CN1876681A - Cancer-suppressing protein and its gene and uses - Google Patents
Cancer-suppressing protein and its gene and uses Download PDFInfo
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- CN1876681A CN1876681A CN 200610019144 CN200610019144A CN1876681A CN 1876681 A CN1876681 A CN 1876681A CN 200610019144 CN200610019144 CN 200610019144 CN 200610019144 A CN200610019144 A CN 200610019144A CN 1876681 A CN1876681 A CN 1876681A
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Abstract
The invention discloses the anticancer protein and its genes and usage. The invention has the advantages of high depression effect, strong specificity and nontoxicity.
Description
Technical field
The present invention relates to biological technical field.Specifically, the present invention relates to human cancer suppressor protein (tumor suppressor protein), this proteic gene of coding, and the application in biological medicine (comprising that human tumor suppresses medicine and biotechnological formulation) of described albumen and gene (and mutant or derivative).
Background technology
Cancer is the malignant disease of serious harm human health, and nearly 2,000 ten thousand people in the whole world suffer from cancer at present.Estimate that the year two thousand twenty will increase to 3,000 ten thousand, annual cancer new cases number will rise to 1,500 ten thousand from 1,000 ten thousand.The whole world has 7,100,000 people die from cancer (account for general mortality rate 12.5%) every year.China increases about 1,060,000 people of malignant tumor patient newly every year at present, and death toll has 1,540,000 approximately, and the cancer mortality number increases with annual 600000 speed, and is sustainable growth trend.Cancer occupies China all kinds of causes of the death first place, in per 5 death tolls, has 1 to die from cancer.Though traditional therapy is better to a lot of early-stage cancer results of treatment, up to the present also there are not effective means to treat middle and terminal cancer.
The traditional therapy of cancer mainly is methods such as surgical operation, radiation cure, chemotherapy, but these methods all have its limitation.Surgical operation can't be treated invasive and metastatic cancer, also may impel cancer metastasis; Radiation cure can cause the normal cell injury, can't pursue-treatment the metastatic carcinoma cell; Chemotherapy toxicity is all too strong usually, and normal cell is damaged.And the gene therapy method that utilizes cancer suppressor gene to treat cancer is the main direction of studying of capturing cancer now in the world, and each state has all dropped into substantial contribution and human and material resources.Gene therapy clinical trial in the world starts from nineteen ninety, and the whole world sets foot in the existing nearly various schools of thinkers of specialized company of field of gene so far, and the gene therapy clinical protocol reaches more than 700.Reach the purpose for the treatment of cancer at cancer suppressor gene by the expression that its product-cancer suppressor protein suppresses oncogene.The effect target of cancer suppressor protein is strong, and to not influence of normal cell, and effect is more satisfactory, so cancer suppressor protein, gene are a kind of rising purposes in tumor suppression medicine and biotechnological formulation.
Summary of the invention
The purpose of this invention is to provide a kind of cancer suppressor protein, this albumen can suppress the growth of hepatocellular carcinoma and colorectal carcinoma effectively.
The invention still further relates to proteins encoded as the application in preparation treatment or the prevention hepatocellular carcinoma medicine.
The invention still further relates to proteins encoded as the application in preparation treatment or the prevention colorectal carcinoma medicine.
A further object of the present invention provides a kind of cancer suppressor gene, and this gene can suppress the growth of hepatocellular carcinoma and colorectal carcinoma effectively.
The invention still further relates to encoding gene as the application in preparation treatment or the prevention hepatocellular carcinoma medicine.
The invention still further relates to encoding gene as the application in preparation treatment or the prevention colorectal carcinoma medicine.
The inventor with hepatitis B virus (HBV) X protein (HBx) as bait, from people's liver cell cDNA library, filter out a kind of and the human protein HBx protein-interacting by yeast-two hybrid technique, by to its research and the determined dna sequence of encoding gene and analyze the back determine this albumen and gene be in the past undiscovered albumen and gene (document Jianlin Zhang sees reference, Weiguang Zhao, KailangWu, Ke Wang, Xue Zhang, Chunfang, Gu, Yah Li, Ying Zhu, and Jianguo Wu. (2004) .Human hepatitis B virus X protein promotes cell proliferation and inhibits cellapoptosis through interacting with a serine protease Hepsin.Archive of virology, 150 (4), 721-741.).By its function discovered that they are a kind of cancer suppressor protein and cancer suppressor gene, the nucleotide sequence and the proteic aminoacid sequence of YueF of the new yueF gene of identifying, liver cell cDNA library with the people is a template, carrying out PCR with the primer that designs comes out yueF gene amplification, and be cloned on the pCMV-flag-2C carrier, send order-checking company to check order.Infer according to the nucleotide sequence of yueF gene and the proteic aminoacid sequence of YueF.
