Application of human EIF3A gene and related drugs
A gene and drug technology, applied in the field of the use of human EIF3A gene and related drugs, can solve the problem of few reports on EIF3A
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Embodiment 1
[0084] Example 1 Preparation of RNAi lentivirus against human EIF3A gene
[0085] 1. Screening for effective siRNA targets against the human EIF3A gene
[0086] Retrieve EIF3A (NM_00375) gene information from Genbank; design effective siRNA targets for EIF3A gene. Table 1 lists one of the effective siRNA target sequences for the EIF3A gene.
[0087] Table 1 is targeted at the siRNA target sequence of human EIF3A gene
[0088]
[0089] 2. Preparation of lentiviral vector
[0090] Aiming at the siRNA target (taking SEQIDNO: 1 as an example) synthetic double-stranded DNA Oligo sequence (table 2) containing AgeI and EcoRI restriction endonuclease at both ends; Acting on the pGCSIL-GFP vector ( Provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and identify the digested fragments by agarose gel electrophoresis.
[0091] Table 2 Double-stranded DNA Oligo with sticky ends containing AgeI and EcoRI restriction sites at both ends
[009...
Embodiment 2
[0110] Example 2 EIF3A expression abundance detection in 5 strains of colon cancer cells
[0111] Five colon cancer cell lines HCT116, RKO, SW480, SW620, and LOVO in the logarithmic growth phase were digested with trypsin to make cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, RKO: 10) value, an appropriate amount of the virus prepared in Example 1 was added, the culture medium was replaced after 24 hours of cultivation, and the cells were collected after the infection time reached 4 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. The specific method is:
[0112] 1) Collect the cells, centrifuge at 1000rpm for 5min, remove the supernatant, add 1ml Trizol to the cell pellet, pipette quickly and let stand at room temperature for 5min, then transfer to a new 1.5mleppendorf tube;
[0113...
Embodiment 3
[0129] Example 3 Real-time fluorescent quantitative RT-PCR method to detect the silencing efficiency of EIF3A gene
[0130] Colon cancer RKO cells in the logarithmic growth phase were digested with trypsin to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, RKO: 10) value, an appropriate amount of the virus prepared in Example 1 was added, the culture medium was replaced after 24 hours of cultivation, and the cells were collected after the infection time reached 4 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. Then, according to the M-MLV instruction manual of Promega Company, the RNA was reverse-transcribed to obtain cDNA (see Table 7 for the reverse transcription reaction system, react at 42°C for 1 hour, and then bathe in a water bath at 70°C for 10 minutes to inactivate the ...
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