CN117106725A - Lung cancer cell strain for stably expressing FAM19A5 gene and construction method and application thereof - Google Patents
Lung cancer cell strain for stably expressing FAM19A5 gene and construction method and application thereof Download PDFInfo
- Publication number
- CN117106725A CN117106725A CN202310895631.3A CN202310895631A CN117106725A CN 117106725 A CN117106725 A CN 117106725A CN 202310895631 A CN202310895631 A CN 202310895631A CN 117106725 A CN117106725 A CN 117106725A
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- cancer cell
- fam19a5
- gene
- stably expressing
- 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
- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 71
- 201000005202 lung cancer Diseases 0.000 title claims abstract description 71
- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 71
- 101000788164 Homo sapiens Chemokine-like protein TAFA-5 Proteins 0.000 title claims abstract description 49
- 238000010276 construction Methods 0.000 title abstract description 7
- 241000700605 Viruses Species 0.000 claims abstract description 17
- 238000012216 screening Methods 0.000 claims abstract description 13
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims abstract description 12
- 229950010131 puromycin Drugs 0.000 claims abstract description 6
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- 208000015181 infectious disease Diseases 0.000 claims description 13
- 239000012634 fragment Substances 0.000 claims description 12
- 210000004556 brain Anatomy 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 238000004321 preservation Methods 0.000 claims description 10
- 206010027476 Metastases Diseases 0.000 claims description 8
- 230000009401 metastasis Effects 0.000 claims description 8
- 239000013598 vector Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 238000011160 research Methods 0.000 claims description 6
- 230000014509 gene expression Effects 0.000 claims description 5
- 230000007246 mechanism Effects 0.000 claims description 5
- 238000011161 development Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 230000004927 fusion Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000013612 plasmid Substances 0.000 abstract description 17
- 102100025942 Chemokine-like protein TAFA-5 Human genes 0.000 abstract description 7
- 238000012258 culturing Methods 0.000 abstract description 7
- 241000713666 Lentivirus Species 0.000 abstract description 4
- 102000003960 Ligases Human genes 0.000 abstract description 4
- 108090000364 Ligases Proteins 0.000 abstract description 4
- 239000012980 RPMI-1640 medium Substances 0.000 abstract description 4
- 238000004806 packaging method and process Methods 0.000 abstract description 4
- 229920006395 saturated elastomer Polymers 0.000 abstract description 4
- 108700026220 vif Genes Proteins 0.000 abstract description 3
- 238000010186 staining Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 74
- 239000000243 solution Substances 0.000 description 23
- 238000006243 chemical reaction Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 206010059866 Drug resistance Diseases 0.000 description 3
- 108091092584 GDNA Proteins 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000003208 gene overexpression Methods 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 3
- 229960004359 iodixanol Drugs 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108010012236 Chemokines Proteins 0.000 description 2
- 102000019034 Chemokines Human genes 0.000 description 2
- 238000010802 RNA extraction kit Methods 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 206010041047 Slow virus infection Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 238000011369 optimal treatment Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- 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
- C12N5/0693—Tumour cells; Cancer cells
-
- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/521—Chemokines
-
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- 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
- C12N15/86—Viral vectors
-
- 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
- C12N5/0688—Cells from the lungs or the respiratory tract
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- 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
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Pulmonology (AREA)
- Food Science & Technology (AREA)
- Oncology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application relates to the technical field of medical molecular biology, and particularly discloses a lung cancer cell strain for stably expressing FAM19A5 genes, a construction method and application thereof. The application prepares a lentivirus solution by chemically synthesizing FAM19A5 gene and transferring the FAM19A5 gene into 293T cells to complete virus packaging after connecting lentivirus plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro with T4 ligase. Subsequently, RPMI1640 medium containing 10% FBS was used at 37℃with 5% CO 2 Culturing human lung cancer cell H460 in saturated humidity incubator, and adding slow virus solution when the cells grow to a certain amountAfter 24h of staining, puromycin is used for screening to obtain a lung cancer cell strain which stably expresses the FAM19A5 gene.
