CN1291017C - Novel gene P40 containing acyl group carrying protein-like structural domain - Google Patents
Novel gene P40 containing acyl group carrying protein-like structural domain Download PDFInfo
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- CN1291017C CN1291017C CN 00113886 CN00113886A CN1291017C CN 1291017 C CN1291017 C CN 1291017C CN 00113886 CN00113886 CN 00113886 CN 00113886 A CN00113886 A CN 00113886A CN 1291017 C CN1291017 C CN 1291017C
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Abstract
The present invention relates to a new gene P40 containing acyl groups and carrying a protein-like structure domain, particularly to molecular biology technology. The obtained cancer suppressor genes and cancer suppressor candidate genes are only more than 20 kinds at present, but the genes do not have the characteristics of cancer suppressor genes of containing acyl groups and carrying a protein-like structure domain. The new gene p40 of the present invention is 2024 bp in the cDNA full length, has a complete reading frame and encodes proteins containing 357 amino acid residues, and furthermore, the molecular weight of the amino acid residues is about 40kD. The present invention is a difference expression gene for high expression of normal brain tissues and low expression or no expression of glioma tissues and glioma cell lines, can inhibit the growth of glioma cells and is a good cancer suppressor gene.
Description
Technical field:
The present invention relates to Protocols in Molecular Biology
Background technology:
100,000 different genes are arranged in the human genome according to estimates, and present known gene has only 9000 kinds, also has most gene and function thereof unknown by the people.Tumour is human second deadly disease, serious threat human beings'health and life.The generation and the development mechanism of tumour still imperfectly understand.But it has been recognized that tumour is because activation and/or the disappearance of cancer suppressor gene or the product of deactivation of proto-oncogene.Therefore, capturing on the road of cancer, scientists is devoted to the research of cancer suppressor gene, and expectation makes the growing multiplication of cell reach new balance by import certain cancer suppressor gene in tumour cell.The cancer suppressor gene of having found at present and press down the cancer candidate gene have only 20 surplus kind, in known gene, also do not contain the gene in acylester albumen (ACP) spline structure territory.
Summary of the invention:
The objective of the invention is to obtain a kind of new cancer suppressor gene-contain gene p40 in acylester albumen (ACP) spline structure territory.The P40 gene is at normal cerebral tissue's high expression level, and samples of human glioma is low expresses (or not expressing), and glioma cell line (BT325) is not expressed.And the p40 gene is that (HL-60, HalaS3, K562 and MOLT4) and the rheumatoid arthritis synovial cell with part neoplasm characteristics all do not have expression lung cancer A549 cell system, colorectal carcinoma SW480 clone, Raji and Daudi lymphoma cell line, 4 kinds of leukemia cells; And expression is all arranged corresponding normal lung tissue, normal colonic tissue, normal lymphoglandula and normal synovial cell.The p40 gene is changed over to BT325 cell, lung cancer A549 cell, rheumatoid arthritis synovial cell and the prostate cancer PC-3 cell of not expressing this gene, have to make these tumour cells be trapped in the tendency of G1 phase, and can cause the apoptosis of lung cancer A549 cell, rheumatoid arthritis synovial cell and prostate cancer PC-3 cell.