A kind of cancer suppressor protein (tumor suppressor protein) is characterized in that having the aminoacid sequence shown in the SEQ ID NO:2 (and mutant nucleotide sequence or derived sequence).This albumen can be used for preparing the medicine that suppresses tumor growth, also can be used for preparing pharmaceutical composition and other biotechnological formulation of suppressing tumor growth.
A kind of cancer suppressor gene has the nucleotide sequence shown in the SEQ ID NO:1 (and mutant nucleotide sequence or derived sequence), the cancer suppressor protein of coding (and mutain or derived protein).This gene can be used for preparing the medicine that suppresses tumor growth, also can be used for preparing pharmaceutical composition and other biotechnological formulation of suppressing tumor growth.
Technical solution of the present invention is as follows:
1, the separation of cancer suppressor gene, evaluation and expression plasmid make up and purifying:
1) clone's design of primers of cancer suppressor gene and synthetic: the X protein (HBx) of using hepatitis B virus (HBV) is as bait, (document Jianlin Zhang sees reference by yeast-two hybrid technique, Weiguang Zhao, Kailang Wu, Ke Wang, Xue Zhang, Chunfang, Gu, Yan Li, Ying Zhu, andJianguo Wu. (2004) .Human hepatitis B virus X protein promotes cellproliferation and inhibits cell apoptosis through interacting with a serine proteaseHepsin.Archive of virology, 150 (4), 721-741.), filter out a kind of from people's liver cell cDNA library and the human protein HBx protein-interacting, this albumen has the aminoacid sequence shown in the SEQ ID NO:2.With this albumen called after Yue Fei (YueF), this proteic gene of coding called after yueF correspondingly.At this gene design one couple of PCR primers: upstream primer 5 '-GCTTAAGCTTCGATGGCTGCAAGTGGCCGAGGTCTC-3 ', downstream primer 5 '-CGTTCGGTACCTCTTCCTGGGTCAGAGCTGGTTC-3 ', and send Dalian precious biosynthesizing.
2) cancer suppressor gene pcr amplification: as template, carry out pcr amplification with a pair of primer of synthetic with human liver cell cDNA library, reaction conditions is: 95 ℃ of pre-sex change 5min; Enter circulation then: 94 ℃ of 1min, 64 ℃ of 1min, 35 circulations of 72 ℃ of 1.5min; 72 ℃ are extended 10min.The dna fragmentation of the proteic whole open reading basket of coding YueF is increased out.With 1% sepharose pcr amplification product is carried out electrophoresis, the DNA purifying that provides with Qiagen company reclaims the test kit purifying.
3) cancer suppressor gene clone and order-checking: the dna fragmentation that reclaims is connected in plasmid pCMV-flag-2C (available from Promega company) after with HindIII and two enzymic digestions of KpnI, obtain the carrier for expression of eukaryon pCMV-YueF plasmid of cancer suppressor gene, transformed competence colibacillus E.coli DH5 α (magnificent Bioisystech Co., Ltd), screening positive clone.Positive strain and plasmid thereof are preserved standby, and serve Hai Huanuo and order-checking.Obtain the nucleotide sequence shown in the SE QID NO:1.
2, the aminoacid sequence of cancer suppressor protein: infer that according to the nucleotide sequence of cancer suppressor gene cancer suppressor protein (YueF protein) has the aminoacid sequence shown in the SEQ ID NO:2 and (accession number: BC0061310) the 33rd open reading basket on the 10th karyomit(e) of the human body of being logined (Homosapiens chromosome 10 open reading frame 33) and encoded protein sequence thereof are identical in GenBank.
3, the carrier for expression of eukaryon pCMV-YueF plasmid purification of cancer suppressor gene: positive colony is inoculated in the LB liquid nutrient medium of ammonia benzyl antibiotic that concentration is 50 μ g/ml, and 37 ℃ of shaking culture are to the ultrapure plasmid extraction kit purifying pCMV-YueF plasmid of 600nm absorbance for providing with vast Imtech in 1～2 o'clock.