Description
Technical Field
The application relates to the technical field of medical molecular biology, in particular to a lung cancer cell strain for stably expressing FAM19A5 genes, a construction method and application thereof.
Background
Lung cancer is one of the common malignant tumors worldwide, and its morbidity and mortality rate are high. The main reason for this research analysis is that the disease is difficult to diagnose early and its high metastatic nature. Among pathological forms of lung cancer, non-small cell lung cancer (non-small cell lung cancer, NSCLC) accounts for about 85% of lung cancer, and the disease has early stage concealment, and patients have advanced stage and middle stage at the time of diagnosis, miss the optimal treatment period and have low survival rate. For this reason, early screening for lung cancer is particularly important.
In recent years, molecular diagnosis technology has become a research hotspot for cancer screening due to its noninvasive and rapid diagnosis characteristics. TAFA chemokine family member 5 (FAM 19 A5) is composed of 5 highly homologous genes encoding small secreted proteins, expressed primarily in specific areas of the brain, and is thought to act as a brain-specific chemokine or neurofactor, as a regulator of immune and neural cells. The study shows that the expression of FAM19A5 in lung cancer patients is remarkably abnormal, but the action mechanism of the FAM19A5 is still yet to be studied intensively.
Disclosure of Invention
The application provides a lung cancer cell strain for stably expressing FAM19A5 genes, a construction method and application thereof, and provides a basic and powerful research tool for further researching the FAM19A5 genes in the occurrence, development, treatment and subsequent drug resistance processes of lung cancer.
In a first aspect, the present application provides a lung cancer cell line stably expressing the FAM19A5 gene.
The application prepares the slow disease by chemically synthesizing FAM19A5 gene and connecting slow virus plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro through T4 ligase and transferring into 293T cells to complete virus packagingA toxic solution. Subsequently, RPMI1640 medium containing 10% FBS was used at 37℃with 5% CO 2 Culturing human lung cancer cells H460 in a saturated humidity incubator, adding a slow virus solution to infect for 24 hours when the cells grow to a certain quantity, and screening by puromycin to obtain a lung cancer cell strain stably expressing FAM19A5 genes.
The lung cancer cell strain is preserved in 2023 and 4 months and 20 days, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation address is North Chenxi Lu No.1 and No.3 in the Korean area of Beijing city, and the preservation number is CGMCC No. 45547. The classification is named as target gene over-expression lung cancer cell strain.
The lung cancer cell strain can stably express FAM19A5-GFP fusion genes.
In a second aspect, the present application provides the use of the above lung cancer cell line stably expressing the FAM19A5 gene.
In a specific embodiment, the lung cancer cell line stably expressing the FAM19A5 gene is used for researching the occurrence, development and drug resistance processes and mechanisms of lung cancer.
In a specific embodiment, the above lung cancer cell line stably expressing the FAM19A5 gene is used as a cell model for early screening of lung cancer and/or research of brain metastasis mechanism of lung cancer.
In a specific embodiment, the use of a lung cancer cell line stably expressing the FAM19A5 gene as described above for screening or predicting molecular markers for early screening and/or brain metastasis of lung cancer.
In a specific embodiment, the use of a lung cancer cell line stably expressing the FAM19A5 gene as described above for the manufacture of a medicament for preventing or treating early lung cancer and/or lung cancer brain metastasis.
In a specific embodiment, the use of a lung cancer cell line stably expressing the FAM19A5 gene as described above for preparing a kit for assessing and detecting early lung cancer and/or lung cancer brain metastasis risk.
In a third aspect, the present application provides a method for constructing the above lung cancer cell line stably expressing the FAM19A5 gene.
Synthesizing a sequence fragment of the FAM19A5 gene chemically, cloning the sequence fragment onto a vector PCDH, and transferring the sequence fragment into 293T cells to prepare a lentiviral solution; and (3) infecting the H460 cells for 24 hours by using the slow virus solution, and screening by using puromycin to obtain a lung cancer cell strain stably expressing the FAM19A5 gene.
In a specific embodiment, the multiplicity of infection (MOI) with optimal infection effect is 10 when the H460 cells are infected with the lentiviral solution.
