CN117126892A - Method for constructing hypopharyngeal carcinoma cell line capable of stably and highly expressing MYCT1 by using lentivirus - Google Patents

Method for constructing hypopharyngeal carcinoma cell line capable of stably and highly expressing MYCT1 by using lentivirus Download PDF

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CN117126892A
CN117126892A CN202311398086.3A CN202311398086A CN117126892A CN 117126892 A CN117126892 A CN 117126892A CN 202311398086 A CN202311398086 A CN 202311398086A CN 117126892 A CN117126892 A CN 117126892A
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董洋
孙媛媛
富伟能
张翼
姜鹏飞
刘为敏
刘冬民
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Shenzhen Huarui Model Organism Technology Co ltd
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Abstract

The invention relates to a method for constructing a hypopharyngeal carcinoma cell line with stable high expression of MYCT1 by using lentivirus, belonging to the technical field of DNA recombination. The invention provides a method for constructing a hypopharyngeal carcinoma cell line with stable and high expression MYCT1 by using slow virus, which aims to solve the problem that plasmid transfection cannot construct the stable and high expression MYCT1 cell line, and comprises the steps of obtaining the whole length of MYCT1 genes, constructing a pLVX-AcGFP1-C1-MYCT1 slow virus plasmid, packaging slow virus, infecting hypopharyngeal carcinoma cells by slow virus, screening positive transformants, and amplifying to obtain the hypopharyngeal carcinoma cell line with stable and high expression MYCT1. The hypopharynx cancer cell line for stably and highly expressing MYCT1 constructed by the invention obviously improves the biological function and the molecular mechanism research accuracy of MYCT1 genes in hypopharynx cancer cells.

