Use of human GSDMB gene and related products
A gene and application technology, applied in the application of human GSDMB gene and related products, can solve the problems that GSDMB has not been reported in bladder cancer research
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Embodiment 1
[0119] Example 1 Preparation of RNAi lentivirus for human GSDMB gene
[0120] 1. Screening for effective siRNA targets against the human GSDMB gene
[0121] Retrieve GSDMB (NM_018530) gene information from Genbank; design effective siRNA targets for GSDMB gene. Table 1-1 lists the screened effective siRNA target sequences against the GSDMB gene.
[0122] Table 1-1 is targeted at the siRNA target sequence of human GSDMB gene
[0123] SEQ ID NO TargetSeq(5'-3') 1 GGAGAGTTGATAGTGAGAT
[0124] 2. Preparation of lentiviral vector
[0125] Aim at the siRNA target (take SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites at both ends; Dicer acts on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragment.
[0126] Table 1-2 Double-stranded DNA Oligo with sticky ends cont...
Embodiment 2
[0143] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0144] The human bladder cancer 5637 and T24 cells in the logarithmic growth phase were respectively trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in 6-well plates, and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, 10 for both cell lines), an appropriate amount of the lentivirus prepared in Example 1 was added, the culture medium was replaced after 24 hours of culture, and the cells were collected after the infection time reached 5 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 2-1 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 in...
Embodiment 3
[0151] Example 3 Celigo detects the proliferation ability of tumor cells infected with GSDMB-shRNA lentivirus
[0152] The human bladder cancer 5637 and T24 cells in the logarithmic growth phase were respectively trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in 6-well plates, and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, 10 for both cell lines), an appropriate amount of virus was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 5 days, the cells in each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 4 / ml), inoculate a 96-well plate at a cell density of about 2000 / well. 5 replicate wells in each group, 100 μl per well. After laying the board, place at 37°C, 5% CO 2 Incubator cultivation. From the second day after plating, the...
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