In a specific embodiment, the infection effect is 30% when the H460 cells are infected with the lentiviral solution for 24H.
In a specific embodiment, when the lentiviral solution is used to infect the H460 cells for 24 hours, the target gene FAM19A5 in the lung cancer cell line stably expressing the FAM19A5 gene is obtained in an amount 1600 times that of the control group.
In a fourth aspect, the present application also provides a primer set for amplifying the FAM19A5 gene in the lung cancer cell strain.
Specifically, the nucleotide sequence of primer F is shown as SEQ ID NO. 3; the nucleotide sequence of primer R is shown as SEQ ID NO. 4.
In summary, the application has the following beneficial effects:
according to the application, through a slow virus infection technology, a PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro vector is utilized to infect H460 cells, and a lung cancer cell strain for stably expressing FAM19A5 genes is established. The cell is detected by using the techniques of cell fluorescence and fluorescence quantitative PCR to prove that the cell strain is successfully constructed. The constructed lung cancer cell strain can stably express the FAM19A5 gene, and provides a basic and powerful research tool for further researching the FAM19A5 gene in the processes of occurrence, development, treatment and subsequent drug resistance of lung cancer.
Drawings
FIG. 1 is an identification chart of recombinant plasmids after double cleavage.
FIG. 2 is a block diagram of the lentiviral master plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro.
FIG. 3 is a graph showing the results of a cell infection experiment (wherein FIG. 3A is a cell map under normal light, and FIG. 3B is a cell fluorescence map after the same field of view of the cell map under normal light is irradiated with a laser having a wavelength of 450 to 490 nm).
Fig. 4 is a graph of the results of a fluorescent quantitative PCR reaction (relative mRNA expression level in ordinate; "indicates p < 0.0001).
Description of the embodiments
The application prepares a lentivirus solution by chemically synthesizing FAM19A5 gene and transferring the FAM19A5 gene into 293T cells to complete virus packaging after connecting lentivirus plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro with T4 ligase. Subsequently, RPMI1640 medium containing 10% FBS was used at 37℃with 5% CO 2 Culturing human lung cancer cells H460 in a saturated humidity incubator, adding a slow virus solution to infect for 24 hours when the cells grow to a certain quantity, and screening by puromycin to obtain a lung cancer cell strain stably expressing FAM19A5 genes.
The lung cancer cell strain is preserved in 2023 and 4 months and 20 days, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation address is North Chenxi Lu No.1 and No.3 in the Korean area of Beijing city, and the preservation number is CGMCC No. 45547. The classification is named as target gene over-expression lung cancer cell strain.
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present application.
The present application will be described in further detail with reference to examples and drawings.
Examples
The present example provides a lung cancer cell line stably expressing the FAM19A5 gene.
The construction method of the lung cancer cell strain specifically comprises the following steps:
(1) Synthesis of FAM19A5 sequence fragment
In the database of national center for biological information gene (NCBI), the full-length sequence of the gene of FAM19A5 was obtained as the complete sequence of the target gene. In addition, restriction enzyme cleavage sites with NheI and NotI were designed at the 5-and 3-ends of the full-length sequence of the gene, respectively, and the gene synthesis was carried out by the company Shanghai, inc. of Biotechnology. The target gene was mounted on a plasmid (pUC 57) to obtain a recombinant plasmid.
The recombinant plasmid is transformed by using escherichia coli (competent cells), and the recombinant plasmid is plated and cultured on a solid medium (formula: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride, 15g/L of agar powder and 1% of Ampicillin) containing Ampicillin to obtain a strain containing a target gene. After overnight culture, single colony is selected and inoculated into a liquid culture medium (formula: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of sodium chloride) for culture, recombinant plasmid is extracted after overnight culture, restriction enzyme NheI and NotI are used for double enzyme digestion, target gene fragments are intercepted, and gel is cut and recovered.
The identification chart of the recombinant plasmid after double enzyme digestion is shown in figure 1. As shown in FIG. 1, the size of the target gene fragment is 399bp, and the target gene fragment is a nucleotide sequence shown as SEQ ID NO.1 through sequencing detection.