Technical scheme of the present invention:
At first, extract the mRNA of normal cerebral tissue and samples of human glioma respectively, then, reverse transcription becomes cDNA respectively, and the mRNA of normal cerebral tissue adds joint in two ends when reverse transcription; The mRNA of samples of human glioma becomes cDNA through common reverse transcription, then by vitamin H on the PCR reaction marking, as subtracted probe.Subsequently, two groups of cDNA are hybridized, with the streptavidin absorption vitamin H that is marked on the magnetic bead, under the effect in magnetic field, remove crossbred and subtracted probe (cDNA of samples of human glioma), resulting promptly is to express in normal cerebral tissue, and at samples of human glioma expression difference expressing gene not.When analyzing these difference expression genes, obtain a gene that contains the acylester protein-like structural domain, this gene has complete frame district; total length is 2024bp; the protein that contains 357 amino-acid residues of encoding, the about 40kD of molecular weight, p40 albumen is positioned at the nucleus celestial being.Base nucleotide sequence and amino acids coding thereof following (the line part is ACP spline structure territory):
1?cgggacgcag?caaagagagg?agagacccca?agtcagaagg?agtgagaaccc?tgaccccta
61?atcccactgc?atccagccaa?taggagccca?gccaccatgg?cggagctgca?ggaggtgcag
M?A E L Q E V Q
121?atcacagagg?agaagccact?gttgccagga?cagacgcctg?aggcggccaa?gactcactct
I T E E K P L L P G Q T P E A A K T H S
181?gtggagacac?catacggctc?tgtcactttc?actgtctatg?gcacccccaa?acccaaacgc
V E T Y P G S V T F T V Y G T P K P K R
241?ccagcgatcc?ttacctacca?cgatgtggga?ctcaactata?aatcttgctt?ccagccactg
P A I L T Y H D V G L N Y K S C F Q P L
301?tttcagttcg?aggacatgca?ggaaatcatt?cagaactttg?tgcgggttca?tgtggatgcc
F Q F E D M Q E I I Q N F V R V H V D A
361?cctggaatgg?aagagggagc?ccctgtgttc?cctttgggat?atcagtaccc?atctctggac
P G M E E G A P V F P L G Y Q Y P S L D
421?cagcttgcag?acatgatccc?ttgcgtcctg?cagtacctaa?atttctctac?aataattgga
Q L A D M I P C V L Q Y L N F S I I G
481?gttggtgttg?gagctggagc?ctacatcctg?gcgagatatg?ctcttaacca?cccggacact
V G V G A G A Y I L A R Y A L N H?P?D?T
541?gttgaaggtc?ttgtcctcat?caacattgat?cccaatgcca?agggttggat?ggattgggca
V E G L V L I N I D P N A K G W M?D?W?A
601?gcccacaagc?taacaggcct?cacctcttcc?attccggaga?tgatccttgg?acatcttttc
A H K L T G L T S S I P E M I L G?H?L?F
661?agccaggaag?agctctctgg?aaattctgag?ttgatacaaa?agtacagaaa?tatcattaca
S Q E E L S G N S E L I Q K Y R N?I?I?T
721?catgcaccca?acctggataa?cattgaattg?tactggaaca?gctacaacaa?ccgccgagac
H A P N L D N I E L Y W N S Y N N?R?R?D
781?ctgaactttg?agcgtggagg?tgatatcacc?ctcaggtgtc?ctgtgatgct?ggtggtagga
L N F E R G G D I T L R C P V M L?V?V G
841?gaccaagcac?ctcatgaaga?tgcagtggtg?gaatgtaact?caaaactgga?ccccacccag
D Q A P H E D A V V E C N S K L D?P?T?Q
901?acctcgttcc?tcaagatggc?tgactccgga?ggtcagcccc?agctgactca?gccaggcaag
T S F L K M A D S G G Q P Q L T Q?P?G?K
961?ctgaccgagg?ccttcaagta?cttcctgcaa?ggcatgggct?acatggcctc?atcctgcatg
L T E A F K Y F L Q G M G Y M A S?S?C M
1021?actcgcctgt?cccggtctcg?tacagcctct?ctgaccagtg?cagcatccgt?tgatggcaac
T R L S R S R T A S L T S A A S V?D?G N
1081?cggtcccgct?ctcgcaccct?gtcccagagc?agcgagtctg?gaactctttc?ttcggggccc
R S R S R T L S Q S S E S G T L S?S?G P
1141?ccggggcaca?ccatggaggt?ctcctgttga?atggcccttg?ttgccctaga?gtgggaccca
P G H T M E V S C
1201?gccctcacct?cccccagagc?taacctggga?ggtgctgaag?gggcattggg?ccaccgtaag
1261?caagggaaaa?agggcagatc?atgcggggag?atgaccttga?tctttgattg?ctaccctaac
1321?cttgaccttt?aacccgtgat?tccccccagc?tcctggaaga?gatgtcctaa?tatctcttag
