CN112522314A - Method for constructing high-expression PTEN vector and application thereof - Google Patents
Method for constructing high-expression PTEN vector and application thereof Download PDFInfo
- Publication number
- CN112522314A CN112522314A CN202011442228.8A CN202011442228A CN112522314A CN 112522314 A CN112522314 A CN 112522314A CN 202011442228 A CN202011442228 A CN 202011442228A CN 112522314 A CN112522314 A CN 112522314A
- Authority
- CN
- China
- Prior art keywords
- pten
- vector
- cells
- bamh
- immune
- 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
Images
Classifications
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- 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
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T 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
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Oncology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for constructing a high expression PTEN carrier and application thereof, wherein the high expression PTEN carrier system is constructed, the high expression PTEN protein is generated by expression in patient autoimmune cells, and the immune cells of the high expression PTEN protein are further conveyed to the body of a patient to achieve the effect of treating tumors, so that the anti-tumor activity of a PTEN gene is more effectively exerted. Meanwhile, the autoimmune cells of the patient are utilized, so that the immunological rejection is reduced, and the anti-tumor activity of the immune cells can be improved.
Description
Technical Field
The invention belongs to the field of molecular cell biology, and particularly relates to a method for constructing a high-expression PTEN vector and application thereof.
Background
The PTEN gene (NC-000010.11) is a cancer suppressor gene discovered in recent years and closely related to tumorigenesis and development after the p53 gene, plays an important role in tumorigenesis and development, and the PTEN protein encoded by the gene can finely regulate the intracellular protein phosphorylation level, thereby influencing the growth, proliferation, differentiation, apoptosis, adhesion and migration of cells. However, a complete method is still lacking for the preparation of immune cells carrying PTEN proteins.
Disclosure of Invention
In order to obtain immune cells carrying PTEN protein, the invention provides a method for constructing a high expression PTEN vector, and the over-expression of the high expression PTEN vector in autoimmune T cells of a patient is adopted to prepare the immune cells of the high expression PTEN protein, the immune cells can be greatly expanded in vitro, the expanded immune T cells are infused back into the body of the patient through intravenous injection, cancer can be well treated, the pain of the patient is relieved, and the aim of radically treating tumors is finally achieved.
In order to achieve the above object, an object of the present invention is to provide a method for constructing a high expression PTEN vector, the method comprising:
s1: acquiring a PTEN gene fragment, and connecting the PTEN gene fragment to an expression vector to obtain a connection product;
s2: mixing the connecting product with competent cells, putting the mixture into a first culture medium to obtain a substance to be transformed, and coating the substance to be transformed on a second culture medium for transformation to obtain transformed colony cells;
s3: and extracting the PTEN plasmid in the transformed colony cell to obtain the high-expression PTEN vector.
As an embodiment of the present invention, in step S1, the method for obtaining PTEN gene fragment is: adopting a BamH I enzyme digestion system to carry out enzyme digestion on a BamH I-PTEN-BamH I gene to obtain a PTEN gene fragment;
the expression vector is a pcDNA 3.1(+) vector; the method for obtaining the pcDNA 3.1(+) vector comprises the following steps: carrying out enzyme digestion recovery on a pcDNA 3.1(+) empty vector by a BamH I enzyme digestion system, and then carrying out dephosphorylation treatment to obtain the pcDNA 3.1(+) vector;
in the BamH I enzyme digestion system, the water content is 60-70 vol%, the Buffer content is 15-25 vol%, the DNA content is 15-25 vol%, and the BamH I enzyme content is 1-5 vol%.
As an embodiment of the present invention, in step S2, the competent cell is Escherichia coli DH5 α competent cell.
As an embodiment of the present invention, in step S2, the first medium is an LB medium;
the second culture medium is an LB culture medium containing ampicillin; the ampicillin content in the LB medium containing ampicillin is 0.01-0.2% by volume.
As an embodiment of the present invention, in step S2, the method of conversion is: adding the ligation product into colony cells, carrying out ice bath for 20-40min, immediately carrying out heat shock in a water bath at 35-45 ℃ for 80-100s, and then moving to ice for ice bath for 2-5 min; the colony cells are escherichia coli colony cells.
As an embodiment of the present invention, in step S3, the extracting method includes: performing the extraction using escherichia coli cultured in the second medium;
the second culture medium is an LB culture medium containing ampicillin; the ampicillin content in the LB medium containing ampicillin is 0.01-0.2% by volume.