(2) Cloning of the Gene of interest into the vector PCDH
The target gene fragment is connected with a lentiviral main plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro (namely, the nucleotide sequence shown as SEQ ID NO.2 and the structure diagram shown as figure 2) by T4 ligase to obtain the vector with the connected structure.
Adding 10 μl of the carrier with the connection completion into 50 μl of competent cells (TOP 10) suspension on ice, standing on ice for 30min after mixing, activating competent cells in a water bath at 42 ℃ for 90s after transformation, plating on solid medium containing ampicillin for culturing to screen strains containing target genes, selecting single colony after overnight culture for inoculating to liquid medium for culturing, extracting plasmids thereof after overnight culture, and obtaining the carrier with target genes.
(3) Preparation of lentiviral solution with target Gene and cell infection experiment
To construct a cell over-expression gene model, 293T cells were first used to prepare lentiviral solutions with the target genes.
The method comprises the following steps: 293T cells were plated on a culture plate having a diameter of 10cm, and after the cells grew to about 8 confluence, vectors carrying the target gene and auxiliary vectors including envelope plasmid (envelop plasmid) and packaging plasmid (packaging plasmid) were co-transfected with lipid nanoparticle (Lipofectamine 3000) to obtain a cell culture solution. After 48h, fluorescence was observed with a fluorescence inversion microscope, and the transfection efficiency was analyzed.
Cell culture broth transfected for 48h was collected and 0.2. Mu.l nuclease was added for standing, reacted at 37℃for 30min, centrifuged at 4℃for 3,000 Xg to remove cells and protein fragments, and the supernatant was transferred to a new centrifuge tube to obtain a virus-containing supernatant. Preparing 15%, 25%, 40% and 60% iodixanol injection and a special ultra-high speed centrifuge tube, respectively adding iodixanol injection with different concentrations into the bottom of the ultra-high speed centrifuge tube by using a needle head (the flow rate is needed to be noted when adding, the mixing is avoided, the generation of bubbles is avoided), carefully adding the supernatant containing viruses slowly into the uppermost layers of the iodixanol injection with different gradients, balancing by using a microbalance, sealing the centrifuge tube by using a sealing tool, and centrifuging for 1h at 18 ℃ and 350,000Xg. After centrifugation, the cap of the tube was pierced with a needle to aspirate the virus solution at the layering site of the lentiviral particles, the aspirated virus solution was placed on ice, the virus was purified using an ultrafiltration tube, and PBS-MK (component: 137mM NaCl,5.2mM KCl,8 mM Na) 2 HPO 4 ，2mM KH 2 PO 4 , 1 mM MgCl 2 ) Recovering to obtain slow virus solution, and storing at-80 ℃.
Cell infection experiments: RPMI1640 medium containing 10% FBS was used at 37℃with 5% CO 2 Culturing H460 cells in saturated humidity incubator, spreading H460 cells into 24-well plate when cells grow to a certain amount, adding slow virus solution into each well with MOI gradient of 0, 10, 20, 40, 80, respectively, performing fluorescent cell count after 24 hr to find out infection complex (MOI) with optimal infection effect as 10, performing infection again in 6cm diameter culture plate with the virus concentration for 24 hr, and completely culturing with puromycin containing 6 μg/mlThe culture medium is screened for 24 hours, washed twice by PBS and placed in a 5% CO2 incubator for growth, and the screened cells are obtained.
And carrying out cell infection experiments on the screened cells. The results of the cell infection experiment are shown in FIG. 3. Fig. 3A is a cell map under normal light, and fig. 3B is a cell fluorescence map after irradiation of the same field of view of the cell map under normal light with laser light having a wavelength of 450 to 490 nm.
As can be seen from FIG. 3, the cells in FIG. 3B emit green fluorescence, which represents that the cell line constructed by the application can stably express the fusion protein with the target gene FAM19A5 and the green fluorescent protein.
