Application of human CFAP65 gene and related products
A gene and application technology, applied in the application of human CFAP65 gene and related products, to achieve the effect of inhibiting proliferation rate, inhibiting proliferation and inhibiting the growth of gastric cancer
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Embodiment 1
[0091] Example 1 Preparation of RNAi lentivirus for human CFAP65 gene
[0092] 1. Screening for effective siRNA targets against the human CFAP65 gene
[0093] Retrieve CFAP65 (NM_194302) gene information from Genbank; design effective siRNA targets for CFAP65 gene. Table 1-1 lists the screened effective siRNA target sequences against CFAP65 gene.
[0094] Table 1-1 siRNA target sequence targeting human CFAP65 gene
[0095] SEQ ID NO TargetSeq(5'-3') 1 CCTTGAAACTCCAGAAGAT
[0096] 2. Preparation of lentiviral vector
[0097] 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.
[0098] Table 1-2 Double-stranded DNA Oligo with sticky ends containing Age...
Embodiment 2
[0116] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0117] Human gastric cancer AGS 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, AGS: 10), 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 inactivate the reverse transcript...
Embodiment 3
[0124] Example 3 Detection of proliferation ability of tumor cells infected with CFAP65-shRNA lentivirus
[0125] Human gastric cancer AGS 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, AGS: 10), 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 of 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 1500 cells / 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 plate was detected and read o...
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