1381?ggacccagac?ccctaaattc?tcctcctccc?ccattttgat?gttaaggtgg?agagggcata
1441?tgcatcctct?gtcctgatct?aggtgtctat?agctgagggg?taagaggttg?ttgtagttgt
1501?cctggtgcct?ccatcagact?ctccctactt?gtcccatatt?tgcaagggga?ggggatttgg
1561?ggctggggct?ccattcacca?aagctgaggt?ggcttctcat?taacccctta?ggactctgaa
1621?gggtatggac?ctacgtgaat?gtgtgtcagg?gggagacttg?ctggtgggtt?agtggtcctc
1681?aggatgtgat?aaaaacatcc?agtgtaaaaa?ggaagttgga?atgggagttg?gcgggcagtg
1741?aacgagtgtg?gggaaggatt?ggtgctgggg?caacaggaag?gggcctgggg?ccgtttggct
1801?gcactaactt?tggtagctca?gtgtgcatct?aaagtgggac?tggggaggga?gctaagcttg
1861?ggctgggctg?cttggggctt?ggcatagggt?ggaaagggct?accctggggc?ttctgacccc
1921?cctgtagtat?gtgtggaggg?tgccctcccg?tctcccacaa?cttctgctat?aacaataaac
1981?tgtagaggaa?tcggaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaa
The concrete steps of technical solution of the present invention are:
1.mRNA separate and reverse transcription
Extract the mRNA of normal cerebral tissue and samples of human glioma respectively, respectively the mRNA reverse transcription of normal cerebral tissue and samples of human glioma is become cDNA with ThermoScript II superscrip II.The mRNA of normal cerebral tissue adds joint in two ends when reverse transcription; The mRNA of samples of human glioma becomes cDNA through common reverse transcription, then by vitamin H on the PCR reaction marking, as subtracted probe.
2. the preparation of subtracted probe
The PCR condition is: 20 μ mol/L biotin-21dUTP, 200 μ mol/L dNTP, 1 μ goligo-dT, 1 μ l reverse transcription template, 50 μ l reaction volumes.The PCR parameter is: 95 ℃, and 20s; 40 ℃, 5 minutes; 72 ℃, 3 minutes; 40 circulations.
3. hybridization
Hybridize with cDNA of normal cerebral tissue and samples of human glioma cDNA.The 5 μ l cDNA of normal cerebral tissue are added in the 100 μ l samples of human glioma cDNA PCR products, under the condition of 6 * SSC and 0.5%SDS, hybridized 18 hours for 65 ℃.
4. desalination reaches and removes small segment
40 μ l hybridization solutions are added to Sephacryl S-400 purification column, centrifugal collection liquid.
5. remove crossbred and remove probe (samples of human glioma cDNA)
Aforesaid liquid (40 μ l) is added in the magnetic bead of 0.1ml streptavidin mark (with 1ml magnetic bead suspension with 0.5 * SSC washing three times, be suspended from again among the 0.1ml 0.1 * SSC), room temperature was in conjunction with 2 minutes, 65 ℃ of incubations 5 minutes, reduce non-specific hybridization, under the action of a magnetic field, isolate supernatant as pcr template.
6. deducted the amplification of the cDNA of normal cerebral tissue of samples of human glioma cDNA
In 50 μ l reaction systems, contain 400nmol/L primer (TACGGCTGCGAGAAGACGACAGAA); 200 μ mol/LdNTP, the supernatant after 40 μ l hybridization separates; Lu Klen Taq enzyme, the PCR parameter is: 95 ℃, 15s; 68 ℃.5 minutes; 25 circulations.
7.PCR the recovery of product and purifying
From glue, reclaim the PCR product.
8.PCR the clone of product and evaluation
The PCR product cloning is gone into T easy carrier, cut evaluation with EcoR I enzyme.
9. sequencing
Require to react and check order according to ABI 310 DNA automatization sequenators.
Gene p40 is at human normal tissue and corresponding tumour cell differential expression and to the checking of tumour cell inhibit feature
One. the in situ hybridization checking
1 usefulness, 3 ' end-labelling, isotropic substance
35The S labeled oligonucleotide probe
The paraffin section of 2 preparation normal cerebral tissues' (postmortem) and samples of human glioma, the glioma cell line BT325 cell that preparation is cultivated drips sheet.