In one embodiment, the method of extracting may be: selecting Escherichia coli monoclonal, adding into LB liquid culture medium containing ampicillin, culturing overnight, and extracting with EndoFree plasmid Maxi Kit (cat.no.12362) plasmid macroextraction Kit according to the instruction procedure when the color of the bacterial liquid is yellow and white.
The second purpose of the invention is to provide an immune cell for high expression of PTEN protein, which contains the high expression PTEN vector constructed by the method of the first purpose of the invention.
As one embodiment of the present invention, the immune cell is an immune T cell.
The third purpose of the invention is to provide a method for preparing immune cells highly expressing PTEN protein, which comprises the following steps:
the immune cell with the high expression PTEN protein is obtained by transfecting the immune cell with the high expression PTEN vector constructed by the method of the first object of the invention.
The fourth purpose of the present invention is to provide an application of the immune cell highly expressing PTEN protein according to the second purpose of the present invention or the immune cell highly expressing PTEN protein prepared by the method according to the third purpose of the present invention in the preparation of a drug for treating tumor.
The technical scheme provided by the invention has the beneficial effects that at least: the invention constructs a high expression PTEN carrier system, expresses in patient autoimmune cells to generate high expression PTEN protein, and further conveys the immune cells of the high expression PTEN protein to the body of a patient to achieve the effect of treating tumors, thereby more effectively exerting the anti-tumor activity of the PTEN gene. Meanwhile, the autoimmune cells of the patient are utilized, so that the immunological rejection is reduced, and the anti-tumor activity of the immune cells can be improved.
Drawings
FIG. 1 is a schematic diagram of PTEN gene obtained by using BamH I enzyme digestion system in preparation example of the present invention;
FIG. 2 is an electrophoretogram of pcDNA 3.1(+) -PTEN vector in the preparation example of the present invention, wherein the control group is an uncut enzyme system;
FIG. 3 is a schematic diagram of the result of detecting the expression of the high expression PTEN vector in the immune T cell by the Western blotting method in the test example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
When the specific experiment temperature is not indicated in the invention, the experiment temperature is 20-25 ℃, and the precooling temperature is 2-5 ℃.
[ PREPARATION EXAMPLES ]
1. Construction of eukaryotic expression vectors
1-1 recovery of the Gene of interest
As shown in FIG. 1, a BamH I-PTEN-BamH I gene (purchased from Kinsley Biotechnology Co., Ltd.) was digested with a BamH I digestion system to recover a gene fragment. The BamH I digestion system consists of:
the enzyme digestion system and the BamH I-PTEN-BamH I gene are put in a water bath kettle at 37 ℃ for enzyme digestion overnight. The enzyme digestion product was purified with a rapid DNA product purification kit (ciskejie DNA purification recovery kit Cat. # AE0301-a, purchased from singapore haosai technologies ltd.), the purification process followed the kit instructions.
1-2 target genes are linked to a vector
The pcDNA 3.1(+) empty vector is subjected to dephosphorylation treatment after enzyme digestion and recovery by a BamH I enzyme digestion system. The recovered PTEN gene fragment was ligated with the digested pcDNA 3.1(+) empty vector using Thermo Fisher T4 DNA ligase (Cat: EL0011) enzyme in a water bath at 16 ℃ overnight.
1-3 ligation vector transformation
mu.L of the ligation product obtained in 1-2 was added to 100. mu.L of E.coli DH 5. alpha. competent cells (purchased from Takara Bio Inc.), ice-washed for 30min, immediately heat-shocked in a water bath at 42 ℃ for 90s, transferred to ice-washed ice for 2min, added to 400. mu.L of LB medium, and cultured at 37 ℃ and 200rpm for 1 hr. 10 mu L of Escherichia coli bacterial liquid is evenly coated to the solution containing 100 mu g/mL-1Ampicillin on solid LB medium, the solid LB medium was cultured at 37 ℃ for approximately 16hr in an inverted incubator.