Above-mentioned LB liquid nutrient medium is: soluble protein peptone 10g in 1 premium on currency, yeast extract 5g, NaCl 10g.
4, the expression and purification of cancer suppressor protein:
1) construction of prokaryotic expression vector of cancer suppressor protein: with BamHI and two restriction endonucleases of XhoI the YueF gene is scaled off and be cloned into the prokaryotic expression carrier pET28a-YueF that prokaryotic expression carrier pET28a (purchasing the company in Novagen) obtains YueF from the pCMV-YueF plasmid, then transformed into escherichia coli BL21 (purchasing company) screening positive clone and serve Hai Huanuo order-checking and confirm in Novagen.
2) preparation of cancer suppressor protein and purifying: the positive strain of above-mentioned pET28a-YueF is inoculated in the YT liquid nutrient medium of ammonia benzyl antibiotic that concentration is 50 μ g/ml, 37 ℃ of shaking culture to 600nm absorbances are 1～2, it is that 0.5mol/L induces that adding isopropylthio β-D galactoside (IPTG) makes its concentration, 37 ℃ of shaking culture 4～6 hours, 5000 rev/mins of centrifugal 10 minutes collection thalline, ultrasonication 1 minute, 12000 rev/mins centrifugal 5 minutes, abandon supernatant, the precipitation with phosphate buffered saline buffer (PBS pH7.4) resuspended, again 12000 rev/mins centrifugal 5 minutes, abandon supernatant and get precipitation, so repetitive scrubbing is 5 times, promptly obtains purifying cancer suppressor protein inclusion body.
Above-mentioned YT liquid nutrient medium is: soluble protein peptone 10g in 1 premium on currency, yeast extract 5g, NaCl 5g.
5, the inhibition hepatocellular carcinoma of cancer suppressor gene and colorectal carcinoma test:
1) the expression plasmid solution of cancer suppressor gene preparation: the expression plasmid of the cancer suppressor gene that purifying is good is mixed with 0.1 μ g-1 μ g/ μ L solution with the deionized water of sterilization.
2) model of nude mice bearing tumor is set up: the SPF level nude mice of choosing 4～6 ages in week (body weight is between 18～20 grams), with the back of liver cancer cell HepG2 (purchasing in China typical culture collection center) that cultivates or colon-cancer cell CT26 (purchasing in China typical culture collection center) injection nude mice, cell HepG2 injection volume is 6 * 10
6Individual/only.The nude mice of handling continues to let alone the injection site and grow tumour after raising for 2 weeks under the SPF condition, forms tumor bearing nude mice.
3) tumor bearing nude mice is divided into 2 groups again, every group is selected 6 animals to press down the knurl experiment.First group, the experiment plasmid (pCMV-YueF) of 30 μ g is injected directly in the tumour body of every tumor bearing nude mice; Second group, the control plasmid (pCMV-flag-2C) of 30 μ g is injected directly in the tumour body of every tumor bearing nude mice.
4) press down the knurl experiment and handle after, tumor bearing nude mice is normally raised and is observed its tumor growth situation.After pressing down the knurl experiment and handling 30 days, kill tumor bearing nude mice, separate tumour and weigh, calculate tumour inhibiting rate by following formula: tumour inhibiting rate=(1-treatment group tumor weight/control group tumor weight) * 100%.The results are shown in Table 1.
Table 1
| Group | Number of animals (only) | Exemplary embodiment lock (gram) | Tumor control rate (%) | |
| Hepatocellular carcinoma | Contrast (empty carrier plasmid) | 6 | 2.23 | |
| The expression of tumor suppressor gene plasmid | 6 | 0.45 | 79.8 | |
| Contrast | 6 | 1.91 | ||
| Cancer suppressor protein | 6 | 0.36 | 81.2 | |
| Colorectal carcinoma | Contrast (empty carrier plasmid) | 6 | 1.15 | |
| The expression of tumor suppressor gene plasmid | 6 | 0.26 | 77.4 | |
| Contrast | 6 | 1.38 | ||
| Cancer suppressor protein | 6 | 0.32 | 76.8 | |
6, the inhibition hepatocellular carcinoma of cancer suppressor protein and colorectal carcinoma test:
1) preparation of cancer suppressor protein solution: the cancer suppressor protein solubilization of inclusion bodies of purifying is mixed with 1g/L cancer suppressor protein solution in the urea soln of 1.5mol/L.
