Applications of human PTP4A3 gene and related drugs thereof
A gene and drug technology, applied in the field of the use of human PTP4A3 gene and related drugs, can solve the problems such as unclear molecular mechanism
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Embodiment 1
[0087] Example 1: Preparation of RNAi lentivirus against human PTP4A3 gene
[0088] 1. Screening for effective siRNA targets against the human PTP4A3 gene
[0089] Retrieve PTP4A3 (NM_032611) gene information from Genbank; design effective siRNA targets for PTP4A3 gene. Table 1 lists 13 effective siRNA target sequences for the PTP4A3 gene.
[0090] Table 1 is targeted at the siRNA target sequence of human PTP4A3 gene
[0091]
[0092] 2. Preparation of lentiviral vector
[0093] For the siRNA target (take SEQ ID NO: 2 as an example), synthesize a double-stranded DNA Oligo sequence containing Age I and EcoR I restriction sites at both ends (Table 2); use Age I and EcoR I restriction endonucleases Act 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.
[0094] Table 2 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I ...
Embodiment 2
[0112] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of PTP4A3 gene
[0113] Human breast cancer MCF-7 cells and lung cancer H1299 cells in logarithmic growth phase were trypsinized to make cell suspension (the number of cells was about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, MCF-7:10, H1299:1) 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 5 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to Promega's M-MLV operating instructions, RNA was reverse-transcribed to obtain cDNA (see Table 6 for the reverse transcription reaction system, react at 42 °C for 1 h, and then bathe in a water bath at 70 °C for 10 min to inactivate...
Embodiment 3
[0121] Example 3: Detection of proliferation ability of tumor cells infected with PTP4A3-siRNA lentivirus
[0122] Human breast cancer MCF-7 cells and lung cancer H1299 cells in logarithmic growth phase were trypsinized to make cell suspension (the number of cells was about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, MCF-7:20, H1299:1), add an appropriate amount of virus, and replace the medium after 24 hours of culture. After the infection time reaches 5 days, collect the cells in each experimental group in the logarithmic growth phase . 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 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 once a...
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