3 in situ hybridizations
Go into to contain interior processing of glass jar of 4% Paraformaldehyde 96,0.1mol/LPBS and hybridization pretreatment liquid (containing 0.9% sodium-chlor, 1.5% trolamine and 0.25% diacetyl oxide) after the section rapid drying successively.During hybridization with 200 μ l hybridization solutions (containing 4 * SSC, 50% methane amide, 0.025%tRNA, 10% sulfuric acid threose, Denhardt liquid (containing 0.02% bovine serum albumin, 0.02%Fical 400,0.02%PVP K-30)), 15 μ l DTT and label probe (6-9 * 106dpm/ slide glass) drip in slice surface, put in the warm box and hatch 48h in 40 ℃.After the hybridization,, at last LM-1 type latex is applied to slice surface, exposes in 4 ℃ of magazines with 1 * SSC (containing 0.15mol/L sodium-chlor and 0.15mol/L Sodium Citrate) flushing (55 ℃, 3 * 20 minutes).After 4 weeks, with Kodak D-19 developing liquid developing, the photographic fixing of F-5 fixer-harderner.Dehydration, transparent, mounting, dark-field is observed under the light microscopic.
4. result:
In situ hybridization is the result show, the p40 gene does not have expression at the extensive high expression level of normal cerebral tissue in samples of human glioma and glioma BT325 clone.
Two. the dotting checking
1. with point the multiple tissue expression Hybond membrane of 84 kinds of mRNA and the dot blot film of oneself preparation are arranged
2. the preheating hybridization solution is to 50-60 ℃, and DNA is heated to 95-100 ℃ with the 1.5mg milt, is put on ice then
3. the milt DNA with thermally denature adds hybridization solution
4. Hybond membrane is put into hybridization bag, add 65 ℃ of prehybridizations of 10ml hybridization solution 30 minutes
5. with isotopic labeling cDNA probe and 30ug C
0T-1DNA, 150ug milt DNA, 50 μ l, 20 * SC mixing, final volume to 200 μ l.Then at 95-100 ℃ of heating 5 minutes, 68 ℃ of incubations 30 minutes
6. will go up the hybridization solution that step liquid adds 5ml step 3, thoroughly mixing
7. pour out prehybridization solution, add step 6 liquid in hybridization bag, be evenly distributed, get rid of bubble, 65 ℃ of hybridization 6h
8. removal hybridization solution shakes with 200ml W1 and to wash film, 65 ℃ 20 minutes * 5 times
9. shake with 200ml W2 and wash film, 55 ℃ 20 minutes * 2 times
10. the taking-up Hybond membrane is sealed with preservative film, presses phosphorus screen 9h, shields scanner scanning with phosphorus, carries out data processing with computer
11. result
Quantitative computer analysis shows that the p40 gene is the highest at sialisterium, cerebral tissue and skeletal muscle expression amount.Cortex and infracortical kankar at human brain are rolled into a ball extensive high expression level.Minimum at thymus gland, marrow and testis expression amount, there is not expression in peripheral blood leucocyte.Brain, the heart, kidney,liver,spleen, thymus gland and lung fetus have expression.Do not have expression at glioma BT325 clone, lung cancer A549 cell system, straight adenocarcinoma of colon SW480 clone, two kinds of Burkitt lymphoma cell lines (Raji and Daudi) and four kinds of leukemia cell system 9 kinds of tumor cell lines such as (HL-60, HelaS3, K562 and MOLT-4), and the expression of different levels is all arranged in corresponding normal cerebral tissue, normal lung tissue, normal colonic tissue and normal Lymphoid tissue.In addition, do not have expression, expression is arranged normal relatively osteoarthritis synovial cell the synovial cell of rheumatoid arthritis.Further having pointed out p40 is the possibility of cancer suppressor gene
Three .P40 genes are in external restraining effect to tumour cell
1. be with the Construction of eukaryotic of HA-tag
At first will be cloned into the PUC19 plasmid of band HA-tag through EcoR I and BamH I restriction enzyme site with the p40 gene coding region of designed restriction enzyme site, with Hind III and BamH I HA-tag and p40 gene coding region are downcut then, again be cloned into the PcDNA3 plasmid that same enzyme is cut, with the carrier transformed into escherichia coli JM109 competent cell that builds, bed board, choose the clone, enzyme is cut evaluation.
2. cell cultures
Ordinary method cultivation, the glioma that goes down to posterity BT325, lung cancer A549, prostate cancer PC-3 and rheumatoid arthritis synovial cell.
3. gene transfection
Go down to posterity and cultivate used cell, cell state is good behind the 24h, and about 60-80% cell remittance sheet is washed 2 times with serum-free DMEM nutrient solution.In advance dilute liposome (5-40ug) and plasmid (2-8ug) respectively with serum-free DMEM nutrient solution, then, the two mixing room temperature placement was added culturing bottle after 20 minutes, cultivation 12-48h takes pictures, collecting cell.Use 0.25% trypsin digestion cell, changed tapered centrifuge tube 1000g over to centrifugal 5 minutes, wash twice, 70% ethanol with PBS and fix 20 minutes, wash twice with PBS again, be used for cell cycle analysis.