2) foundation of model of nude mice bearing tumor: the SPF level nude mice of choosing 4～6 ages in week (body weight is between 18～20 grams), with the back of liver cancer cell HepG2 (purchasing in China typical culture collection center) that cultivates or colon-cancer cell CT26 (purchasing in China typical culture collection center) injection nude mice, cell HepG2 injection volume is 6 * 10
6Individual/only.The nude mice of handling continues to let alone the injection site and grow tumour after raising for 2 weeks under the SPF condition, forms tumor bearing nude mice.
3) knurl that presses down of cancer suppressor protein is tested: tumor bearing nude mice is divided into 2 groups, and every group of 6 animals press down the knurl experiment.First group, the amount of injecting the 0.1g cancer suppressor protein by the body weight per kilogram of tumor bearing nude mice is injected directly into cancer suppressor protein in the tumour body of every tumor bearing nude mice; Second group, be injected directly into by the urea soln of the body weight per kilogram of tumor bearing nude mice injection 0.1L in the tumour body of every tumor bearing nude mice in contrast.Injection is once injected 4 times altogether weekly later on.
4) observation and result: after pressing down knurl experiment processing, tumor bearing nude mice is normally raised and observed its tumor growth situation.The expression of tumor suppressor gene vector plasmid of purifying and the cancer suppressor protein of purifying are mixed with certain density preparation, nude mice model with said preparation direct injection hepatocellular carcinoma and intestinal cancer, after normally raising 30 days, kill tumor bearing nude mice, separating tumour weighs, and the calculating tumour inhibiting rate, tumour inhibiting rate=(1-treatment group tumor weight/control group tumor weight) * 100%.The results are shown in Table 1.
Table 1
| Group | Number of animals (only) | Exemplary embodiment lock (gram) | Tumor control rate (%) | |
| Hepatocellular carcinoma | Contrast (empty carrier plasmid) | 6 | 2.23 | |
| The expression of tumor suppressor gene plasmid | 6 | 0.45 | 79.8 | |
| Contrast | 6 | 1.91 | ||
| Cancer suppressor protein | 6 | 0.36 | 81.2 | |
| Colorectal carcinoma | Contrast (empty carrier plasmid) | 6 | 1.15 | |
| The expression of tumor suppressor gene plasmid | 6 | 0.26 | 77.4 | |
| Contrast | 6 | 1.38 | ||
| Cancer suppressor protein | 6 | 0.32 | 76.8 | |
Invention advantage and effect
The invention provides a kind of new cancer suppressor gene and cancer suppressor protein, they can effectively suppress growth of tumor such as the hepatocellular carcinoma and the rectum cancer.Its biggest advantage is to press down knurl efficient height, and high specificity to the normal cell nontoxicity, is the biotechnological formulation that has potential clinical value, for effective treatment of cancer provides new medicine and method.
Embodiment
Embodiment:
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal experiment condition such as Sambrook, molecular cloning, laboratory manual (third edition) (NewYork:Cold Spring Harbor Laboratory Press, 2002) condition described in, or the condition of advising according to manufacturer.
The separation of embodiment 1. cancer suppressor genes, evaluation and expression plasmid make up and purifying:
1) clone's design of primers of cancer suppressor gene and synthetic: the X protein (HBx) of using hepatitis B virus (HBV) is as bait, (document Jianlin Zhang sees reference by yeast-two hybrid technique, Weiguang Zhao, Kailang Wu, Ke Wang, Xue Zhang, Chunfang, Gu, Yan Li, Ying Zhu, andJianguo Wu. (2004) .Human hepatitis B virus X protein promotes cellproliferation and inhibits cell apoptosis through interacting with a serine proteaseHepsin.Archive of virology, 150 (4), 721-741.), filter out a kind of from people's liver cell cDNA library and the human protein HBx protein-interacting, this albumen has the aminoacid sequence shown in the SEQ ID NO:2.The applicant is with this albumen called after Yue Fei (YueF), this proteic gene of coding called after yueF correspondingly.At this gene design one couple of PCR primers: upstream primer 5 '-GCTTAAGCTTCGATGGCTGCAAGTGGCCGAGGTCTC-3 ', downstream primer 5 '-CGTTCGGTACCTCTTCCTGGGTCAGAGCTGGTTC-3 ', and send Dalian precious biosynthesizing.
