CN108060135A - A kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins - Google Patents
A kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins Download PDFInfo
- Publication number
- CN108060135A CN108060135A CN201711434089.2A CN201711434089A CN108060135A CN 108060135 A CN108060135 A CN 108060135A CN 201711434089 A CN201711434089 A CN 201711434089A CN 108060135 A CN108060135 A CN 108060135A
- Authority
- CN
- China
- Prior art keywords
- cell
- cancer
- expression
- carrier
- high efficient
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Abstract
The present invention provides a kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins, and the T cell of high efficient expression P53 cancer suppressor proteins of the present invention is obtained by p53 eukaryotic expression vector transfection T cells；The p53 carrier for expression of eukaryon, be p53 genes are connected to it is built-up in 3.1 carriers of pcDNA.The present invention establishes the carrier system of high efficient expression p53, using immune t-cell as expression vector, with high efficient expression different subtype p53, can more effectively play the antitumor activity of p53.The T cell of high efficient expression P53 cancer suppressor proteins of the present invention can be used for preparing the drug for treating tumour.
Description
Technical field
The present invention relates to the application and conversion in molecular cytobiology field, more particularly to P53 cancer suppressor proteins, Yi Jiyong
P53 cancer suppressor proteins treat the immunotherapy of blood and solid tumor.
Background technology
P53 albumen is to be found by David Lane within 1979 and named according to its molecular size range, it is nucleoprotein four
Aggressiveness is acted on by several posttranscriptional modifications, such as phosphorylation, acetylation, ubiquitination, ubiquitin-like, methylate modification, can be with
It interacts with multiple proteins and DNA[1], influence fine, complicated cell regulation process.Multi-signal can induce p53 to swash
It is living, such as DNA damage, nutrition starvation, heat shock, virus infection, pH changes, anoxic and protooncogene activation.Particularly, p53
As transcription factor, specificity binding p53- response elements (p53REs) activation or inhibition of gene expression can be passed through[2]。p53
On the galianconism of No. 17 chromosome (17p 13.1)[3], contain 11 extrons, 10 intrones.Wherein remove 1 exon
Outer to be not intended to encode, 2-11 exons encode the albumen of 393 amino acid.In p53 amino acid sequences, 93-292 amino acid area
Domain is the high generating region of mutation in DNA calmodulin binding domain CaMs and human tumor[4].95% p53 base substitution mutations are happened at amino
On sour conserved sequence (codon 117-142,171-181,234-258 and 270-286)[5,6].The N- ends of p53 albumen are rich in a large amount of
Amino acid residue, activating transcription factor can be used as[7]；C- ends oligomerization area includes DNA non-specific bindings region, Ke Yishi
DNA that is other and combining damage[8].If p53 gene missense mutations are happened at specific N- C- end regions, it will influence base
Because inhibiting the function of tumour.
P53 genes be have found so far the highest tumor suppressor gene of correlation occurs with human tumor, it is but this
There is neoplastic transformations for the mutation of gene[9].It is reported that it is since P53 genes are dashed forward more than 50% human malignancies
Caused by change, it is happened at more than 90%P53 point mutations on central core structure domain[10].Wherein about 73% mutation belong to by
The change of protein conformation caused by missense mutation[11,12].On the one hand, the p53 of point mutation occurs to tumour associated signal paths
Inactivation is adjusted, then is prone to canceration；On the other hand, it is mutated even if p53 is not present, but the signal path quilt that p53 is participated in
Interference can still make human body suffer from cancer[13].P 53 gene mutation in human ovarian carcinoma rate highest, predominantly point mutation, 29%~
74%.The reports such as Okamoto, 80% oophoroma have p53 allelic losses, and 29% oophoroma has p53 gene mutations.Intrauterine
In film cancer, 32% patient is with p53 allelic losses.Mutation rate in cervical carcinoma is relatively low, and about 3%~7%.In addition,
There is also p53 gene mutations for 70%~80% patients with lung cancer.The p53 albumen of mutation can be detected by means such as immunohistochemistry
Largely it is gathered in nucleus[14,15]。
Then the study found that p53 is not only inhibited, while the performance of various cell functions can also be maintained, because
And it is referred to as " defender of genome "[16,17].Be presently believed that tumor suppressor gene mutation be compared to the mutation of oncogene for
The generation of cancer is more dangerous.By using appropriate tumor suppressor gene or get rid of carcinogenic corresponding oncogene be induction cancer
A kind of antineoplaston strategy got a good chance of of cell death.The ectopic expression of exogenous p53 genes can effectively induce perhaps
The death of the tumour cell of more p53 inactivations.In order to induce the expression of exogenous p53 genes, in the clinical precursor of kinds cancer perhaps
In outer and experiment in vivo p53 is expressed commonly using adenovirus vector.These cancers include：Non-small cell lung cancer, head and neck cancer are disliked
Property brain tumor, oophoroma, carcinoma of urinary bladder, cervix cancer, colorectal cancer and the cancer of the esophagus etc..