(4) Detection of expression of target gene by fluorescent quantitative PCR method
And taking out the screened cells, sucking the complete culture medium, and dissolving the cells by using a Lysis Buffer containing 1% of 2-mercaptoethanol, wherein the obtained cell lysate is used for extracting total RNA according to the standard operation flow of Biospin Total RNA Extraction Kit (Biospin total RNA extraction kit, bori technology Co., hangzhou).
Removing gDNA: mu.g of total RNA was mixed with 2. Mu.l of 5 XgDNA Buffer and the volume was homogenized with ddH 2 O was added to 10. Mu.l, and the mixture was incubated at 42℃for 3 minutes to obtain a reaction solution from which gDNA was removed, and the reaction solution was placed on ice.
Reverse transcription: mu.l of 10 XKing RT Buffer, 1. Mu. l FastKing RT Enzyme Mix, 2. Mu.l of FQ-RQ Primer Mix were mixed and the volume was mixed with ddH 2 Supplementing O to 10ul, mixing with the reaction solution after gDNA removal, incubating at 42 ℃ for 15min, incubating at 95 ℃ for 3min, and placing on ice to obtain cDNA product.
Fluorescent quantitative PCR reaction: according to the instruction manual (instruction manual of qPCR premix reagent, bai Meng' S medicine, cat# BM 60304S), 10 mu l Taq SYBR Green qPCR Premix, 1 mu l cDNA, 0.5 mu l primer F, 0.5 mu l primer R, ddH was used for volume 2 O was fed to 20ul, the reaction conditions were pre-denatured at 95℃for 5min, denatured at 95℃for 20s in cycles, annealed at 60℃for 15s, 40 cycles were set, and fluorescence signals were collected at the end of extension of each cycle.
The results of the fluorescent quantitative PCR reaction are shown in FIG. 4. As shown in FIG. 4, the expression level of the target gene FAM19A5 is 1600 times that of the control group, and the obtained lung cancer cell strain can stably and highly express the FAM19A5-GFP fusion gene, which indicates that the construction of the required lung cancer cell strain is successful.
The lung cancer cell strain is preserved in 2023 and 4 months for 20 days, wherein the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation address is North Chengxi Lu No.1 and No.3 in the Korean region of Beijing city, and the preservation number is CGMCC No. 45547. The classification is named as target gene over-expression lung cancer cell strain.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and are not limiting; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims (10)
1. A lung cancer cell strain for stably expressing FAM19A5 gene is characterized in that the preservation number of the lung cancer cell strain is CGMCC NO. 45547.
2. The lung cancer cell line of claim 1, wherein the lung cancer cell line stably expresses a FAM19A5-GFP fusion gene.
3. Use of a lung cancer cell line stably expressing the FAM19A5 gene of claim 1 in the study of lung cancer development, progression and resistance processes and mechanisms.
4. Use of a lung cancer cell line stably expressing the FAM19A5 gene of claim 1 as a cell model for early screening of lung cancer and/or research of brain metastasis mechanism of lung cancer.
5. Use of a lung cancer cell line stably expressing the FAM19A5 gene of claim 1 in screening or predicting molecular markers for early screening and/or brain metastasis of lung cancer.
6. Use of a lung cancer cell line stably expressing the FAM19A5 gene of claim 1 in the manufacture of a medicament for preventing or treating early lung cancer and/or lung cancer brain metastasis.
7. Use of a lung cancer cell line stably expressing the FAM19A5 gene according to claim 1 in the preparation of a kit for assessing and detecting early lung cancer and/or lung cancer brain metastasis risk.
8. A method of constructing a lung cancer cell line stably expressing the FAM19A5 gene of claim 1, comprising:
synthesizing a sequence fragment of the FAM19A5 gene chemically, cloning the sequence fragment onto a vector PCDH, and transferring the sequence fragment into 293T cells to prepare a lentiviral solution; and (3) infecting the H460 cells for 24 hours by using the slow virus solution, and screening by using puromycin to obtain a lung cancer cell strain stably expressing the FAM19A5 gene.