2) cancer suppressor gene pcr amplification: as template, carry out pcr amplification with a pair of primer of synthetic with human liver cell cDNA library, reaction conditions is: 95 ℃ of pre-sex change 5min; Enter circulation then: 94 ℃ of 1min, 64 ℃ of 1min, 35 circulations of 72 ℃ of 1.5min; 72 ℃ are extended 10min.The dna fragmentation of the proteic whole open reading basket of coding YueF is increased out.With 1% sepharose pcr amplification product is carried out electrophoresis, the DNA purifying that provides with Qiagen company reclaims the test kit purifying.
3) cancer suppressor gene clone and order-checking: the dna fragmentation that reclaims is connected in plasmid pCMV-flag-2C (available from Promega company) after with HindIII and two enzymic digestions of KpnI, obtain the carrier for expression of eukaryon pCMV-YueF plasmid of cancer suppressor gene, transformed competence colibacillus E.coli DH5 α (magnificent Bioisystech Co., Ltd), screening positive clone.Positive strain and plasmid thereof are preserved standby, and serve Hai Huanuo and order-checking.Obtain the nucleotide sequence shown in the SEQID NO:1.
Separation, the evaluation of embodiment 2. cancer suppressor proteins
1, infers that according to the nucleotide sequence of cancer suppressor gene cancer suppressor protein (YueF protein) has the aminoacid sequence shown in the SEQ IDNO:2 and (accession number: BC0061310) the 33rd open reading basket on the 10th karyomit(e) of the human body of being logined (Homo sapienschromosome 10 open reading frame 33) and encoded protein sequence thereof are identical in GenBank.
The preparation and the purifying of embodiment 3. cancer suppressor proteins
1) construction of prokaryotic expression vector of cancer suppressor protein: with BamHI and two restriction endonucleases of XhoI the YueF gene is scaled off and be cloned into the prokaryotic expression carrier pET28a-YueF that prokaryotic expression carrier pET28a (Novagen) obtains YueF from the pCMV-YueF plasmid, then transformed into escherichia coli BL21 (purchasing company) screening positive clone and serve Hai Huanuo order-checking and confirm in Novagen
2) preparation of cancer suppressor protein and purifying: the positive strain of above-mentioned pET28a-YueF is inoculated in the YT liquid nutrient medium of ammonia benzyl antibiotic that concentration is 50 μ g/ml, 37 ℃ of shaking culture to 600nm absorbances are 1～2, it is that 0.5mol/L induces that adding isopropylthio β-D galactoside (IPTG) makes its concentration, 37 ℃ of shaking culture 4～6 hours, 5000 rev/mins of centrifugal 10 minutes collection thalline, ultrasonication 1 minute, 12000 rev/mins centrifugal 5 minutes, abandon supernatant, the precipitation with phosphate buffered saline buffer (PBS pH7.4) resuspended, again 12000 rev/mins centrifugal 5 minutes, abandon supernatant and get precipitation, so repetitive scrubbing is 5 times, promptly obtains purifying cancer suppressor protein inclusion body.
Above-mentioned YT liquid nutrient medium is: soluble protein peptone 10g in 1 premium on currency, yeast extract 5g, NaCl5g.
The inhibition test of embodiment 4. cancer suppressor genes
1) the expression plasmid solution of cancer suppressor gene preparation: the expression plasmid of the cancer suppressor gene that purifying is good is mixed with 0.1 μ g-1 μ g/ μ L solution with the deionized water of sterilization.
2) model of nude mice bearing tumor is set up: the SPF level nude mice of choosing 4～6 ages in week (body weight is between 18～20 grams), with the back of liver cancer cell HepG2 (purchasing in China typical culture collection center) that cultivates or colon-cancer cell CT26 (purchasing in China typical culture collection center) injection nude mice, cell HepG2 injection volume is 6 * 10
6Individual/only.The nude mice of handling continues to let alone the injection site and grow tumour after raising for 2 weeks under the SPF condition, forms tumor bearing nude mice.
3) tumor bearing nude mice is divided into 2 groups again, every group is selected 6 animals to press down the knurl experiment.First group, the experiment plasmid (pCMV-YueF) of 20 μ g-30 μ g is injected directly in the tumour body of every tumor bearing nude mice; Second group, the control plasmid (pCMV-flag-2C) of 20 μ g-30 μ g is injected directly in the tumour body of every tumor bearing nude mice.