Generally existing oncogene and tumor suppressor gene in body including people, their dysfunction can result in cancer
The occurrence and development of disease.It is presently believed that the mutation of tumor suppressor gene is compared to the mutation of oncogene and more endangers for the generation of cancer
Danger.By using appropriate tumor suppressor gene or get rid of carcinogenic corresponding oncogene be inducing cancer cell death it is a kind of very
Promising antineoplaston strategy[6,18].The ectopic expression of exogenous p53 genes can effectively induce the swollen of many p53 inactivations
The death of oncocyte.In order to induce the expression of exogenous p53 genes, outside the clinical precursor of kinds cancer perhaps and in experiment in vivo
P53 is expressed commonly using adenovirus vector.These cancers include[15,19,20]：Non-small cell lung cancer, head and neck cancer, pernicious brain swell
Knurl, oophoroma, carcinoma of urinary bladder, cervix cancer, colorectal cancer and cancer of the esophagus etc..
Gene therapy for cancer carrier mediated adenovirus-p53 (Ad-p53) has been applied to clinical treatment.Outside clinical precursor
Research with experiment in vivo shows that the gene therapy of Ad-p53- mediations can induce extensive anti-tumor effect in tumour cell,
And there is low cytotoxicity in normal cell.The tumour cell of p53 function damages is carried usually to traditional chemotherapy radiotherapy
Caused genetoxic pressure has very strong tolerance[21,22].Research shows adenovirus mediated p53 wild type genes expression
Sensibility of these tumour cells to traditional chemotherapy and radiation can be enhanced.Recently, in the clinical trial of kinds of tumors patient,
The effect of the combination treatment combined to Ad-p53 carriers as monotherapy or with traditional chemotherapy and radiation is commented
Estimate.Although clinical research shows in various tumor patients, replication defect type Ad-p53 carriers are safe and feasibles, and are had
Good tolerance, but can not possibly be induced by this Ad-p53 carriers in each tumour cell deep exogenous
P53 protein expressions.In order to improve treatment late tumor patient effect, the relatively low transgene efficiency of Ad-p53 carriers be one must
The main problem that must be overcome.On the other hand, Ad-p53 on the market mainly passes through Lump body injection, behaviour for the treatment of solid tumor
Make complicated and have certain danger.Explore it is a kind of can effectively high expression p53 but also simple operations tumor therapeuticing method tool
There is broader prospect.
Application number 02815303.0, the entitled T cell receptor molecule combined with p53 with and application thereof invention it is special
A kind of T cell receptor (TCR) molecule is provided in profit, it can be combined with the peptide derived from p53 protein, particularly the mankind
P53 protein.The TCR molecules include heterodimeric molecule and single chain molecule, and the TCR molecular specificities be bound to and be revealed in
One tract of the p53 protein in the HLA molecular systems of context, particularly HLA-A2.1, especially amino acid 264-
273 position.The invention further discloses manufacture and the method using this kind of TCR molecules.Invention tool has been widely used, including
The purposes of the cell of therapeutical uses and detection expression p53 protein.
But the patent of invention only expresses the epitope of p53 protein sequences, without certain space conformation；In addition,
The invention is served only for the position of identification p53 264-273 rather than the full amino acid sequence of p53 albumen.The invention can be used for TCR
To meeting the identification of 264-273 peptide fragments, for combining the p53 of the p53 comprising the peptide fragment or identification containing the peptide fragment, not with table
Up to for the purpose of p53 holoproteins.
The content of the invention
For more than technological deficiency, the present invention establishes the carrier system of high efficient expression p53, is carried by expression of immune t-cell
Body with high efficient expression different subtype p53, can more effectively play the antitumor activity of p53.
The first object of the present invention is intended to build the carrier of high expression p53, further prepares high efficient expression P53 cancer suppressor proteins
T cell.
Therefore, the present invention provides a kind of T cell of high efficient expression P53 cancer suppressor proteins, by p53 eukaryotic expression vector transfections T
Cell obtains；The p53 carrier for expression of eukaryon, be p53 genes are connected to it is built-up in 3.1 carriers of pcDNA.
The second object of the present invention is the preparation method for providing the T cell of the high efficient expression P53 cancer suppressor proteins.This hair
The preparation method of the T cell of bright high efficient expression P53 cancer suppressor proteins, step are：
(1) structure stablizes expression p53 carrier for expression of eukaryon：P53 genes are connected with 3.1 carriers of pcDNA, structure p53 is true
Nuclear expression carrier pcDNA 3.1-p53；
(2) immune t-cell is transfected：Fresh T cell is added in 1640 complete mediums of RPMI containing 10%FBS,
T cell, the 3rd day pcDNA 3.1- that will be built with lipofectamine 2000 are stimulated with AntiCD3 McAb, CD28 antibody, IL-2
P53 carrier transfecting T cells, screening obtain stablizing the T cell strain p53-T cells for being overexpressed p53.