Description

Method for constructing hypopharyngeal carcinoma cell line capable of stably and highly expressing MYCT1 by using lentivirus
Technical Field
The invention belongs to the technical field of DNA recombination, and particularly relates to a method for constructing a stable and high-expression MYCT1 hypopharyngeal carcinoma cell line by using slow viruses.
Background
MYCT1 (Mye target 1), a c-Myc target gene in laryngeal cancer cells, has been named as a novel gene cloned in laryngeal cancer cells, and has been attracting attention as a downstream target gene of transcription factor c-Myc, which plays a role in the progress of tumor development. Earlier studies have found that MYCT1 is involved in the life process and functional maintenance of normal cells, but that there is abnormal expression in many types of tumors, involved in the occurrence and progression of the tumor. Studies have demonstrated that MYCT1 shows an oncogene inhibiting effect in laryngeal cancer, liver cancer and esophageal cancer, but the exact molecular mechanism has not yet been elucidated. It is also considered that it shows different functions in gastric cancer and acute myeloid leukemia.
The biological function and molecular mechanism by which the MYCT1 gene plays in hypopharynx cancer is still unclear. The research strategy mainly comprises knocking down and over-expressing MYCT1 genes in a cell line, but common plasmid transfection is only a transient effect, so that the biological functions of the MYCT1 genes are difficult to clearly and accurately reveal, and in-vivo function research cannot be performed.
Disclosure of Invention
The invention provides a method for constructing a hypopharynx cancer cell line stably and highly expressing MYCT1 by using slow viruses, which aims to solve the problem that plasmid transfection cannot construct a cell line stably and highly expressing MYCT1.
The technical scheme of the invention is as follows:
a method for constructing a stable and high-expression MYCT1 hypopharynx cancer cell line by using lentivirus comprises the following steps:
step one, amplifying a full-length MYCT1 gene;
step two, constructing pLVX-AcGFP1-C1-MYCT1 lentiviral plasmid;
packaging slow viruses;
step four, infecting hypopharyngeal carcinoma cells by slow viruses;
and fifthly, screening positive transformants, and obtaining a hypopharynx cancer cell line with stable and high expression of MYCT1 after amplification.
Further, the nucleotide sequence of the MYCT1 gene in the first step is shown as SEQ ID No. 1.
Further, the step one of amplifying the full-length MYCT1 gene is to amplify cDNA of the full-length MYCT1 gene by PCR, the primer sequences for PCR amplification are shown as SEQ ID No.2 and SEQ ID No.3, the amplification conditions are 94 ℃ for 5-8 min,94 ℃ for 20-50 s,55 ℃ for 30-40 s and 72 ℃ for 1-5 min, and after 25 cycles, the primer sequences are extended for 6-12 min at 72 ℃.
And further, constructing the pLVX-AcGFP1-C1-MYCT1 lentiviral plasmid in the second step, namely connecting the MYCT1 gene fragment obtained in the first step with a linearized pLVX-AcGFP1-C1 lentiviral vector, transforming competent cells of escherichia coli by using the obtained connection product, performing dual screening of blue white spots and antibiotics on the competent cells obtained by transformation, picking white colonies, performing PCR amplification and identification, performing amplification culture on positive clones, and extracting the pLVX-AcGFP1-C1-MYCT1 lentiviral plasmid.
Further, the packaging lentivirus in the third step is to fully and uniformly mix the pLVX-AcGFP1-C1-MYCT1 lentivirus plasmid solution obtained in the second step with Package Plasmid Mix, and then to perform transfection on 293T cells after standing for 15-30 min at room temperature; and collecting culture medium supernatant after 48 hours to obtain a packaged lentivirus solution.
Further, the step four of infecting the hypopharyngeal carcinoma cells by the lentivirus is to transfer the hypopharyngeal carcinoma cells with good state into an orifice plate for culturing for 24 hours, and then adding the packaged lentivirus solution obtained in the step three, uniformly mixing and culturing for 48 hours.
Further, the transfer of the hypopharynx cancer cells in good condition to the well plate is performed by 1X10 hypopharynx cancer cells 4 Density was transferred to 24-well plates.
Further, the concentration of the packaged lentiviral solution is 1×10 7 TU/ml, add-on2 uL/well.
And step five, screening positive transformants, namely adding puromycin into the cell culture solution obtained in step four, then continuously culturing for 7 days, and collecting the obtained cells to obtain the positive transformants.
Further, the concentration of puromycin was 2. Mu.g/mL.
The invention has the beneficial effects that:
the invention realizes stable and high expression of MYCT1 genes in hypopharyngeal carcinoma cells for the first time by means of lentiviral vectors, constructs a hypopharyngeal carcinoma cell line for stable and high expression of MYCT1, can be widely applied to related researches on biological functions and molecular mechanisms of MYCT1 genes in hypopharyngeal carcinoma cells, and can obviously improve the accuracy of the researches.
Drawings
FIG. 1 is a comparative chart showing the PCR identification result of positive transformants obtained in example 1;
FIG. 2 is a fluorescence field micrograph of the lentivirus of example 1 after infection of hypopharynx cancer cells for 72 hours;
FIG. 3 is a bright field micrograph of the lentivirus of example 1 after 72h infection of hypopharynx cancer cells;
FIG. 4 is a graph showing the relative expression level of MTCT 1mRNA after stable and high expression of MYCT1 by Real-time PCR in example 1;
FIG. 5 is a graph showing the results of Real-time PCR detection of the relative expression amount of MYCT1mRNA after transient transfection of MYCT1 plasmid in comparative example 1.
Detailed Description
The following embodiments are used for further illustrating the technical scheme of the present invention, but not limited thereto, and all modifications and equivalents of the technical scheme of the present invention are included in the scope of the present invention without departing from the spirit and scope of the technical scheme of the present invention. The process equipment or apparatus not specifically noted in the following examples are all conventional equipment or apparatus in the art, and the raw materials and the like used in the examples of the present invention are commercially available unless otherwise specified; unless specifically indicated, the technical means used in the embodiments of the present invention are conventional means well known to those skilled in the art.
Examples
The present example provides a method for constructing a hypopharynx cancer cell line that stably expresses MYCT1 in high levels using lentiviruses.
The materials used in this example were pLVX-AcGFP1-C1 lentiviral vector from Biyun Tian Biotechnology Co, packaging cell 293T cell of lentivirus from Wuhan Punuo Siro Life technologies Co., ltd, plasmid extraction kit from Tiangen Biochemical technologies (Beijing); lentivirus packaging kit model L00002M was purchased from bi yun Tian biotechnology company.
The method for constructing the hypopharynx cancer cell line capable of stably and highly expressing MYCT1 by using the lentivirus specifically comprises the following steps:
step one, amplifying a full-length MYCT1 gene;
the cDNA of the full-length MYCT1 gene was amplified by PCR, and the primer sequences for PCR amplification were as follows:
F-CGCAAATGGGCGGTAGGCGTG；R-CGTCGCCGTCCAGCTCGACCAG。
the amplification system is shown in Table 1:
TABLE 1
PrimeSTAR Max Premix（2X） 25 μl
Primer F 3 μl
Primer R 3 μl
Template 100 ng
Sterilizing water Up to 50 μl
The amplification conditions are 94 ℃ for 5-8 min,94 ℃ for 20-50 s,55 ℃ for 30-40 s, and 72 ℃ for 1-5 min, and after 25 cycles, the amplification is performed for 6-12 min at 72 ℃.
Separating the PCR product from the amplified PCR product by agarose gel electrophoresis, and recovering and collecting the purified MYCT1 gene fragment by gel, wherein the nucleotide sequence is shown as follows:
TAGTGaACCGTCAGATCCGCTAGCGCTACCGGACTCAGATCTCGAGATGCGAACACAAGTATATGAGGGGTTGTGTAAAAATTATTTTTCTCTTGCTGTACTACAAAGAGATAGAATCAAACTGCTTTTTTTCGACATACTGGTTTTTCTTTCTGTTTTTCTTCTCTTTCTTCTATTTCTTGTGGATATTATGGCTAATAACACAACAAGTTTAGGGAGTCCATGGCCAGAAAACTTTTGGGAGGACCTTATCATGTCCTTCACTGTATCCATGGCAATCGGGCTGGTACTTGGAGGATTTATTTGGGCTGTGTTCATTTGTCTGTCTCGAAGAAGAAGAGCCAGTGCTCCCATCTCACAGTGGAGTTCAAGCAGGAGATCTAGGTCTTCTTACACCCACGGCCTCAACAGAACTGGATTTTACCGCCACAGTGGCTGTGAACGTCGAAGCAACCTCAGCCTGGCCAGTCTCACCTTCCAGCGACAAGCTTCCCTGGAACAAGCAAATTCCTTTCCAAGAAAATCAAGTTTCAGAGCTTCTACTTTCCATCCCTTTCTGCAATGTCCACCACTTCCTGTGGAAACTGAGAGTCAGCTGGTGACTCTCCCTTCTTCCAATATCTCTCCCACCATCAGCACTTCCCACAGTCTGAGCCGTCCTGACTACTGGTCCAGTAACAGTCTTCGAGTGGGCCTTTCAACACCGCCCCCACCTGCCTATGAGTCCATCATCAAGGCATTCCCAGATTCCACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAA。
step two, constructing pLVX-AcGFP1-C1-MYCT1 lentiviral plasmid;
and (3) connecting the purified MYCT1 gene fragment obtained in the step (I) with a linearized pLVX-AcGFP1-C1 lentiviral vector, wherein the molar ratio of the MYCT1 gene fragment to the linearized pLVX-AcGFP1-C1 lentiviral vector is 2:1, and the connecting temperature is 25 ℃ and the time is 30min.
The obtained ligation product is used for transforming competent cells DH5 alpha of the escherichia coli, the competent cells obtained by transformation are subjected to dual screening of blue white spots and antibiotics, the culture is carried out for 12 hours at 37 ℃, white colonies are picked for PCR amplification and identification, and the amplified primer sequences are shown as follows:
F-CGCAAATGGGCGGTAGGCGTG；R-CGTCGCCGTCCAGCTCGACCAG。