4) press down the knurl experiment and handle after, tumor bearing nude mice is normally raised and is observed its tumor growth situation.After pressing down the knurl experiment and handling 30 days, kill tumor bearing nude mice, separate tumour and weigh, calculate tumour inhibiting rate by following formula: tumour inhibiting rate=(1-treatment group tumor weight/control group tumor weight) * 100%.The results are shown in Table 1.
The application of embodiment 5. cancer suppressor genes (composition)
1) preparation of cancer suppressor protein solution: the cancer suppressor protein solubilization of inclusion bodies of purifying is mixed with 1g-10g/L cancer suppressor protein solution in the urea soln of 1.5mol/L.
2) preparation of tumor bearing nude mice: the SPF level nude mice of choosing 12 4～6 ages in week (body weight is between 18～20 grams), with the back of liver cancer cell HepG2 (purchasing in China typical culture collection center) that cultivates or colon-cancer cell CT26 (purchasing in China typical culture collection center) injection nude mice, cell HepG2 injection volume is 6 * 10
6Individual/only.The nude mice of handling continues to let alone the injection site and grow tumour after raising for 2 weeks under the SPF condition, forms tumor bearing nude mice.
3) knurl that presses down of cancer suppressor protein is tested: tumor bearing nude mice is divided into 2 groups, and every group of 6 animals press down the knurl experiment.First group, the amount of injecting the 0.01g cancer suppressor protein by the body weight per kilogram of tumor bearing nude mice is injected directly into cancer suppressor protein in the tumour body of every tumor bearing nude mice; Second group, be injected directly into by the urea soln of the body weight per kilogram of tumor bearing nude mice injection 0.01L in the tumour body of every tumor bearing nude mice in contrast.Injection is once injected 4 times altogether weekly later on.
4) observation and result: after pressing down knurl experiment processing, tumor bearing nude mice is normally raised and observed its tumor growth situation.After pressing down the knurl experiment and handling 30 days, kill tumor bearing nude mice, separate tumour and weigh, calculate tumour inhibiting rate by following formula: tumour inhibiting rate=(1-treatment group tumor weight/control group tumor weight) * 100%.The results are shown in Table 1.
SEQUENCE LISTING
＜110〉Wuhan University
＜120〉cancer suppressor protein and gene thereof and purposes
＜130〉cancer suppressor protein and gene thereof and purposes
<160>2
<170>PatentIn version 3.3
<210>1
<211>1746
<212>DNA
＜213〉intestinal bacteria
<400>1
atggctgcaa gtggccgagg tctctgcaag gctgtggccg cctctccctt cccggcgtgg 60
agacgagata acacggaagc caggggaggt ctgaagcctg agtatgatgc ggtggtgata 120
ggagcaggac acaacggact ggtggctgca gcgtacctgc agagactggg ggtgaacacc 180
gccgtcttcg agaggcgcca tgtgatcggg ggtgcagctg tcactgagga gatcatccca 240
gggtttaagt tctcccgtgc gtcctacctg ctcagcctgc tgaggccgca gatttacact 300
gatctggagc tgaagaaaca tgggctgagg cttcatcttc gaaaccccta ctccttcacc 360
cccatgctgg aagagggtgc aggcagcaag gtgcccaggt gccttctgct gggcacagac 420
atggcagaaa accagaagca gatcgcccag ttctcccaga aggatgccca ggtctttccc 480
aaatatgagg agttcatgca tcgcttggca ttagccattg accctctgct ggatgcggcc 540
cccgtggaca tggcggcctt ccagcatggc tccttgctgc aaaggatgag gtcgctctcc 600