T cell prepared by the present invention can be frozen with -80 DEG C, long-term to preserve.In use, recovery cell, and it is a large amount of in vitro
Amplification, makes it respectively reach 108The order of magnitude, by being injected intravenously independent or inputting patient body jointly with other auxiliary elements
It is interior, have the function that alleviate cancer patient illness.
Therefore, the T cell the present invention also provides the high efficient expression P53 cancer suppressor proteins is preparing to treat the medicine of tumour
The application in object space face.
The tumour includes：Liver cancer, non-small cell lung cancer, head and neck cancer, malignant brain tumor, oophoroma, carcinoma of urinary bladder, cervix
Cancer, colorectal cancer and the cancer of the esophagus.
The present invention has the advantages that：
(1) the T cell not only production cell as P53, but also be the immunocyte that can kill tumour cell has wide
Compose the effect for the treatment of cancer.
(2) a large amount of immunocyte T cells are defeated to after in patient body, can promote the immunocompetence of patient, stimulate and activate disease
The immune system of people, so as to further enhance killing ability of the patient itself to cancer cell.
(3) the P53 albumen that T cell generates can be secreted into patient blood, and reaches body everywhere with blood circulation, therefore
Local entity tumor can not only be killed, and also plays an important role of to kill transferred tumour cell.
(4) present invention can express the p53 of different subtype, achieve the effect that treat for individual different symptoms.P53 genes
Different hypotypes is not only expressed in different tumours, and there is also different transcriptional activities and tumor suppression for different hypotypes
Because of subfunction.Due to the importance and complexity of p53 subtype expressions, the present invention is using T cell as carrier, and production is containing natural
, the p53 holoproteins of tripe systems elephant, for the treatment of tumor patient, curative effect is more definite.
Description of the drawings
Fig. 1 is the p53 carrier for expression of eukaryon structure diagrams that the present invention is built.
Fig. 2 is expression vector pcDNA 3.1-p53 agarose gel electrophoresis figures；Wherein 1, BamH I digestion carriers；2, Not
I digestion carriers；3, control group.
Fig. 3 is that western blotting detect expression of the p53 over-express vectors in T cell.
Specific embodiment
Technical scheme and its generated technique effect are done further with reference to specific test method
It illustrates.It should be appreciated that the following description is only intended to explain the invention, but is not in any way any limitation as the present invention, it is based on
Any conversion or replacement that the present invention is made, all belong to the scope of protection of the present invention.The method of the invention unless otherwise specified,
It is this field conventional method.
The preparation method of the T cell of 1. high efficient expression P53 cancer suppressor proteins of embodiment
1. the structure of eukaryotic expression vector
1-1 recycles target gene
The synthesis of BamH I-p53-BamH I genes is synthesized by Jin Sirui bio tech ltd, such as Fig. 1.What is obtained contains
The carrier of purposeful gene after BamH I digestions by recycling genetic fragment.Target gene digestion system is as follows：
The digestion system is placed in 37 DEG C of water-baths, and digestion is overnight.Digestion products are pure with rapid DNA Product Purification Kit
Change.The process is carried out in accordance with kit specification.
1-2 target gene is connected with carrier
PcDNA 3.1 (+) empty carrier dephosphorylation process after BamH I digestions recycling.Use DNA ligation kit
The p53 segments of recycling are connected by Ligation high Ver.2 with pcDNA 3.1 (+) zero load of digestion, 16 DEG C of water-baths
Night.
1-3 connections carrier converts
The above-mentioned connection products of 16 μ L are all added in 100 μ L bacillus coli DH 5 alpha competent cells, ice bath 30min is stood
Ice bath 2min on ice is moved to after thermal shock 90s in 42 DEG C of water-baths.400 μ L LB culture mediums are added in, 37 DEG C, 200rpm cultivates 1h.
10 μ L bacterium solutions are added dropwise and extremely contain 100 μ gmL-1On the LB solid plates of ampicillin, even spread.Culture dish is in 37 DEG C of trainings
It supports and culture about 16h is inverted in case.
1-4 positives bacterium colony screens
Escherichia coli clones are selected, adds in the LB fluid nutrient mediums that 4ml contains ampicillin and trains after lable number
It supports overnight.Next day takes 2ml bacterium solutions, and conversion plasmid is extracted with the small extraction reagent kit of plasmid (Kang Wei centuries).Agarose gel electrophoresis is examined
Survey result.Control group be the corresponding plasmid of non-digestion, such as Fig. 2.Sequencing compares correct positive colony bacterium to contain pcDNA 3.1-
The plasmid of p53 carriers.