positive clones were cultivated in an expanded manner to extract pLVX-AcGFP1-C1-MYCT1 lentiviral plasmids.
Packaging slow viruses;
fully and uniformly mixing the pLVX-AcGFP1-C1-MYCT1 lentiviral plasmid solution obtained in the step II with Package Plasmid Mix in a lentivirus packaging kit, standing at room temperature for 15-30 min, and then transfecting 293T cells; the medium formulation for 293T cells was dmem+10% fbs with fresh medium replacement every 6h of culture. Collecting culture medium supernatant after 48h under the condition of 5% CO2 at 37 ℃, filtering by a 0.45 mu m filter, and concentrating the filtered virus solution to obtain a packaged lentivirus solution.
And performing quality control on the obtained packaged lentivirus, wherein the quality control comprises physical state detection, sterile detection and virus titer detection.
Step four, infecting hypopharyngeal carcinoma cells by slow viruses;
hypopharyngeal carcinoma cells with good status are treated with 1X10 4 Transferring the density to a 24-well plate, and culturing for 24 hours at 37 ℃; diluting the packaged lentivirus solution obtained in the step three to a virus concentration of 1×10 7 TU/ml, 2. Mu.L of virus solution was added to each well, shaking and mixing in a rice shape, and culturing at 37℃for 48 hours.
And fifthly, screening positive transformants, and obtaining a hypopharynx cancer cell line with stable and high expression of MYCT1 after amplification.
And (3) adding puromycin with the concentration of 2 mug/mL into the cell culture solution obtained in the step (IV), continuously culturing for 7 days, collecting the obtained cells as positive transformants, and amplifying the obtained positive transformants to obtain the hypopharynx cancer cell line with stable and high expression of MYCT1.
PCR was performed on the positive transformants obtained in this example, and the results were as shown in FIG. 1, FIG. 1#: negative control (ddH 2O); 2#: negative control (empty self-linked control group); 3#: positive control (GAPDH); 4#: marker is 5Kb, 3Kb, 2Kb, 1.5Kb, 1Kb, 750bp, 500bp, 250bp, 100bp from top to bottom in sequence; 5# to 7#: positive transformants. As can be seen from FIG. 1, the present example successfully constructed hypopharynx cancer cell lines containing MYCT1 gene.
The infection efficiency of lentivirus in this example was observed by fluorescence microscopy, and the results are shown in FIGS. 2 and 3. FIGS. 2 and 3 are fluorescence and bright field micrographs, respectively, of example 1 after 72h infection of hypopharynx cancer cells with lentivirus; as can be seen from FIGS. 2 and 3, the green fluorescent protein on the lentiviral vector is well expressed, and the lentiviral infection efficiency is higher.
And detecting the expression level of MYCT1 in a hypopharynx cancer cell line with stable and high expression of MYCT1 by using GAPDH as an internal reference protein through a Real-time PCR test, so as to evaluate the efficiency of high expression of MYCT1. Fig. 4 is a comparative graph of the relative expression level of MTCT 1mRNA after Real-time PCR detection of lentivirus to stably and highly express MYCT1, and the ordinate represents the relative expression level RQ value, and it can be seen from the graph that the expression level of MYCT1 in the hypopharynx cancer cell line constructed in this embodiment is significantly higher than that in the empty vector control group, so that MYCT1 can be stably and highly expressed.
Comparative example 1
The comparative example adopts transient transfection MYCT1 over-expression plasmid to construct a hypopharynx cancer cell line, and the specific method is as follows:
hypopharyngeal carcinoma cells with good status are treated with 1X10 4 Transferring the density to a 24-well plate, and culturing for 24 hours at 37 ℃; the pLVX-AcGFP1-C1-MYCT1 lentiviral plasmid obtained in the step II of example 1 was added to hypopharyngeal carcinoma cells, 2. Mu.g of plasmid was added to each well, and after mixing, the cells were cultured at 37℃for 48 hours, and total RNA of the cells was extracted, and the MYCT1 expression level was detected by PCR.
And detecting the MYCT1 expression level in the constructed hypopharynx cancer cell line by using GAPDH as an internal reference protein through Real-time PCR experiments. FIG. 5 is a comparative graph of the relative expression level of MYCT1mRNA after transient transfection of MYCT1 plasmid by Real-time PCR, and the ordinate shows the relative expression level RQ value, from which it can be seen that the expression level of MYCT1 in the hypopharynx cancer cell line constructed by transient transfection of MYCT1 over-expression plasmid is higher than that in the empty vector control group, but the relative expression level RQ value is significantly lower than that in the hypopharynx cancer cell line constructed by lentivirus of example 1 and stably and highly expressing MYCT1.