accctcaagc ccctgctgaa ggcaggccgc atcctgggag cccagcttcc ccgatattat 660
gaggtcctca cagctcccat taccaaggtg ctggatcagt ggttcgagtc tgagccttta 720
aaagccactc tagccacaga tgcagtgatt ggagccatga caagtcccca cactccgggg 780
agtgggtatg tgctgctgca ccatgtgatg gggggcctgg agggaatgca gggggcctgg 840
ggctacgtcc aggggggcat gggtgccctc tctgatgcga tcgcaagctc agccaccaca 900
catggagcaa gcatcttcac tgaaaagaca gtggcgaagg tgcaggtgaa cagtgaaggc 960
tgtgttcaag gagttgtgct ggaagatggc acagaggtga gaagcaaaat ggtgctgtcc 1020
aacacatcac cgcagatcac cttcctgaag ctgacgccac aggagtggct tcctgaggag 1080
ttcctggaga gaatctctca gctggacacc cggtcgcctg tcaccaagat caatgtggcc 1140
gtagacaggc tgcccagctt cctggcggcc cccaatgctc ccaggggcca gccgctgccc 1200
catcaccaat gctccatcca cctgaactgt gaagacaccc tcctccttca tcaggccttt 1260
gaagatgcca tggatggcct gtcttcccac aggcctgtga ttgagctctg catcccttcc 1320
tcgctggacc ccaccctggc tccccctggc tgccatgtag tctccctctt cactcagtac 1380
acgccctata cgctggctgg aggcaaggcc tgggacgagc aggagagaga cgcttatgca 1440
gacagagtgt ttgattgcat cgaggtctat gcccctggct tcaaggactc tgtggttggc 1500
agagacatcc tcacaccacc agatttggag agaatcttcg ggcttcctgg agggaacata 1560
ttccactgcg ccatgtccct ggaccagctc tacttcgccc gccccgtgcc cctgcattct 1620
ggctaccgct gccctctcca gggcctgtat ctctgtggaa gtggggctca tcctggagga 1680
ggtgtgatgg gagctgctgg gcgaaatgca gcacatgtgg cctttaggga cctcaagagc 1740
atgtga 1746
<210>2
<211>581
<212>PRT
＜213〉intestinal bacteria
<400>2
Met Ala Ala Ser Gly Arg Gly Leu Cys Lys Ala Val Ala Ala Ser Pro
1 5 10 15
Phe Pro Ala Trp Arg Arg Asp Asn Thr Glu Ala Arg Gly Gly Leu Lys
20 25 30
Pro Glu Tyr Asp Ala Val Val Ile Gly Ala Gly His Asn Gly Leu Val
35 40 45
Ala Ala Ala Tyr Leu Gln Arg Leu Gly Val Asn Thr Ala Val Phe Glu
50 55 60
Arg Arg His Val Ile Gly Gly Ala Ala Val Thr Glu Glu Ile Ile Pro
65 70 75 80
Gly Phe Lys Phe Ser Arg Ala Ser Tyr Leu Leu Ser Leu Leu Arg Pro
85 90 95
Gln Ile Tyr Thr Asp Leu Glu Leu Lys Lys His Gly Leu Arg Leu His
100 105 110
Leu Arg Asn Pro Tyr Ser Phe Thr Pro Met Leu Glu Glu Gly Ala Gly
115 120 125
Ser Lys Val Pro Arg Cys Leu Leu Leu Gly Thr Asp Met Ala Glu Asn
130 135 140
Gln Lys Gln Ile Ala Gln Phe Ser Gln Lys Asp Ala Gln Val Phe Pro
145 150 155 160
Lys Tyr Glu Glu Phe Met His Arg Leu Ala Leu Ala Ile Asp Pro Leu
165 170 175
Leu Asp Ala Ala Pro Val Asp Met Ala Ala Phe Gln His Gly Ser Leu
180 185 190
Leu Gln Arg Met Arg Ser Leu Ser Thr Leu Lys Pro Leu Leu Lys Ala
195 200 205
Gly Arg Ile Leu Gly Ala Gln Leu Pro Arg Tyr Tyr Glu Val Leu Thr
210 215 220
Ala Pro Ile Thr Lys Val Leu Asp Gln Trp Phe Glu Ser Glu Pro Leu
225 230 235 240
Lys Ala Thr Leu Ala Thr Asp Ala Val Ile Gly Ala Met Thr Ser Pro
245 250 255
His Thr Pro Gly Ser Gly Tyr Val Leu Leu His His Val Met Gly Gly
260 265 270
Leu Glu Gly Met Gln Gly Ala Trp Gly Tyr Val Gln Gly Gly Met Gly
275 280 285
Ala Leu Ser Asp Ala Ile Ala Ser Ser Ala Thr Thr His Gly Ala Ser
290 295 300
Ile Phe Thr Glu Lys Thr Val Ala Lys Val Gln Val Asn Ser Glu Gly
305 310 315 320
Cys Val Gln Gly Val Val Leu Glu Asp Gly Thr Glu Val Arg Ser Lys
325 330 335
Met Val Leu Ser Asn Thr Ser Pro Gln Ile Thr Phe Leu Lvs Leu Thr
340 345 350
Pro Gln Glu Trp Leu Pro Glu Glu Phe Leu Glu Arg Ile Ser Gln Leu
355 360 365
Asp Thr Arg Ser Pro Val Thr Lys Ile Asn Val Ala Val Asp Arg Leu
370 375 380
Pro Ser Phe Leu Ala Ala Pro Asn Ala Pro Arg Gly Gln Pro Leu Pro
385 390 395 400
His His Gln Cys Ser Ile His Leu Asn Cys Glu Asp Thr Leu Leu Leu
405 410 415