4. genetic expression is identified
The cell suspension of collecting is dropped on the slide glass, fix 30 minutes with phosphoric acid acetone-formaldehyde damping fluid behind the airing, row ABC method immunohistochemical staining.
5. cell cycle analysis
Cell after fixing is used for DNA-Prep Reagent dyeing 15-20 minute, carries out the cell cycle with flow cytometer and detects.
P40 gene transient transfection is in the glioma BT325 cell of not expressing the p40 gene, lung cancer A549 cell and rheumatoid arthritis synovial cell and prostate cancer PC-3 cell, its form is changed, part cell rounding or smaller volume, the cell refractivity is poor, and the part cell is floating etc.Cell cycle analysis shows, the p40 gene makes that G1 phase cell proportion increases, S phase cell or G2 phase cell proportion downward trend.The P40 gene overexpression can cause that also apoptotic peak appears in lung cancer A549 cell, rheumatoid arthritis synovial cell and prostate cancer PC-3 cell.
Advantage of the present invention:
1.p40 albumen enters nucleus and plays a role, propagation capable of inhibiting cell, but and inducing apoptosis of tumour cell.
2. the technical scheme that clone of the present invention presses down the cancer candidate gene obtains full-length cDNA easily.
Embodiment
Embodiments of the invention
With transfection BT325 cell and rheumatoid arthritis synovial cell is example, and 1ug is contained the DNA plasmid of p40 gene, uses liposome mediated-method, changing over to goes down to posterity cultivate about 10
6Individual BT325 cell or rheumatoid arthritis synovial cell, after 48 hours, detect the cell cycle with flow cytometer, the p40 genome has 49.1% cell to be in G1 (before DNA the is synthetic) phase, 30.5% cell is in S (DNA the is synthetic) phase, and the empty carrier control group has only 39.7% cell to be in the G1 phase, and 41.7% cell is in the S phase.The rheumatoid arthritis synovial cell of transfection p40 gene has 2.8-6.3% apoptosis to occur.
Claims (1)
1, a kind of gene P40 that contains the acylester protein-like structural domain has purposes in the medicine of cancer suppressing action in preparation, and the nucleotide sequence and the amino acid sequence coded thereof of described gene are as follows:
1 cgggacgcag?caaagagagg?agagacccca?agtcagaagg?agtgagaacc?ctgaccccta
61 atcccactgc?atccagccaa?taggagccca?gccaccatgg?cggagctgca?ggaggtgcag
M?A E L Q E V Q
121?atcacagagg?agaagccact?gttgccagga?cagacgcctg?aggcggccaa?gactcactct
I T E E K P L L P G Q T P E A A K T H S
181?gtggagacac?catacggctc?tgtcactttc?actgtctatg?gcacccccaa?acccaaacgc
V E T Y P G S V T F T V Y G T P K P K R
241?ccagcgatcc?ttacctacca?cgatgtggga?ctcaactata?aatcttgctt?ccagccactg
P A I L T Y H D V G L N Y K S C F Q P L
301?tttcagttcg?aggacatgca?ggaaatcatt?cagaactttg?tgcgggttca?tgtggatgcc
F Q F E D M Q E I I Q N F V R V H V D A
361?cctggaatgg?aagagggagc?ccctgtgttc?cctttgggat?atcagtaccc?atctctggac
P G M E E G A P V F P L G Y Q Y P S L D
421?cagcttgcag?acatgatccc?ttgcgtcctg?cagtacctaa?atttctctac?aataattgga
Q L A D M I P C V L Q Y L N F S I I G
481?gttggtgttg?gagctggagc?ctacatcctg?gcgagatatg?ctcttaacca?cccggacact
V G V G A G A Y I L A R Y A L N H?P?D?T