1-4 Positive colony screening
Coli monoclonals were selected, and the number of the selected monoclonals was marked and then cultured overnight in 4ml of liquid LB medium containing ampicillin. The next day, 2ml of the bacterial solution was taken, and the transformed plasmid was extracted using a Tiangen plasmid miniprep kit (Cat. # DP 103-02). And (5) detecting results by agarose gel electrophoresis. The control group was the corresponding plasmid that was not digested, as shown in FIG. 2. The positive clone with correct sequencing alignment is a plasmid containing pcDNA 3.1(+) -PTEN.
2. Immune T cell harvesting and transfection
10ml of blood was drawn from the patient, transferred to an anticoagulation tube, and mixed with 10ml of PBS buffer. Two 15ml centrifuge tubes were taken and 5ml of Ficoll solution (concentration 1.077. + -. 0.0001g/ml) was added first. Then, 10ml of diluted blood is respectively and lightly added to the upper layers of the Ficoll solutions of the two centrifuge tubes, so that the two solutions are prevented from being mixed together; after centrifugation at 2000rpm for 20min, the white cell layer was aspirated and added to a clean 15ml centrifuge tube to obtain peripheral blood mononuclear cells. PBS buffer was added to a solution volume of 10-15ml, centrifugation was continued at 1500rpm for 10min and the supernatant was removed, the remaining solution was added to LB medium and washed at 1500rpm for 10min, RPMI1640 complete medium (Cat: BC-M-017) with FBS concentration of 10% was added, T cells were stimulated with CD3 antibody, CD28 antibody, IL-2, and cells were transfected with lipofectamine 2000 (model invitrogen 11668019, from Jinan Aizoon Biotechnology Co., Ltd.) on the third day. On the fifth day, the RPMI1640 complete medium containing IL-2 with the FBS concentration of 10% is replaced, the amplification culture is continued for about 10 days, and immune T cells with high expression of PTEN protein are collected.
[ test examples ]
1. Western blotting method for detecting expression of high-expression PTEN vector in immune T cell
1-1Western blot related reagent
(1)5 XSDS-PAGE running buffer: weighing 15.1g of Tris, 5.0g of SDS, 94g of glycine and deionized water to a constant volume of 1L for later use. Working concentration was 1 ×.
(2)5 XSDS-PAGE Loading buffer: 0.5g SDS and 25mg bromophenol blue are weighed, 1.25mL 1M Tris-HCl (pH 6.8), 2.5mL glycerol and deionized water are added to the solution to a constant volume of 5mL, and the solution is stored for later use.
(3) Membrane transfer buffer: weighing 2.9g of glycine, 5.8g of Tris, 0.37g of SDS, dissolving with a proper amount of deionized water, adding 200mL of methanol, diluting the volume of the deionized water to 1L, and storing at 4 ℃ for later use.
(4) TBST buffer (purchased from solibao): weighing 8.8g of NaCl, adding 20mL of 1M Tris-HCl (pH8.0), then diluting with deionized water to constant volume of 1L, adding 200.5 mL of Tween, and mixing uniformly for later use.
(5) 5% skim milk blocking solution: it is prepared by TBST.
(6) RIPA lysate: 150mM NaCl, 5mM EDTA, 5mM sodium pyrophosphate, 25mM Tris (pH 7.4), 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate. When in use, 1% of PMSF and 0.1% of aprotinin are added.
1-2 extraction of high expression PTEN protein
(1) The cell culture dish in step 2 of preparation example was placed on ice, and the culture solution was discarded. After washing with pre-cooled PBS 3 times, the residual liquid was blotted clean. Adding into pre-cooled RIPA lysate (700. mu.L/10-cm culture dish; 100. mu.L/6-well plate), scraping adherent cells with a cell scraper, and ice-cooling for 30 min. The lysate was collected and vortexed, and centrifuged at 12000rpm for 15min at 4 ℃. Carefully pipette the supernatant into a clean 1.5mL centrifuge tube and place on ice. Taking a part of protein solution for measuring protein concentration, adding 5 × sample buffer solution (final concentration of 1 ×) into the rest protein solution, mixing, and boiling for 5 min. The obtained protein is frozen at-20 ℃ for later use. Control groups were T cells transfected without added vector.
(2) Protein electrophoresis: loading the protein to be detected into a polyacrylamide gel protein sample application hole, carrying out electrophoresis for 30min by adding electrophoresis buffer solution at 80V, and then carrying out electrophoresis at the voltage of 160V until bromophenol blue reaches the bottom of the separation gel.
