CN101143222A - Application of p53 expression product in preparing medicine for treating tumor - Google Patents
Application of p53 expression product in preparing medicine for treating tumor Download PDFInfo
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- CN101143222A CN101143222A CNA2006101160520A CN200610116052A CN101143222A CN 101143222 A CN101143222 A CN 101143222A CN A2006101160520 A CNA2006101160520 A CN A2006101160520A CN 200610116052 A CN200610116052 A CN 200610116052A CN 101143222 A CN101143222 A CN 101143222A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention belongs to the biomedical field and relates to the application of p53 in preparing for a drug preparation for remedying the tumor. A p53 gene coding product is a transcription regulator which can induce the apoptosis or the block of the cell cycle by regulating lower target gene. An over-expression of the p53 can very effectively induce the apoptosis of tumor cells. Attenuated salmonella typhimurium can be used as a carrier to introduce the p53 to express inside the tumor cells and kill the tumor cells. The development of a novel treatment drug which considers the salmonella with the p53 as orally taken gene not only solves the problem of non-injection path of a protein polypeptide drug but also more importantly provides the tumor treatment with a safe, effective, peculiar and economical method.
Description
Technical field
The invention belongs to biomedicine field, relate to the application of p53 expression product in the agent of preparation medicine for treating tumor.
Background technology
Tumor disease has now risen to No. the 2nd, the world " killer ", and its death toll is only second to the cardiovascular diseases.In the past few years, domestic and international medical circle has had new understanding again for the pathogeny of tumor disease on cell base.Based on the further understanding to tumor invasion mechanism, people utilize various approach to develop and develop can be special, effectively killing tumor cell and to the avirulent medicine of normal cell.At present, for treatment for cancer is first-selection with chemotherapy and radiotherapy still, though both have obtained suitable curative effect to tumor treatment, but since lack to the specificity of tumor cell thus have bigger toxic and side effects and some tumor cell to chemotherapy and radiation handle insensitive, therefore limited their application in clinical to a great extent.In recent years, in order to develop killing tumor cell specifically and normal cell is not had the medicine of toxic side effect, from cell, paid much attention to and huge investment by the research on the molecular level to the pathogenesis of cancer for people.
The coded product of antioncogene p53 is that a kind of transcription regulatory factor can come cell death inducing or cell cycle arrest and then suppress tumor cell external by the target gene of regulating the downstream, intravital growth, thereby be considered to the new point of a therapy of tumor.
Attenuated salmonella typhimurium is proved it and not only can gathers propagation and suppress tumor cell in the intravital growth of mice at the tumor cell camber specifically, simultaneously also can be used as the engineering bacteria transport agent comes expression alien gene and has good tumor cell targeting, thereby the therapy of tumor of attenuation salmonella typhi mediation will be opened up a new approach for treatment for cancer.
Summary of the invention
The purpose of this invention is to provide the application of p53 expression product in the agent of preparation medicine for treating tumor, comprise the tumor target gene therapy technology of mediation.
The invention provides the application of p53 polypeptide in the agent of preparation medicine for treating tumor, described medicine for treating tumor agent is the tumor target gene therapy medicine.Mainly being based on attenuated salmonella typhimurium can be specifically gather propagation and can be used as the engineering bacteria transport agent at the tumor cell camber and express the p53 gene, so by behind the oral administration, attenuated salmonella typhimurium can arrive tumor locus and then continuous expression p53 by different approaches by intestinal, thereby improves the tumor-targeting and the efficient of medicine.
Among the present invention, the p53 polypeptid coding sequence is to obtain from the screening of people's kidney cDNA expression library, and the length of cDNA sequence is 1182bp, utilizes technique for gene engineering to express this albumen, and corresponding polypeptide molecular weight is 53KD.