2. stablize the screening of the T cell of expression p53 albumen
Fresh T cell is added in 1640 complete mediums of RPMI containing 10%FBS, with AntiCD3 McAb, CD28 antibody,
IL-2 stimulates T cell, the 3rd day pcDNA 3.1-p53 carrier transfectional cell that will be built with lipofectamine 2000.
The T cell strain for being overexpressed p53, p53-T cells are stablized in screening, and are detected for western blotting.
3.Western blotting detect expression of the p53 over-express vectors in T cell
3-1Western blot related reagents
(1) 5 × SDS-PAGE electrophoretic buffers：Tris 15.1g, SDS 5.0g, glycine 94g are weighed, deionized water is fixed
Hold spare to 1L.Working concentration for 1 ×.
(2) 5 × SDS-PAGE sample-loading buffers：SDS 0.5g, bromophenol blue 25mg are weighed, adds in 1M Tris-HCl (pH
6.8) 1.25mL, glycerine 2.5mL, deionized water are settled to 5mL, save backup.
(3) film transfer buffer solution：Glycine 2.9g, Tris 5.8g, SDS 0.37g is weighed, after appropriate amount of deionized water dissolving
Methanol 200mL is added in, deionized water is settled to after 1L and saves backup for 4 DEG C.
(4) TBST buffer solutions：NaCl 8.8g are weighed, add in deionized water constant volume after 1M Tris-HCl (pH8.0) 20mL
1L, adds in polysorbas20 0.5mL, and mixing is spare.
(5) 5% skim milk confining liquids：It is prepared with TBST.
(6) RIPA lysates：150mM NaCl, 5mM EDTA, 5mM sodium pyrophosphates, 25mM Tris (pH 7.4), 1%
Triton X-100,0.1%SDS, 0.5% NaTDCs.1%PMSF, 0.1% Aprotinin are added during use.
The extraction of 3-2 total proteins
(1) Tissue Culture Dish in step 2 is taken to be placed on ice, abandons culture solution.By Liquid Residue after the PBS cleaning 3 times of precooling
Body blots only.Add in RIPA lysates (the 700 μ L/10-cm culture dishes of precooling；100 μ L/6 orifice plates) after, take cell spatula that will paste
Parietal cell scrapes, ice bath 30min.It collects lysate and is vortexed and shake, 12000rpm, 4 DEG C of centrifugation 15min.It is careful to draw supernatant extremely
In clean 1.5mL centrifuge tubes, it is placed on ice.A part of protein liquid is taken for measuring protein concentration, residual protein liquid adds in 5 ×
(5min is boiled after mixing in final concentration of 1 ×) to sample-loading buffer.Obtained protein-20 DEG C freezes spare.Control group is to be added without
The T cell of carrier transfection.
(2) protein electrophoresis：Testing protein loading adds electrophoretic buffer into polyacrylamide gel albumen loading wells,
80V, 30min set 160V to bromophenol blue arrival separation gel bottom afterwards.
(3) transferring film：Take the filter paper 8 piece identical with being soaked in separation gel size in transferring film buffer solution, pvdf membrane 1.It uses
Preceding pvdf membrane, which is placed in methanol solution, impregnates 10s.Transferring film instrument uses Trans-Blot Turbo albumen transferring systems.In transferring film
Patch speckles with filter paper, pvdf membrane, separation gel, the filter paper of transferring film buffer solution from the bottom to top on electrode plate under instrument.Pay attention to operating
The bubble in transferring film system is excluded in journey.Transferring film condition：25V constant pressures, 5-10min (are determined) according to transferring film molecular weight of albumen.
(4) close：The pvdf membrane for being printed on albumen is placed in 5% skim milk, 37 DEG C of incubation 2hr.
(5) antibody incubation：Pvdf membrane is placed in TBST buffer solutions after closing, and primary antibody is added in accordance with specification concentration：
Anti-p53 (abcam, ab1101,43.6kDa), shakes overnight incubation slowly by 4 DEG C.TBST cleanings pvdf membrane 3 times, 10min/ times.Add
Enter corresponding secondary antibody, 37 DEG C of slow shake are incubated 1hr, repeat above step, cleaned 3 times in TBST.
(6) develop the color：Pro-Light HRP chemiluminescence detection reagents are prepared in accordance with specification, and is added dropwise to and waits to develop the color
On pvdf membrane, ChemiDoc is usedTMXRS+ imaging systems exposure acquisition image.The results are shown in Figure 3.