9. The method according to claim 8, wherein when the H460 cells are infected with the lentiviral solution, the optimal multiplicity of infection (MOI) is 10;
preferably, when the H460 cells are infected with the lentiviral solution for 24 hours, the infection effect is 30%;
preferably, when the lentiviral solution is used for infecting the H460 cells for 24 hours, the obtained lung cancer cell strain stably expressing the FAM19A5 gene has the target gene FAM19A5 of about 1600 times of the expression level of a control group.
10. A primer set for amplifying the FAM19A5 gene in the lung cancer cell line of claim 1, wherein the nucleotide sequence of primer F is shown in SEQ ID No. 3; the nucleotide sequence of primer R is shown as SEQ ID NO. 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310895631.3A CN117106725A (en) | 2023-07-20 | 2023-07-20 | Lung cancer cell strain for stably expressing FAM19A5 gene and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310895631.3A CN117106725A (en) | 2023-07-20 | 2023-07-20 | Lung cancer cell strain for stably expressing FAM19A5 gene and construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117106725A true CN117106725A (en) | 2023-11-24 |
Family
ID=88799272
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310895631.3A Pending CN117106725A (en) | 2023-07-20 | 2023-07-20 | Lung cancer cell strain for stably expressing FAM19A5 gene and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117106725A (en) |
-
2023
- 2023-07-20 CN CN202310895631.3A patent/CN117106725A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105524924B (en) | Cyclic RNA circ-ZKSCAN1 use | |
| CN107502608A (en) | Construction method and application for sgRNA, ALDH2 gene delection cell line for knocking out people's ALDH2 genes | |
| CN105176981A (en) | DNA (deoxyribonucleic acid) sequence used for circular RNA (ribonucleic acid) expression, expression vector and applications of DNA sequence and expression vector | |
| CN111172114B (en) | Humanized pre-intestinal cancer lesion immortalized epithelial cell line, construction method and application thereof | |
| CN102827254B (en) | Cell penetrating peptide hPP10 and use thereof | |
| CN107663539A (en) | Circular rna circ PTGR1 purposes | |
| CN112029803A (en) | Lentiviral overexpression viral vector and preparation method and application thereof | |
| CN114432452B (en) | Medicine for resisting esophageal squamous cell carcinoma | |
| CN112143704B (en) | Cell strain capable of indicating ACE2 expression level and construction method and application thereof | |
| US12013390B2 (en) | EBIV nucleic acid composition and application thereof | |
| CN105039342A (en) | siRNA capable of inhibiting MAT2A genetic expression and application of siRNA | |
| CN113462655A (en) | Reagent and method for detecting novel coronavirus | |
| CN117106725A (en) | Lung cancer cell strain for stably expressing FAM19A5 gene and construction method and application thereof | |
| CN116694634A (en) | Nucleic acid molecule for improving protein expression and application of nucleic acid molecule in mRNA vaccine | |
| CN110804621A (en) | Preparation method of escherichia coli extracellular vesicle with endogenous high-expression miRNA (micro ribonucleic acid) | |
| CN116875558A (en) | Lung cancer cell strain for stably expressing VGLL3 gene, construction method and application thereof | |
| CN116987671A (en) | Lung cancer cell strain for stably expressing MFGE8 gene, construction method and application thereof | |
| CN112226460B (en) | Screening and breeding method of cell line for stably expressing porcine host restriction factor A3Z2 molecule and application thereof | |
| CN115779084A (en) | Application of preparation for activating pig TUSC1 gene expression in preparation of pig pseudorabies virus infection resisting medicine | |
| CN114457158A (en) | Application of Hsa _ circ _0006867 serving as esophageal cancer molecular target in preparation of drugs and kits | |
| CN110904056B (en) | Infectious bronchitis virus rH120-YZS1 delta 5a and construction method and application thereof | |
| CN110117585B (en) | Bacterial RNase E truncation and application thereof | |
| CN109112129B (en) | Specific sgRNA for targeted knockout of human OC-2 gene and application | |
| CN112961841A (en) | Packaging method for rapidly obtaining high-titer lentiviruses | |
| CN112063620A (en) | shRNA for inhibiting expression of porcine epidemic diarrhea virus M gene |
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 |