541?gttgaaggtc?ttgtcctcat?caacattgat?cccaatgcca?agggttggat?ggattgggca
V E G L V L I N I D P N A K G W M?D?W?A
601?gcccacaagc?taacaggcct?cacctcttcc?attccggaga?tgatccttgg?acatcttttc
A H K L T G L T S S I P E M I L G?H?L?F
661?agccaggaag?agctctctgg?aaattctgag?ttgatacaaa?agtacagaaa?tatcattaca
S Q E E L S G N S E L I Q K Y R N?I?I?T
721?catgcaccca?acctggataa?cattgaattg?tactggaaca?gctacaacaa?ccgccgagac
H A P N L D N I E L Y W N S Y N N?R?R?D
781?ctgaactttg?agcgtggagg?tgatatcacc?ctcaggtgtc?ctgtgatgct?ggtggtagga
L N F E R G G D I T L R C P V M L?V?V G
841 gaccaagcac?ctcatgaaga?tgcagtggtg?gaatgtaact?caaaactgga?ccccacccag
D Q A P H E D A V V E C N S K L D?P?T?Q
901 acctcgttcc?tcaagatggc?tgactccgga?ggtcagcccc?agctgactca?gccaggcaag
T S F L K M A D S G G Q P Q L T Q?P?G?K
961 ctgaccgagg?ccttcaagta?cttcctgcaa?ggcatgggct?acatggcctc?atcctgcatg
L T E A F K Y F L Q G M G Y M A S?S?C M
1021?actcgcctgt?cccggtctcg?tacagcctct?ctgaccagtg?cagcatccgt?tgatggcaac
T R L S R S R T A S L T S A A S V?D?G N
1081?cggtcccgct?ctcgcaccct?gtcccagagc?agcgagtctg?gaactctttc?ttcggggccc
R S R S R T L S Q S S E S G T L S?S?G P
1141?ccggggcaca?ccatggaggt?ctcctgttga?atggcccttg?ttgccctaga?gtgggaccca
P G H T M E V S C
1201?gccctcacct?cccccagagc?taacctggga?ggtgctgaag?gggcattggg?ccaccgtaag
1261?caagggaaaa?agggcagatc?atgcggggag?atgaccttga?tctttgattg?ctaccctaac
1321?cttgaccttt?aacccgtgat?tccccccagc?tcctggaaga?gatgtcctaa?tatctcttag
1381?ggacccagac?ccctaaattc?tcctcctccc?ccattttgat?gttaaggtgg?agagggcata
1441?tgcatcctct?gtcctgatct?aggtgtctat?agctgagggg?taagaggttg?ttgtagttgt
1501?cctggtgcct?ccatcagact?ctccctactt?gtcccatatt?tgcaagggga?ggggatttgg
1561?ggctggggct?ccattcacca?aagctgaggt?ggcttctcat?taacccctta?ggactctgaa
1621?gggtatggac?ctacgtgaat?gtgtgtcagg?gggagacttg?ctggtgggtt?agtggtcctc
1681?aggatgtgat?aaaaacatcc?agtgtaaaaa?ggaagttgga?atgggagttg?gcgggcagtg
1741?aacgagtgtg?gggaaggatt?ggtgctgggg?caacaggaag?gggcctgggg?ccgtttggct
1801?gcactaactt?tggtagctca?gtgtgcatct?aaagtgggac?tggggaggga?gctaagcttg
1861?ggctgggctg?cttggggctt?ggcatagggt?ggaaagggct?accctggggc?ttctgacccc
1921?cctgtagtat?gtgtggaggg?tgccctcccg?tctcccacaa?cttctgctat?aacaataaac
1981 tgtagaggaa tcggaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa line part is ACP spline structure territory.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00113886 CN1291017C (en) | 2000-07-21 | 2000-07-21 | Novel gene P40 containing acyl group carrying protein-like structural domain |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00113886 CN1291017C (en) | 2000-07-21 | 2000-07-21 | Novel gene P40 containing acyl group carrying protein-like structural domain |
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| CN1291017C true CN1291017C (en) | 2006-12-20 |
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| CN1304573C (en) * | 2004-01-16 | 2007-03-14 | 中国药品生物制品检定所 | NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour |
| CN101265471B (en) * | 2007-04-29 | 2012-01-11 | 北京华大基因研究中心 | Method for simultaneously amplifying and connecting one or multiple aim genome region |
| CN101555469B (en) * | 2009-03-13 | 2012-03-21 | 中国人民解放军第四军医大学 | High-efficiency recombined adenovirus containing ndrg2 gene and pharmic purpose thereof |
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