In the present invention, term " p53 gene open reading frame sequence " comprises the open reading frame sequence (its full length nucleotide sequence is seen NCBI NM000546) of p53 gene or its degenerate sequence.This degenerate sequence is meant, in the p53 gene order (open reading frame), having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, so be low to moderate about 70% the degenerate sequence proteic sequence of p53 of also encoding out with the ORF nucleotide sequence homology of p53 gene order.This term comprises that also its expression product has anti-tumor activity and its sequence and has variant form as the nucleic acid molecule of p53 gene order.These variant forms do not influence the anti-tumor activity of its expression product, comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of nucleotide, and (being generally in 60, preferably is in 30 to add several at 5 ' and/or 3 ' end, more preferably being in 10, is in 5 best) nucleotide.
P53 of the present invention can come cell death inducing or cell cycle arrest and then suppress tumor cell external, intravital growth by the target gene of regulating the downstream.
Among the present invention, p53 is cloned in the eukaryotic vector transfection human colon carcinoma (HCT116) and human lung carcinoma cell (H1299) then, collecting cell after 48 hours, flow cytometer testing result prompting p53 overexpression is cell death inducing preferably.
The present invention detects the expression of p53 in tumor cell that attenuated salmonella typhimurium mediates with the method for Western blot and PCR, and results suggest and blank bacterial strain compare, and p53 can express in tumor cell, and its protein product is 53KD.After expressing the attenuated salmonella typhimurium infected person tumor cell of p53, can induce tangible apoptosis of tumor cells; Along with the increase of infection time, more tumor cell generation apoptosis.P53 of the present invention can be used to induce the apoptosis of tumor cells such as human colon cancer cell, human pancreatic cancer cell, hepatoma carcinoma cell, human cervical carcinoma cell, people's non-small cell lung cancer cell, osteosarcoma cell, the acute T lymphocytic leukemia cells of people and people's acute promyelocytic leukemia cell.
The attenuated salmonella typhimurium of expressing p53 can suppress tumor cell in the intravital growth of mice: with the attenuated salmonella typhimurium oral administration feeding tumor-bearing mice of expressing p53, compare with the blank bacterial strain, can suppress the growth cycle of tumor cell significantly at intravital growth of mice and prolongation tumor-bearing mice.
P53 polypeptide of the present invention, p53 gene and analog thereof etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can be with being changed to some extent by preparation Substance Properties and disease to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, Intradermal or topical.
With p53 polypeptide of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains protein and the pharmaceutically acceptable carrier or the excipient for the treatment of effective dose.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.P53 of the present invention can be made into the injection form, for example is prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as tablet and capsule can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component is the treatment effective dose, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, p53 of the present invention also can use with the other treatment agent.
When p53 polypeptide of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to mammal, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 20 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Among the present invention, will contain the p53 polypeptide (its corresponding full length nucleotide sequence is seen NCBINM000546) of effect consumption and carrier pharmaceutically or excipient and promptly form the medicine for treating tumor agent.
Preferably, the present invention is transformed into the virus of attenuation with p53 gene open reading frame sequence, extracts positive colony; To contain the positive colony of effect consumption and carrier pharmaceutically or excipient then and form described medicine for treating tumor agent.
Furthermore, the present invention modifies p53 gene open reading frame sequence, is transformed into the viral vector of attenuation again and extracts the positive colony that process is modified; To contain the positive colony of effect consumption and carrier pharmaceutically or excipient at last and form described medicine for treating tumor agent.
Better, the present invention is connected p53 gene open reading frame sequence with carrier for expression of eukaryon, modify and transform the virus of attenuation then, and separation and purification obtains positive colony again; To contain the positive colony of effective therapeutic dose and carrier pharmaceutically or excipient at last and form described medicine for treating tumor agent.
Particularly, the present invention is connected p53 gene open reading frame sequence with carrier for expression of eukaryon, then with recombinant plasmid transformed prokaryotic hosts LB5000, extracts the virus that plasmid transforms attenuation from LB5000, and separation and purification obtains positive colony again; To contain the above-mentioned clone of effective therapeutic dose and carrier pharmaceutically or excipient at last and form described medicine for treating tumor agent.