(3) Film transfer: taking 8 pieces of filter paper with the same size as the separation gel soaked in the membrane transferring buffer solution and 1 piece of PVDF membrane. The PVDF membrane is soaked in a methanol solution for 10s before use. The membrane transfer instrument uses a Trans-Blot Turbo protein transfer system. And a filter paper sheet, a PVDF membrane, separation glue and a filter paper sheet which are stained with a membrane conversion buffer solution are attached to the lower electrode plate of the membrane converter from bottom to top. Care was taken to exclude bubbles from the rotary film system during operation. Film transferring conditions: constant pressure of 25V, 5-10min (determined according to the molecular weight of transmembrane protein).
(4) And (3) sealing: the PVDF membrane printed with the protein was placed in 5% skim milk and incubated at 37 ℃ for 2 hr.
(5) Antibody incubation: after blocking, the PVDF membrane was placed in TBST buffer and primary antibody was added at the concentrations used according to the instructions: Anti-PTEN (abcam, ab32199, 42KDa), incubated overnight at 4 ℃ with slow shaking. The PVDF membrane is washed 3 times and 10 min/time by adopting TBST buffer solution. Adding corresponding secondary antibody, incubating at 37 deg.C with slow shaking for 1hr, repeating above steps, and washing with TBST buffer for 3 times.
(6) Color development: Pro-Light HRP chemiluminescent detection reagent (Cat: PA112 from Tiangen Biochemical technology (Beijing) Ltd.) was formulated according to the instructions and dropped onto the PVDF membrane to be developed using ChemiDocTMThe XRS + imaging system exposes the acquired image.
The expression of the high-expression PTEN vector in immune T cells is detected by a Western blotting method, and the result is shown in figure 3, and the expression quantity of PTEN protein in the PTEN-transferred T cells is obviously higher than that of PTEN protein in ordinary T cells.
[ Experimental example ]
In-vitro killing experiment of immune T cells with high expression of PTEN protein on SMMC-7721 liver cancer cells
Thawing frozen immune T cells with high expression PTEN protein for resuscitation, adding into 10% FBS RPMI1640 complete culture medium, stimulating T cells with anti-CD 3, CD28 antibody and IL-2, culturing for 3 days, changing liquid, and supplementing nutritional factors for continuous amplification culture. On the fifth day, tumor antigen HSP70 was added and cultured for 24 hours (no antigen was added to the control group), and SMMC-7721 cells cultured to the exponential phase were plated in 96-well plates at 5000 cells/well after trypsinization. After 24 hours, prepared PTEN-T cells were added to plated SMMC-7721 cells. The ratio of effective targets is 50:1, the experimental groups are shown in table 1, and each group is subjected to 5 parallel experiments. After the cells are co-cultured for 24 hours, adding MTT (methyl thiazolyl tetrazolium) in 20 mu L/hole, continuously culturing for 6 hours, absorbing the supernatant, adding 150 mu L DMSO in each hole, shaking and uniformly mixing, measuring an OD (optical density) value at 492nm of an ELISA (enzyme labeling instrument) and recording. The in vitro killing activity of immune T cells highly expressing PTEN protein on SMMC-7721 cells is calculated according to the following formula: killing activity was 100- ((experimental OD value-effector OD value)/target OD value × 100%). The data were analyzed by T-test and the results are shown in table 1, where PTEN-T cells represent immune T cells highly expressing PTEN proteins of the present invention.
TABLE 1 in vitro killing Activity assay of PTEN-T cells on SMMC-7721 hepatoma cells
[ application example ]
Clinical treatment of patients with abdominal metastasis of colon cancer by PTEN-T cells
Separating immune T cells from patient blood, amplifying, transferring the constructed high expression PTEN carrier into the amplified immune T cells by using liposome lipofectamine 2000, stimulating the transfected immune T cells by using anti-CD 3, CD28 antibody and IL-2, culturing for 3 days, and then culturing at the rate of 10 days7The number of cells of the order of magnitude is input into the body of a patient, and the change of the tumor marker before and after the cell input is observed. After the PTEN-T cells are used for treating the tumor cells, the results of the tumor markers are shown in Table 2, and the detection results show that the values of CEA and glycoprotein 19-9 are obviously reduced, which shows that the PTEN-T cells have the treatment effect on the tumors. Wherein, the CEA and glycoprotein 19-9 level is correlated with the disease condition, and the serum CEA concentration is reduced to indicate the disease condition is improved.
