Method for building overexpression MGST1 gene human lung adenocarcinoma cells and application thereof

[0036] Embodiment 1: Construction of recombinant eukaryotic expression vector pcDNA3-MGST1

[0037] 1. Cloning of MGST1 target gene.

[0038] (1) Design primers for MGST1 overexpression.

[0039] According to the coding sequence of Gene Bank, use primer premier 5 software to design the upstream primer of MGST1 overexpression primer: 5'-CCCAAGCTTGGCTTGCTGCTTCCTCCTC-3' (containing Hind III restriction enzyme cutting site CCCAAGCTT),

[0040] And downstream primer: 5'-CGGGGTACCCCCTCTGCTCCCCTCCTACCT-3' (contains Kpn I restriction site CGGGGTACC)

[0041] (2) The MGST1 target gene was synthesized by PCR.

[0042] The conditions and system of the PCR reaction were set as follows:

[0043] Reaction conditions: pre-denaturation: 98°C 10s; denaturation: 98°C 10s, annealing: 60°C 15s,

[0044] Extension: 72°C for 45s, a total of 33 cycles; store at 4°C.

[0045] reaction system HS DNA Polymerase (Takara)

[0046] The substance of the reaction system

corresponding volume

[0083] Embodiment 2: Construction of human lung adenocarcinoma cells stably overexpressing MGST1 gene

[0084] 1. Cell culture.

[0085] Use RPMI-1640 Medium medium (purchased from GIBCO) containing 10% fetal bovine serum at 37°C, 5% CO 2 The human lung adenocarcinoma cell line SPC-A-1 (purchased from ATCC, USA) was cultured in a cell culture incubator until the culture flask was confluent.

[0086] 2. Eukaryotic expression plasmid pcDNA3-MGST1 was transfected into human lung adenocarcinoma SPC-A-1 cell line.

[0087] Step1: after the SPC-A-1 cells in the logarithmic growth phase were digested with trypsin, a single cell suspension was prepared, and the cells were counted.

[0088] Step2: Seed SPC-A-1 cells in a 6-well plate (approximately 5.0×10 per well 5 Cells) were used for transfection after 12-24 h of inoculation, and were divided into liposome negative control group labeled Lip2000, pcDNA3 plasmid transfection control group (pcDNA3) and pcDNA3-MGST1 recombinant plasmid

[0128] Specific implementation case 3: Detection of MGST1 target gene expression by Western bolt

[0129] 1. Total protein extraction (operated on ice).

[0130] Collect the cells in a centrifuge tube and centrifuge at 2000rpm for 4min, wash twice with pre-cooled PBS, add an appropriate volume of RIPA (10μl / ml protease inhibitor and phosphatase inhibitor) according to the cell volume, mix by pipetting, and seal the EP tube After the membrane was sealed, put it in a cell disruption ultrasonic instrument at 4°C, 500watt, lyse for 20min; centrifuge at 14000g, 20min, to precipitate cell debris, extract the supernatant and transfer it to an EP tube, and store it at -80°C for later use.

[0131] 2. BCA protein quantification method was used to determine the protein content.

[0132] Dilute the 2mg / ml protein standard stock solution in multiple ratios, and the diluted protein standard concentrations are: 2mg / ml, 1mg / ml, 0.5mg / ml, 0.25mg / ml, 0.125mg / ml; in the BCA kit Sol