Among the present invention, the virus of described attenuation is attenuated salmonella typhimurium.
Among the present invention, the virus of described attenuation is attenuated salmonella typhimurium final host SL3261.
Among the present invention, described effective application quantity is 1 microgram/kg body weight-Yue 5 mg/kg body weight.
Among the present invention, described medicine for treating tumor agent is a medicinal preparation for oral administration.
It is that facultative anaerobe can be in the growth of the anoxic zone of tumor tissues simultaneously because attenuated salmonella typhimurium can gather propagation at the tumor cell camber specifically.So by behind the oral administration, attenuated salmonella typhimurium can arrive tumor locus and then continuous expression p53 by different approaches by intestinal, thereby improves the tumor-targeting and the efficient of medicine.
Conventional chemotherapy and radiotheraping method since their toxic and side effects and some tumor cell to chemotherapy and radiation handle insensitive, therefore limited their application in clinical to a great extent.This protein p53 provided by the invention has high lethal effect to tumor cell.Therefore, be a cancer therapy drug that has very much DEVELOPMENT PROSPECT.The novel form of genetically engineered drug and the development of novel drug-supplying system are the directions that field of medicaments is sought to innovate.Because genetically engineered drug is polypeptide drug mostly, generally has only a kind of approach of drug administration by injection, this is extremely inconvenient for the patient of long term administration.The nasal-cavity administration of protein polypeptide medicine, pulmonary administration, oral administration etc. will become the alternative route of drug administration by injection; Gene drug delivery then need solve a series of ten minutes complicated technology difficult problems such as targeting.Attenuated salmonella typhimurium is proved it and not only can gathers propagation and suppress tumor cell in the intravital growth of mice at the tumor cell camber specifically, simultaneously also can be used as the engineering bacteria transport agent comes expression alien gene and has good tumor cell targeting, thereby the therapy of tumor of attenuation salmonella typhi mediation will be opened up a new approach for treatment for cancer.Be that the technology that carrier imports tumor cell specifically with p53 and then suppresses the growth of tumor cell all belongs to initiative at home and abroad with the attenuated salmonella typhimurium.Exploitation the more important thing is will be that oncotherapy provides a kind of safety with the Salmonella of carrying p53 for the difficult problem that oral gene therapy new drug has not only solved the non-injection administration approach of protein and peptide medicine, effectively, and special and economic method.
The specific embodiment
Embodiment 1p53 induces human lung carcinoma cell (H1299) apoptosis
The acquisition of p53 gene: amplify the full-length cDNA fragment of KILLER with PCR from people's kidney cDNA expression library, length is 1182bp; Go in the carrier for expression of eukaryon through order-checking affirmation rear clone.
Human lung carcinoma cell (H1299) is provided by ATCC.Cell strain uses the GIBCO RPMI1640 of company complete medium to cultivate the H1299 human lung carcinoma cell.With the plasmid transfection tumor cell of expressing p53 48 hours, carry out PI dyeing behind the collecting cell, utilize flow cytometer to detect apoptosis.
The result shows that p53 of the present invention can induce human lung carcinoma cell (H1299) apoptosis.
Embodiment 2 p53 induce human colon cancer cell HCT116 apoptosis
The acquisition of p53 gene: amplify the full-length cDNA fragment of KILLER with PCR from people's kidney cDNA expression library, length is 1182bp; Go in the carrier for expression of eukaryon through order-checking affirmation rear clone.
Human colon cancer cell HCT116 is provided by ATCC.Cell strain uses GIBCO Macoy ' the s 5A of company complete medium (containing 10% calf serum, 100U/ml penicillin and 100U/ml streptomycin) to cultivate.With the plasmid transfection tumor cell of expressing p53 48 hours, carry out PI dyeing behind the collecting cell, utilize flow cytometer to detect apoptosis.
The result shows that p53 of the present invention increases promoting human colon carcinoma (HCT116) apoptotic cell quantity.