TABLE 2 clinical treatment results of PTEN-T cells on patients with abdominal metastasis of colon cancer
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A method for constructing a high expression PTEN vector, which is characterized by comprising the following steps:
s1: acquiring a PTEN gene fragment, and connecting the PTEN gene fragment to an expression vector to obtain a connection product;
s2: mixing the connecting product with competent cells, putting the mixture into a first culture medium to obtain a substance to be transformed, and coating the substance to be transformed on a second culture medium for transformation to obtain transformed colony cells;
s3: and extracting the PTEN plasmid in the transformed colony cell to obtain the high-expression PTEN vector.
2. The method as claimed in claim 1, wherein in step S1, the method for obtaining PTEN gene fragment is: adopting a BamH I enzyme digestion system to carry out enzyme digestion on a BamH I-PTEN-BamH I gene to obtain a PTEN gene fragment;
the expression vector is a pcDNA 3.1(+) vector; the method for obtaining the pcDNA 3.1(+) vector comprises the following steps: carrying out enzyme digestion recovery on a pcDNA 3.1(+) empty vector by a BamH I enzyme digestion system, and then carrying out dephosphorylation treatment to obtain the pcDNA 3.1(+) vector;
in the BamH I enzyme digestion system, the water content is 60-70 vol%, the Buffer content is 15-25 vol%, the DNA content is 15-25 vol%, and the BamH I enzyme content is 1-5 vol%.
3. The method of claim 1, wherein in step S2, the competent cell is an E.coli DH5 a competent cell.
4. The method according to claim 1, wherein in step S2, the first medium is LB medium;
the second culture medium is an LB culture medium containing ampicillin; the ampicillin content in the LB medium containing ampicillin is 0.01-0.2% by volume.
5. The method of claim 1, wherein in step S2, the transformation method is: adding the ligation product into colony cells, carrying out ice bath for 20-40min, immediately carrying out heat shock in a water bath at 35-45 ℃ for 80-100s, and then moving to ice for ice bath for 2-5 min; the colony cells are escherichia coli colony cells.
6. The method according to claim 1, wherein in step S3, the extracting method is: performing the extraction using escherichia coli cultured in the second medium;
the second culture medium is an LB culture medium containing ampicillin; the ampicillin content in the LB medium containing ampicillin is 0.01-0.2% by volume.
7. An immune cell highly expressing PTEN protein, which contains a high expression PTEN vector constructed by the method of any one of claims 1-6.
8. The immune cell of claim 7, wherein the immune cell is an immune T cell.
9. A method for preparing immune cells highly expressing PTEN protein, comprising:
transfecting an immune cell with the high expression PTEN vector constructed by the method of any one of claims 1-6 to obtain the immune cell with high expression PTEN protein.
10. Use of the immune cell highly expressing PTEN protein of claim 7 or 8 or the immune cell highly expressing PTEN protein prepared by the method of claim 9 in the preparation of a medicament for treating tumor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011442228.8A CN112522314A (en) | 2020-12-11 | 2020-12-11 | Method for constructing high-expression PTEN vector and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011442228.8A CN112522314A (en) | 2020-12-11 | 2020-12-11 | Method for constructing high-expression PTEN vector and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112522314A true CN112522314A (en) | 2021-03-19 |
Family
ID=74999903
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011442228.8A Pending CN112522314A (en) | 2020-12-11 | 2020-12-11 | Method for constructing high-expression PTEN vector and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112522314A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020150954A1 (en) * | 2000-05-30 | 2002-10-17 | Durden Donald L. | Compositions and methods for identifying agents which modulate PTEN function and PI-3 kinase pathways |
| CN103468725A (en) * | 2013-09-27 | 2013-12-25 | 黑龙江八一农垦大学 | Construction and application of PTEN gene overexpression recombinant adenoviral vectors |
| CN107034235A (en) * | 2017-05-19 | 2017-08-11 | 尹荣 | Joint targeting PD 1 and EGFR mosaic antigens T cells tumour immunity method |