Claims (10)

1. A method for constructing a hypopharynx cancer cell line stably and highly expressing MYCT1 by using slow viruses, which is characterized by comprising the following steps:
step one, amplifying a full-length MYCT1 gene;
step two, constructing pLVX-AcGFP1-C1-MYCT1 lentiviral plasmid;
packaging slow viruses;
step four, infecting hypopharyngeal carcinoma cells by slow viruses;
and fifthly, screening positive transformants, and obtaining a hypopharynx cancer cell line with stable and high expression of MYCT1 after amplification.
2. The method for constructing a hypopharynx cancer cell line stably and highly expressing MYCT1 by using lentivirus according to claim 1, wherein the nucleotide sequence of the MYCT1 gene in step one is shown in SEQ ID No. 1.
3. The method for constructing a hypopharynx cancer cell line stably and highly expressing MYCT1 by using lentiviruses according to claim 1 or 2, wherein in the first step, the full-length MYCT1 gene is amplified by PCR, cDNA of the full-length MYCT1 gene is amplified by PCR, the amplified primer sequences are shown as SEQ ID No.2 and SEQ ID No.3, the amplification conditions are 94 ℃ for 5-8 min,94 ℃ for 20-50 s,55 ℃ for 30-40 s, and 72 ℃ for 1-5 min, and after 25 cycles, the amplification is performed for 6-12 min at 72 ℃.
4. The method for constructing a hypopharynx cancer cell line stably and highly expressing MYCT1 by using lentiviruses according to claim 3, wherein in the second step, the plasmid of pLVX-AcGFP1-C1-MYCT1 is constructed by connecting the MYCT1 gene fragment obtained in the first step with a linearized pLVX-AcGFP1-C1 lentiviral vector, transforming competent cells of escherichia coli with the obtained connection product, performing dual screening of blue and white spots and antibiotics on the competent cells obtained by the transformation, picking white colonies, performing PCR amplification and identification, performing amplification culture on positive clones, and extracting the plasmid of pLVX-AcGFP1-C1-MYCT1 lentivirus.
5. The method for constructing a hypopharynx cancer cell line stably and highly expressing MYCT1 by using lentivirus according to claim 4, wherein, step three, packaging lentivirus is to fully mix the pLVX-AcGFP1-C1-MYCT1 lentivirus plasmid solution obtained in step two with Package Plasmid Mix, and then to transfect 293T cells after standing at room temperature for 15-30 min; and collecting culture medium supernatant after 48 hours to obtain a packaged lentivirus solution.
6. The method for constructing a hypopharynx cancer cell line stably and highly expressing MYCT1 by using lentiviruses according to claim 5, wherein, in the fourth step, the hypopharynx cancer cells infected by lentiviruses are transferred into a well-conditioned hypopharynx cancer cell to be cultured for 24 hours, and the packaged lentivirus solution obtained in the third step is added, and is cultured for 48 hours after being uniformly mixed.
7. The method for constructing a stable high expression MYCT1 hypopharynx cancer cell line using lentivirus according to claim 6, wherein said transferring hypopharynx cancer cells in good condition to an orifice plate is by 1X10 4 Density was transferred to 24-well plates.
8. The method of claim 7, wherein the concentration of the packaged lentiviral solution is 1x10 7 TU/ml was added in an amount of 2 uL/well.
9. The method for constructing a hypopharynx cancer cell line stably and highly expressing MYCT1 by using lentiviruses according to claim 8, wherein, in the fifth step, the positive transformants are selected by adding puromycin to the cell culture solution obtained in the fourth step, and then culturing for 7 days, and the obtained cells are collected as positive transformants.
10. The method of claim 9, wherein the puromycin concentration is 2 μg/mL.
CN202311398086.3A 2023-10-26 2023-10-26 Method for constructing hypopharyngeal carcinoma cell line capable of stably and highly expressing MYCT1 by using lentivirus Pending CN117126892A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2962694A1 (en) * 2014-07-04 2016-01-06 Université de Lausanne Novel polypeptides and their use
CN109825527A (en) * 2018-10-23 2019-05-31 深圳市慧思基因科技有限公司 A kind of building of MYCT-1 gene Lentiviral and identification method
US20220143220A1 (en) * 2020-10-19 2022-05-12 Washington University Compositions and methods for modulating myc target protein 1

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2962694A1 (en) * 2014-07-04 2016-01-06 Université de Lausanne Novel polypeptides and their use
CN109825527A (en) * 2018-10-23 2019-05-31 深圳市慧思基因科技有限公司 A kind of building of MYCT-1 gene Lentiviral and identification method
US20220143220A1 (en) * 2020-10-19 2022-05-12 Washington University Compositions and methods for modulating myc target protein 1

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
SHUANG FU ET AL.: "Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo", 《FRONTIERS IN PHARMACOLOGY》, vol. 9, pages 2 *
张昊 等: "MYCT-1基因慢病毒表达载体的构建及鉴定", 《国际检验医学杂志》, vol. 34, no. 12, pages 1503 *
李彦仕: "探究RAF1在下咽癌淋巴转移中的表达及作用机制", 《中国博士学位论文全文数据库 医药卫生科技辑》, no. 1, pages 072 - 344 *
李艳青 等: "携带人c-Myc和EGFP基因慢病毒表达载体的构建", 《热带医学杂志》, vol. 11, no. 4, pages 369 - 371 *
毛侃琅 等: "三氯乙烯对CYP1A2高表达肝细胞株凋亡基因和癌基因的影响", 《癌变・畸变・突变》, vol. 25, no. 1, pages 7 - 12 *
赵谦 等: "N-myc下游调控基因2蛋白在喉鳞状细胞癌中的表达及对人喉表皮样癌细胞生物学特性的影响", 《安徽医药》, vol. 24, no. 10, pages 1933 - 1937 *

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