctt 7 311
8 168 tcctccttcttgccttccagTCTCTCGACT CTTCCCACAGgtaactgcaggcaacatggt 8 515
9 72 atgccttgcttttcccccagACTGCCCTAG GAATGAAGTGgtgagtattgagaacaacag 9 373
10 63 tgtccttctctttctcatagCAGAAAAGTA TAGTTCAAAGgtatggtgtggcactgggca 10 655
11 123 cgtgctgtctgtactcctagGACTCTCTGG GCCTGAGCGGgtaaggcagagcctttcttt 11 778
12 97 gtcttgactgtggcttatagCTGTATATCC CAGCAGTTGGgtaagtagacctcattcaac 12 1892
13 120 aaaaatgtttcctgtagcagGGAAGGTGGT AAAAAGGCAGgtatgaacctaagaggttgg 13 1994
14 200 ttacttttcctttacaccagATGGCTTGCT CTCAGGCAAGgtgagtggggtttaagctcc 14 162
15 150 agtatttgtttatcttgcagCTTTTTGGCA GAAGGACAAGgtgaggcatccagctctgac 15 1201
16 162 tgacagtttgctcattttagGAGTCGGGAA TGAATACAAGgtactgtcttcagaggggat 16 297
17 120 ggctctctgggtttttcaagGTCACAGCTT GCTTCGAAAGgtaggaaggcatgtcctggc 17 3393
18 138 cttttttgcccctgccctagGATCTCACCA GCTCTACAAGgtacatacagccagctgtct 18 3743
19 174 ggttttctctctctctccagAGCCTGAACC GAACTGGGTGgtggtaagcggtcactacca 19 561
20 211 gccacctcctatccctgcagTACCAGTACT CACCTGCTGAgtgagtgactggggagcctc 20 1775
21 85 attcacttcctcctgtgtagTTGGACTACC AACAAAGCAGgtgagaccctcatgggcagt 21 1065
22 130 tcccggctggtgttttgcagAGAGGAGAAC CACTTGTTTGgtgagtagagcccacagctg 22 86
23 1949 ttattccatctgccttccagGTTGTGGCCT CTCAAAGCAGgtgctttttttttttttgag 23 815
24 908 agcctgggcaacagagtgagACCCTGGCTC GTAATCCCAGctacttgggaggctgtgagg 24 916
25 310 aagcgaattctctgcctcagCCTCCCGAGT GGTAGACAACatcctgtgctctttcatttt
The homologous gene of table 2. PPO
species gene chromosome size(aa) similarity
region ％
Drosophilia CG2943 3R 915 363-915 44.4
Anopheles agCP7856 shortgun 943 378-943 42.9
gambia
Caenorhabditis?CE27186 2 946 378-946 30.0
elegans
melanogaste
cerevisia
Conserved amino acid in table 3. homologous gene
Function?domain Sequence Location?in?KIAA0090
Protein?kinase?C?phosphorylation?site TEK 58-60
TER 853-855
TSR 858-860
TKR 979-981
Casein?kinase?II?phosphorylation?site SREESLAE 443-450
SSLD 824-827
SAME 845-848
SGLE 927-930
cAMP-and?cGMP-dependent?protein KRSS 712-715
kinase?phosphorlation?site
EFTVLELYEGTEQY 804-817
Tyrosine?sulfation?site VLKDDYDYVLISSV 954-968
Claims (7)
1, a kind of human body gene is positioned at the lp36.13 district, and its cDNA nucleotide sequence is as described in the SEQ ID NO.1.
2, a kind of human body gene is positioned at the lp36.13 district, and its aminoacid sequence is as described in the SEQ ID NO.2..
3, the expression product that comprises the cDNA of claim 1.
4, a kind of PPO gene test probe, it has the sequence that comprises from least 15 continuous nucleotides of nucleotide sequence shown in the SEQ ID NO.1.
5, the medicinal use of PPO gene is characterized in that with the PPO gene being that active ingredient is prepared into cancer diagnosis agent or test kit.
6, the medicine and pharmacology purposes of PPO gene is characterized in that the antibody with the expression product of the described separated DNA of claim 1 is activeconstituents, is prepared into pharmaceutical formulations or thinner with suitable pharmaceutical carrier.
7, the medicinal use of PPO gene is characterized in that being prepared into pharmaceutical preparation as activeconstituents with medicine effective quantity and suitable pharmaceutical carrier or thinner with the antisense nucleotide transfer vector of PPO gene or with its antisense nucleotide conversion/cells transfected system.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101275134B (en) * | 2007-04-20 | 2010-06-02 | 清华大学深圳研究生院 | Medicament for curing breast carcinoma and special antisense oligonucleotide therefor |
| CN102277414A (en) * | 2010-06-10 | 2011-12-14 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method and kit for detecting polymorphism of region 1p36.22 associated to liver cell carcinogenesis and application thereof |
| CN106918703A (en) * | 2017-02-23 | 2017-07-04 | 中国人民解放军第三0九医院 | Application of the PPO genes in gland cancer diagnostic reagent is prepared |
| CN107233353A (en) * | 2017-06-13 | 2017-10-10 | 河北医科大学第三医院 | A kind of reagent for being used to treat gland cancer |
-
2004
- 2004-05-26 CN CNB200410012307XA patent/CN100497625C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101275134B (en) * | 2007-04-20 | 2010-06-02 | 清华大学深圳研究生院 | Medicament for curing breast carcinoma and special antisense oligonucleotide therefor |
| CN102277414A (en) * | 2010-06-10 | 2011-12-14 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method and kit for detecting polymorphism of region 1p36.22 associated to liver cell carcinogenesis and application thereof |
| CN102277414B (en) * | 2010-06-10 | 2014-10-29 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method and kit for detecting polymorphism of region 1p36.22 associated to liver cell carcinogenesis and application thereof |
| CN106918703A (en) * | 2017-02-23 | 2017-07-04 | 中国人民解放军第三0九医院 | Application of the PPO genes in gland cancer diagnostic reagent is prepared |
| CN107233353A (en) * | 2017-06-13 | 2017-10-10 | 河北医科大学第三医院 | A kind of reagent for being used to treat gland cancer |
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