Experimental example 1.P53-T cells are to the killing experiments in vitro of SMMC-7721 liver cancer cells
The P53-T cells that freeze thaw after recovery, add in 1640 complete mediums of RPMI of 10%FBS, with AntiCD3 McAb,
CD28 antibody, IL-2 stimulate T cell, after cultivating 3 days, change liquid, the extra-nutrition factor continues to expand culture.It 5th day, adds in swollen
Tumor antigen HSP70 culture 24 it is small when standby use (control group is not added with antigen), meanwhile, by culture to exponential phase SMMC-7721 it is thin
Born of the same parents, 96 orifice plate of bed board after pancreatin digestion, 5000 cells/wells.24 it is small when after, ready P53-T cells are added in into bed board
In SMMC-7721 cells.Target is imitated than 50:1, experiment packet is shown in Table 1, every group of parallel laboratory test 5 times.After when cell co-cultivation 24 is small,
Add in MTT, 20 μ l/ holes, continue culture 6 it is small when after, suck supernatant, per hole add in 150 μ l DMSO, shake mixing, microplate reader
OD values, record are surveyed at 492nm.Cytotoxicity of the P53-T cells to SMMC-7721 cells is calculated as follows：
Killing activity=100- ((experimental group OD values-effector cell OD values)/target cell OD value × 100%)
Data are divided using t inspections.Analysis result is shown in Table 1.
Table 1P53-T cells analyze SMMC-7721 liver cancer cells cytotoxicity
Table 1 the result shows that, compared with simple T cell killing activity, the anti-tumor activity of P53-T improves 12.5
Percentage point.Since the present invention is generated containing natural, tripe systems elephant p53 holoproteins, therefore, is used for using T cell as carrier
The treatment of tumor patient, curative effect are more definite.
It discusses：1984, Matlashewski was found that the hypotype of p53 for the first time[23].After 1 year, the researchs such as Rotter
Variant after mouse p53C- ends alternative splicings, and be confirmed quickly in human cell[24].According to C- ends (α, β,
γ) with the difference at N- ends (△ 40p53, △ 133p53, △ 160p53), theoretically, p53 can be translated by posttranscriptional modification
12 kinds of subtype proteins (p53 α, p53 β, p53 γ, △ 40p53 α, △ 40p53 β, △ 40p53 γ, △ 133p53 α, △ 133p53 β,
△ 133p53 γ, △ 160p53 α, △ 160p53 β, and △ 160p53 γ)[18].P53 genes are not only expressed in different tumours
Different hypotypes, and there is also different transcriptional activities and tumor inhibiting factor subfunction, this staggeredly complexity for different hypotypes
Structure, joint effect the biological function of cell.Due to the importance and complexity of p53 subtype expressions, have very much
Scholar is directed to the clinical research of p53 different subtypes, it is intended to the hypotype and tumour of different p53 is illustrated on mRNA and protein level
The correlation of type[25].Fujita etc. is the study found that can be by reporting the different proportion of p53 β and △ 133p53 α, prediction knot
Carcinoma of the rectum adenoma is to the development grade of malignant tumour[25].In clear-cell carcinoma, the mRNA and protein expression level of p53 β increase indication
The development process of cancer[26].The expression of △ 40p53 α in mucin oophoroma, improves patient without the survival under recurrence
Rate[27].To 30 primary breast cancers the study found that wherein 18 expression deletions with p53 β and p53 γ[28]；12 are deposited
In the overexpression of △ 133p53[28].In cholangiocarcinoma, the expression of △ 133p53 has poor prognosis[29].Acute myelocytic leukemia
The overexpression of middle p53 β and p53 γ is the important indicator that sb.'s illness took a favorable turn after chemotherapy[30].Roundup is as a result, it was confirmed that different sub-
Expression of the p53 of type in different type tumour indicates classification, clinical response and the prognosis situation of tumour.
Research show by overexpression p53 albumen can effective induced various types of tumors cell death[31].So far,
Although being made great progress in terms of p53 targeted therapy of cancer, it is also deposited in terms of effectively treatment human cancer at present
In huge deficiency.For better treating cancer, mitigate the pain of patient, and be finally reached the purpose of radical cure cancer, more
Effective p53 tumor suppressor genes ideas of cancer therapy is urgently further researched and developed.
Based on above analysis, our designs have developed the new targeted therapy more effectively based on human tumor suppressor gene p53 and swell
The strategy of knurl.It can be seen that in being tested from it to tumoricidal compared with simple T cell killing activity, the tumour of P53-T
Killing activity improves 12.5 percentage points.
It is attached：Bibliography
1.Meek,D.W.and C.W.Anderson,Posttranslational modification of p53:
cooperative integrators of function.Cold Spring Harb Perspect Biol,2009.1(6):
p.a000950.
2.Appella,E.and C.W.Anderson,Post-translational modifications and
activation of p53 by genotoxic stresses.Eur J Biochem,2001.268(10):p.2764-72.
3.Isobe,M.,et al.,Localization of gene for human p53tumour antigen to
band 17p13.Nature,1986.320(6057):p.84-5.
4.Cho,Y.,et al.,Crystal structure of a p53 tumor suppressor-DNA
complex:understanding tumorigenic mutations.Science,1994.265(5170):p.346-55.
5.Hollstein,M.,et al.,p53mutations in human cancers.Science,1991.253
(5015):p.49-53.