His Gln Ala Phe Glu Asp Ala Met Asp Gly Leu Ser Ser His Arg Pro
420 425 430
Val Ile Glu Leu Cys Ile Pro Ser Ser Leu Asp Pro Thr Leu Ala Pro
435 440 445
Pro Gly Cys His Val Val Ser Leu Phe Thr Gln Tyr Thr Pro Tyr Thr
450 455 460
Leu Ala Gly Gly Lys Ala Trp Asp Glu Gln Glu Arg Asp Ala Tyr Ala
465 470 475 480
Asp Arg Val Phe Asp Cys Ile Glu Val Tyr Ala Pro Gly Phe Lys Asp
485 490 495
Ser Val Val Gly Arg Asp Ile Leu Thr Pro Pro Asp Leu Glu Arg Ile
500 505 510
Phe Gly Leu Pro Gly Gly Asn Ile Phe His Cys Ala Met Ser Leu Asp
515 520 525
Gln Leu Tyr Phe Ala Arg Pro Val Pro Leu His Ser Gly Tyr Arg Cys
530 535 540
Pro Leu Gln Gly Leu Tyr Leu Cys Gly Ser Gly Ala His Pro Gly Gly
545 550 555 560
Gly Val Met Gly Ala Ala Gly Arg Asn Ala Ala His Val Ala Phe Arg
565 570 575
Asp Leu Lys Ser Met
580
Claims (6)
1. isolating protein, it has the aminoacid sequence shown in the SEQ ID NO:2.
2. an isolating gene has the nucleotide sequence shown in the SEQ ID NO:1.
3. the application of isolating protein in preparation treatment or prevention hepatocellular carcinoma medicine.
4. the application of isolating protein in preparation treatment or prevention colorectal carcinoma medicine.
5. the application of isolating gene in preparation treatment or prevention hepatocellular carcinoma medicine.
6. the application of isolating gene in preparation treatment or prevention colorectal carcinoma medicine.
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| CNB2006100191447A CN100410273C (en) | 2006-05-26 | 2006-05-26 | Cancer-suppressing protein and its gene and uses |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105418745A (en) * | 2014-05-15 | 2016-03-23 | 马恒标 | Tumor suppressor protein variant DV347 and application thereof |
| CN113637052A (en) * | 2021-08-16 | 2021-11-12 | 牡丹江医学院 | Cancer suppressor protein for treating colon cancer and pharmaceutical composition thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1363596A (en) * | 2001-01-05 | 2002-08-14 | 上海博德基因开发有限公司 | Polypeptide-cancer suppressor protein-20.68 and polynucleotide for coding it |
| CN1316515A (en) * | 2001-02-15 | 2001-10-10 | 国家人类基因组南方研究中心 | Human liver cancer related cancer suppressing protein and its coding sequence |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105418745A (en) * | 2014-05-15 | 2016-03-23 | 马恒标 | Tumor suppressor protein variant DV347 and application thereof |
| CN105418743A (en) * | 2014-05-15 | 2016-03-23 | 马恒标 | Tumor suppressor protein variant DV322 and application thereof |
| CN105418747A (en) * | 2014-05-15 | 2016-03-23 | 马恒标 | Tumor suppressor protein variant DV225 and application thereof |
| CN105418744A (en) * | 2014-05-15 | 2016-03-23 | 马恒标 | Tumor suppressor protein variant DV232 and application thereof |
| CN105418746A (en) * | 2014-05-15 | 2016-03-23 | 马恒标 | Tumor suppressor protein variant DV253 and application thereof |
| CN105440121A (en) * | 2014-05-15 | 2016-03-30 | 马恒标 | Tumor suppressor protein variant DV271 and application thereof |
| CN105461795A (en) * | 2014-05-15 | 2016-04-06 | 马恒标 | Tumor arrestin variant DV379 and application thereof |
| CN113637052A (en) * | 2021-08-16 | 2021-11-12 | 牡丹江医学院 | Cancer suppressor protein for treating colon cancer and pharmaceutical composition thereof |
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| CN100410273C (en) | 2008-08-13 |
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