But embodiment 3 expresses the attenuated salmonella typhimurium inducing tumor cell generation apoptosis of p53
With non-resistant complete medium culture of tumor cell, with the expression p53 of 250MOI (ratio of virion number and subject cell number) and negative control attenuated salmonella typhimurium infection cell 3 hours, come cultured cell with the complete medium that contains antibiotic and 100 μ g/ml G418 subsequently.Come infection cell respectively at different time points, detect attenuated salmonella typhimurium the inducing of expressing p53 apoptosis of tumor cells.
The result shows, but the attenuated salmonella typhimurium inducing tumor cell generation apoptosis of expression p53.
The attenuated salmonella typhimurium that embodiment 4 expresses p53 can suppress tumor cell in the intravital growth of mice
With expressing p53 and negative control attenuated salmonella typhimurium oral administration feeding tumor-bearing mice, detect respectively at different time points the attenuated salmonella typhimurium of expressing p53 to tumor cell in the intravital growth of mice and to the influence of tumor-bearing mice growth cycle.
The result shows that the attenuated salmonella typhimurium of expressing p53 can suppress the growth cycle of tumor cell at intravital growth of mice and prolongation tumor-bearing mice.
Claims (10)
1.p53 the application of polypeptide in the agent of preparation medicine for treating tumor is characterized in that described medicine for treating tumor agent is the tumor target gene therapy medicine.
2. an application as claimed in claim 1 is characterized in that, will contain p53 polypeptide and the carrier pharmaceutically or the agent of excipient composition medicine for treating tumor of effect consumption.
3. an application as claimed in claim 1 is characterized in that, p53 gene open reading frame sequence is transformed into the virus of attenuation, extracts positive colony; To contain the positive colony of effect consumption and carrier pharmaceutically or excipient then and form described medicine for treating tumor agent.
4. an application as claimed in claim 1 is characterized in that, p53 gene open reading frame sequence is modified, and is transformed into the viral vector of attenuation again and extracts the positive colony that process is modified; To contain the positive colony of effect consumption and carrier pharmaceutically or excipient at last and form described medicine for treating tumor agent.
5. an application as claimed in claim 1 is characterized in that, p53 gene open reading frame sequence is connected with carrier for expression of eukaryon, modifies and transform the virus of attenuation then, and separation and purification obtains positive colony again; To contain the positive colony of effective therapeutic dose and carrier pharmaceutically or excipient at last and form described medicine for treating tumor agent.
6. application as claimed in claim 1, it is characterized in that, p53 gene open reading frame sequence is connected with carrier for expression of eukaryon, then with recombinant plasmid transformed prokaryotic hosts LB5000, extract the virus that plasmid transforms attenuation from LB5000, separation and purification obtains positive colony again; To contain the above-mentioned clone of effective therapeutic dose and carrier pharmaceutically or excipient at last and form described medicine for treating tumor agent.
7. as described any one application of claim 2-6, it is characterized in that the virus of described attenuation is attenuated salmonella typhimurium.
8. as described any one application of claim 2-6, it is characterized in that the virus of described attenuation is attenuated salmonella typhimurium final host SL3261.
9. an application as claimed in claim 2 is characterized in that, described effective application quantity is 1 microgram/kg body weight-Yue 5 mg/kg body weight.
10. an application as claimed in claim 1 is characterized in that, described medicine for treating tumor agent is a medicinal preparation for oral administration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006101160520A CN101143222A (en) | 2006-09-14 | 2006-09-14 | Application of p53 expression product in preparing medicine for treating tumor |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2006101160520A CN101143222A (en) | 2006-09-14 | 2006-09-14 | Application of p53 expression product in preparing medicine for treating tumor |
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| CN101143222A true CN101143222A (en) | 2008-03-19 |
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| CNA2006101160520A Pending CN101143222A (en) | 2006-09-14 | 2006-09-14 | Application of p53 expression product in preparing medicine for treating tumor |
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Open date: 20080319 |