6.Caron de Fromentel,C.and T.Soussi,TP53 tumor suppressor gene:a
model for investigating human mutagenesis.Genes Chromosomes Cancer,1992.4(1):
p.1-15.
7.Fields,S.and S.K.Jang,Presence of a potent transcription activating
sequence in the p53 protein.Science,1990.249(4972):p.1046-9.
8.Iwabuchi,K.,et al.,Use of the two-hybrid system to identify the
domain of p53 involved in oligomerization.Oncogene,1993.8(6):p.1693-6.
9.Jenkins,J.R.,K.Rudge,and G.A.Currie,Cellular immortalization by a
cDNA clone encoding the transformation-associated phosphoprotein p53.Nature,
1984.312(5995):p.651-4.
10.Olivier,M.,M.Hollstein,and P.Hainaut,TP53 mutations in human
cancers:origins,consequences,and clinical use.Cold Spring Harb Perspect Biol,
2010.2(1):p.a001008.
11.Hainaut,P.and M.Hollstein,p53 and human cancer:the first ten
thousand mutations.Adv Cancer Res,2000.77:p.81-137.
12.Kropveld,A.,et al.,Sequencing analysis of RNA and DNA of exons
1through 11shows p53 gene alterations to be present in almost 100%of head
and neck squamous cell cancers.Lab Invest,1999.79(3):p.347-53.
13.Brown,C.J.,et al.,Awakening guardian angels:drugging the p53
pathway.Nat Rev Cancer,2009.9(12):p.862-73.
14.Inoue,K.,et al.,Overexpression of p53 protein in human tumors.Med
Mol Morphol,2012.45(3):p.115-23.
15.Iggo,R.,et al.,Increased expression of mutant forms of p53
oncogene in primary lung cancer.Lancet,1990.335(8691):p.675-9.
16.Linzer,D.I.and A.J.Levine,Characterization of a 54K dalton
cellular SV40 tumor antigen present in SV40-transformed cells and uninfected
embryonal carcinoma cells.Cell,1979.17(1):p.43-52.
17.Lane,D.P.and L.V.Crawford,T antigen is bound to a host protein in
SV40-transformed cells.Nature,1979.278(5701):p.261-3.
18.Flaman,J.M.,et al.,The human tumour suppressor gene p53 is
alternatively spliced in normal cells.Oncogene,1996.12(4):p.813-8.
19.Cutilli,T.,et al.,p53 as a prognostic marker associated with the
risk of mortality for oral squamous cell carcinoma.Oncol Lett,2016.12(2):
p.1046-1050.
20.Drosten,M.and M.Barbacid,Ras and p53:An unsuspected liaison.Mol
Cell Oncol,2016.3(2):p.e996001.
21.Kung,C.P.and M.E.Murphy,The role of the p53 tumor suppressor in
metabolism and diabetes.J Endocrinol,2016.
22.Lane,D.P.and S.Benchimol,p53:oncogene or anti-oncogeneGenes Dev,
1990.4(1):p.1-8.
23.Matlashewski,G.,et al.,Isolation and characterization of a human
p53 cDNA clone:expression of the human p53 gene.Embo j,1984.3(13):p.3257-62.
24.Wolf,D.,et al.,Isolation of a full-length mouse cDNA clone coding
for an immunologically distinct p53molecule.Mol Cell Biol,1985.5(1):p.127-32.
25.Fujita,K.,et al.,p53 isoforms Delta133p53 and p53beta are
endogenous regulators of replicative cellular senescence.Nat Cell Biol,
2009.11(9):p.1135-42.
26.Song,W.,et al.,Expression of p53 isoforms in renal cell
carcinoma.Chin Med J(Engl),2009.122(8):p.921-6.
27.Hofstetter,G.,et al.,The N-terminally truncated p53 isoform
Delta40p53 influences prognosis in mucinous ovarian cancer.Int J Gynecol
Cancer,2012.22(3):p.372-9.
28.Bourdon,J.C.,et al.,p53 isoforms can regulate p53 transcriptional
activity.Genes Dev,2005.19(18):p.2122-37.
29.Nutthasirikul,N.,et al.,Ratio disruption of the 133p53 and TAp53
isoform equilibrium correlates with poor clinical outcome in intrahepatic
cholangiocarcinoma.Int J Oncol,2013.42(4):p.1181-8.
30.Anensen,N.,et al.,A distinct p53 protein isoform signature
reflects the onset of induction chemotherapy for acute myeloid leukemia.Clin
Cancer Res,2006.12(13):p.3985-92.
31.Oren,M.,P.Tal,and V.Rotter,Targeting mutant p53 for cancer
therapy.Aging(Albany NY),2016.8(6):p.1159-60.
Claims (5)
1. a kind of T cell of high efficient expression P53 cancer suppressor proteins, it is characterized in that, it is obtained by p53 eukaryotic expression vector transfection T cells；
The p53 carrier for expression of eukaryon, be p53 genes are connected to it is built-up in 3.1 carriers of pcDNA.