| CN108060135A (en) * | 2017-12-26 | 2018-05-22 | 东莞赛尔生物科技有限公司 | A kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins |
| CN110804625A (en) * | 2019-11-21 | 2020-02-18 | 安徽大学 | Method for increasing production of therapeutic antibody by over-expressing PTEN C124S in CHO (Chinese hamster ovary) cells |
| CN111808881A (en) * | 2020-08-25 | 2020-10-23 | 海南医学院 | Method for efficiently expressing AFP3-PTEN fusion protein |
-
2020
- 2020-12-11 CN CN202011442228.8A patent/CN112522314A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020150954A1 (en) * | 2000-05-30 | 2002-10-17 | Durden Donald L. | Compositions and methods for identifying agents which modulate PTEN function and PI-3 kinase pathways |
| CN103468725A (en) * | 2013-09-27 | 2013-12-25 | 黑龙江八一农垦大学 | Construction and application of PTEN gene overexpression recombinant adenoviral vectors |
| CN107034235A (en) * | 2017-05-19 | 2017-08-11 | 尹荣 | Joint targeting PD 1 and EGFR mosaic antigens T cells tumour immunity method |
| CN108060135A (en) * | 2017-12-26 | 2018-05-22 | 东莞赛尔生物科技有限公司 | A kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins |
| CN110804625A (en) * | 2019-11-21 | 2020-02-18 | 安徽大学 | Method for increasing production of therapeutic antibody by over-expressing PTEN C124S in CHO (Chinese hamster ovary) cells |
| CN111808881A (en) * | 2020-08-25 | 2020-10-23 | 海南医学院 | Method for efficiently expressing AFP3-PTEN fusion protein |
Non-Patent Citations (1)
| Title |
|---|
| 赵航: "抑癌基因PTEN对结肠癌细胞的抑制作用", 《中国优秀博硕士学位论文全文数据库(博士) 医药卫生科技辑》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20210154231A1 (en) | Method for producing t cells modified by chimeric antigen receptor | |
| CN110684120B (en) | Chimeric antigen receptor targeting GPC3 and application thereof | |
| CN108486060B (en) | Exosome for treating tumors and preparation method and application thereof | |
| CN109111525B (en) | HLA-G chimeric antigen receptor, coding sequence, expression vector and application | |
| CN115029351B (en) | Application of shRNA or BACH 1-deleted macrophage-derived EVs in preparation of medicine for treating hypertension | |
| CN110358737A (en) | A method of Chimeric antigen receptor T lymphocyte is prepared using excretion body | |
| CN109536451B (en) | Application of RTL1 gene in regulation and control of myoblast proliferation and differentiation | |
| CN112522314A (en) | Method for constructing high-expression PTEN vector and application thereof | |
| CN108465108B (en) | Specific gene target for preventing or treating brain glioma | |
| CN114712392B (en) | Immune cell preparation from autologous blood separation and application thereof | |
| CN101705227B (en) | SiRNA for inhibiting human AP-2alpha gene expression and anti-cervical cancer application thereof | |
| CN116751310A (en) | Chimeric antigen receptor targeting CD19 and GPRC5D ligand and application thereof | |
| CN114672460B (en) | Preparation method and application of CD 44-targeted heterogeneous CIC cell model | |
| CN114703190B (en) | Application of ShRNA for targeted inhibition of KIAA1429 gene expression in chronic myelocytic leukemia | |
| CN109837276B (en) | Long-chain non-coding RNA lncRNA-3608 and antibody and application thereof | |
| CN113265428A (en) | Detection system for constructing copper change in living cells by utilizing metallothionein and application | |
| CN114262689A (en) | Method for rapidly detecting activity of CD19/CD20-CAR-T cells | |
| CN107789624B (en) | Application of KIF15 inhibitor in preparation of lung cancer treatment drug | |
| CN106480022B (en) | Interference segment and use thereof | |
| CN114164210B (en) | Long-chain non-coding RNA for regulating and controlling protooncogene MYB and application thereof | |
| CN113817733B (en) | LncRNA transcribed from 34kb region upstream of MYB gene, expression vector and application thereof | |
| CN111549033B (en) | Lentiviral-infected human epidermal keratinocyte strain and construction method and application thereof | |
| CN114410689B (en) | Preparation method for enhancing lethality of tumor infiltrating lymphocytes | |
| CN116813794B (en) | Chimeric antigen receptor for coexpression of SPI1 and application thereof | |
| CN114075548B (en) | AXL-targeted CAR-T cell, and preparation method and application thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210319 |