2. the preparation method of the T cell of high efficient expression P53 cancer suppressor proteins described in claim 1, it is characterized in that, step is：
(1) structure stablizes expression p53 carrier for expression of eukaryon：P53 genes with 3.1 carriers of pcDNA are connected, build p53 eucaryon tables
Up to carrier pcDNA 3.1-p53；
(2) immune t-cell is transfected：Fresh T cell is added in 1640 complete mediums of RPMI containing 10%FBS, with anti-
CD3, CD28 antibody, IL-2 stimulate T cell, the 3rd day pcDNA 3.1-p53 that will be built with lipofectamine 2000
Carrier transfecting T cells, screening obtain stablizing the T cell strain p53-T cells for being overexpressed p53.
3. the preparation method of the T cell of high efficient expression P53 cancer suppressor proteins as claimed in claim 2, it is characterized in that, the step
(1) structure stablizes expression p53 carrier for expression of eukaryon, comprises the concrete steps that：
1-1 target gene obtains
The BamH I-p53-BamH I genes of synthesis are spare by recovery purifying p53 genetic fragments after BamH I digestions；
1-2 target gene is connected with carrier
PcDNA 3.1 (+) empty carrier dephosphorylation process after BamH I digestions recycling；By the p53 segments of recycling and digestion
The unloaded connections of pcDNA3.1 (+), 16 DEG C of water-baths stay overnight；
1-3 connections carrier converts
The above-mentioned connection products of 16 μ L are all added in 100 μ L bacillus coli DH 5 alpha competent cells, ice bath 30min exists immediately
In 42 DEG C of water-baths ice bath 2min on ice is moved to after thermal shock 90s；400 μ L LB culture mediums are added in, 37 DEG C, 200rpm cultivates 1h；It is added dropwise
10 μ L bacterium solutions extremely contain 100 μ gmL-1On the LB solid plates of ampicillin, even spread, culture dish is in 37 DEG C of incubators
It is middle to be inverted culture about 16h；
1-4 positives bacterium colony screens
Escherichia coli clones are selected, add in overnight incubation in the LB fluid nutrient mediums that 4ml contains ampicillin, next day takes
2ml bacterium solutions, extraction conversion plasmid, it is the plasmid containing pcDNA 3.1-p53 carriers that sequencing, which compares correct positive colony bacterium,.
4. the T cell of high efficient expression P53 cancer suppressor proteins described in claim 1 is in terms of the drug for treating tumour is prepared
Using.
5. application as claimed in claim 4, it is characterized in that, the tumour includes：Liver cancer, non-small cell lung cancer, head and neck cancer, evil
Property brain tumor, oophoroma, carcinoma of urinary bladder, cervix cancer, colorectal cancer and the cancer of the esophagus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711434089.2A CN108060135A (en) | 2017-12-26 | 2017-12-26 | A kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711434089.2A CN108060135A (en) | 2017-12-26 | 2017-12-26 | A kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108060135A true CN108060135A (en) | 2018-05-22 |
Family
ID=62140420
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711434089.2A Pending CN108060135A (en) | 2017-12-26 | 2017-12-26 | A kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108060135A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593693A (en) * | 2018-08-07 | 2019-04-09 | 湖南师范大学 | One plant of 1917 antineoplastic target engineering bacteria of Escherichia coli Nissle and its construction method and application |
| CN112522314A (en) * | 2020-12-11 | 2021-03-19 | 山东新医学中西医结合医学研究院有限公司 | Method for constructing high-expression PTEN vector and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040005711A1 (en) * | 2000-10-06 | 2004-01-08 | Regts Djoeke Geesje | Genetic immunization against cervical carcinoma |
| CN1597939A (en) * | 2001-11-15 | 2005-03-23 | 杭州中肽生化有限公司 | Multifunctional anticancer recombined adenovirus |
| CN1692124A (en) * | 2001-06-05 | 2005-11-02 | 阿尔特生物科学公司 | P53 binding T cell receptor molecules and uses thereof |
| CN103038343A (en) * | 2010-03-23 | 2013-04-10 | 英特瑞克斯顿股份有限公司 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof. |
-
2017
- 2017-12-26 CN CN201711434089.2A patent/CN108060135A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040005711A1 (en) * | 2000-10-06 | 2004-01-08 | Regts Djoeke Geesje | Genetic immunization against cervical carcinoma |
| CN1692124A (en) * | 2001-06-05 | 2005-11-02 | 阿尔特生物科学公司 | P53 binding T cell receptor molecules and uses thereof |
| CN1597939A (en) * | 2001-11-15 | 2005-03-23 | 杭州中肽生化有限公司 | Multifunctional anticancer recombined adenovirus |
| CN103038343A (en) * | 2010-03-23 | 2013-04-10 | 英特瑞克斯顿股份有限公司 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof. |
Non-Patent Citations (5)
| Title |
|---|
| H SAITO: "Combination of p53-DC vaccine and rAd-p53 gene therapy induced CTLs cytotoxic against p53-deleted human prostate cancer cells in vitro", 《CANCER GENE THERAPY》 * |
| 彭黎明: "《细胞凋亡的基础与临床》", 31 July 2000, 人民卫生出版社 * |
| 杨晓红: "重组P53基因转染的肝癌细胞ICAM-1分子的表达", 《细胞与分子免疫学杂志》 * |
| 程宏: "CD3ε和5-氟尿嘧啶在T淋巴细胞凋亡中的协同作用及P53的表达", 《中华医学杂志》 * |
| 蒋国梁: "《临床肿瘤学概论》", 31 January 2013, 复旦大学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593693A (en) * | 2018-08-07 | 2019-04-09 | 湖南师范大学 | One plant of 1917 antineoplastic target engineering bacteria of Escherichia coli Nissle and its construction method and application |
| CN109593693B (en) * | 2018-08-07 | 2022-08-05 | 湖南师范大学 | Escherichia coli Nissle 1917 anti-tumor targeting engineering bacterium and construction method and application thereof |
| CN112522314A (en) * | 2020-12-11 | 2021-03-19 | 山东新医学中西医结合医学研究院有限公司 | Method for constructing high-expression PTEN vector and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shabgah et al. | CXC chemokine ligand 16: a Swiss army knife chemokine in cancer | |
| Wang et al. | CXCL17 promotes cell metastasis and inhibits autophagy via the LKB1-AMPK pathway in hepatocellular carcinoma | |
| CN105435228B (en) | New anti-tumor application of arsenic trioxide and anti-tumor preparation | |
| JP2021508253A (en) | Nucleotide sequence encoding CAR, ROBO1 CAR-NK cells expressing this CAR, preparation and use thereof | |
| WO2013026334A1 (en) | Cell dna synthesis prevention and cell proliferation inhibition polypeptide and use thereof | |
| CN105802909B (en) | T cell preparation with HER2 specific TCR and uses thereof | |
| Cui et al. | The glycosyltransferase ST6Gal-I is enriched in cancer stem-like cells in colorectal carcinoma and contributes to their chemo-resistance | |
| JP2021525283A (en) | A pharmaceutical composition for preventing or treating cancer, which comprises an expression inhibitor or activity inhibitor of CD300c. | |
| Li et al. | PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer | |
| CN111303300A (en) | Polypeptide inhibitor for degrading CREPT (cell-specific oncogene receptor potential) and application of polypeptide inhibitor in inhibiting pancreatic cancer cell proliferation and tumorigenesis | |
| Cresswell et al. | Biochemical and growth regulatory activities of the HIN-200 family member and putative tumor suppressor protein, AIM2 | |
| Li et al. | Effect of overexpression of PTEN on apoptosis of liver cancer cells | |
| CN108060135A (en) | A kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins | |
| CN110157686B (en) | Replication type oncolytic adenovirus activated by immune checkpoint and immune co-stimulation and construction method and application thereof | |
| AU2016366515A1 (en) | Combined preparations of PKM2 modulators and HMGB1 | |
| Li et al. | TES inhibits colorectal cancer progression through activation of p38 | |
| Teng et al. | The Complexity of DEK signaling in cancer progression | |
| Chen et al. | Promotion of proliferation and metastasis of hepatocellular carcinoma by LncRNA00673 based on the targeted-regulation of notch signaling pathway. | |
| Feng et al. | Effect of solanum lyratum polysaccharide on malignant behaviors of lung cancer cells by regulating the circ_UHRF1/miR-513b-5p axis | |
| CN113528528B (en) | shRNA for promoting apoptosis of imatinib-resistant chronic myelocytic leukemia cell K562/G01 and application thereof | |
| CN111333710B (en) | C20orf24 protein deletion mutant and application thereof | |
| Yu et al. | Herpes simplex virus type 1 VP22-mediated intercellular delivery of PTEN increases the antitumor activity of PTEN in esophageal squamous cell carcinoma cells in vitro and in vivo | |
| CN112516286B (en) | Application of polypeptide coded by annular RNAcircMAPK14 in preparation of anti-cancer drugs | |
| WO2021043341A2 (en) | Novel micropeptide hmmw and application thereof | |
| Sultan et al. | Exploring Small Nucleolar RNA Host Gene 3 as a Therapeutic Target in Breast Cancer Through Metabolic Reprogramming |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 523000 401, room 4, building 10, innovation and Technology Park, Songshan Lake high tech Industrial Development Zone, Dongguan, Guangdong, China Applicant after: Guangdong cel Biotechnology Co.,Ltd. Address before: 523808 Songshan Lake innovation technology 10 401, Dongguan City, Guangdong Province Applicant before: DONGGUAN CELL BIO-TECHNOLOGY Co.,Ltd. |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180522 |