Use of human pak7 gene and related medicines
A gene and drug technology, applied in the field of human PAK7 gene and its related drugs
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Embodiment 1
[0065] Example 1: Preparation of RNAi lentivirus against human PAK7 gene
[0066] 1. Screening for effective siRNA targets against the human PAK7 gene
[0067] The PAK7 (NM_177990) gene information was retrieved from Genbank; an effective siRNA target for the PAK7 gene was designed using the design software Genechem of Shanghai Jikai Gene Chemical Technology Co., Ltd. In the coding sequence (CDS) region of the PAK7 gene, a sequence of 21 bases is obtained every other base, and Table 1 lists 50 effective siRNA target sequences for the PAK7 gene; through the Genbank database BLAST analysis to confirm that it does not interfere with any other genes.
[0068] Table 1 is targeted at the siRNA target sequence of human PAK7 gene
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[0071] Aiming at the siRNA target (taking SEQIDNO:35 as an example), the double-stranded DNA Oligo sequence (table 2) containing AgeI and EcoRI enzyme cutting site sticky ends at both ends of synthesis; Act on the pGCSIL-GFP vector...
Embodiment 2
[0089] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of PAK7 gene
[0090] Human lung cancer H1299 cells, human gastric cancer SGC7901 cells and glioma U251 cells in the logarithmic growth phase were trypsinized 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 (the MOI of H1299 and U251 cells is 10, and the MOI of SGC7901 is 20), an appropriate amount of the virus prepared in Example 1 was added, and the culture medium was replaced after 24 hours of cultivation. After the infection time reached 5 days, the cells were collected. 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 7 for the reverse transcription reaction system, reac...
Embodiment 3
[0098] Example 3: Detection of proliferation ability of tumor cells infected with PAK7shRNA lentivirus
[0099] Human lung cancer H1299 cells, gastric cancer SGC7901 cells and glioma U251 cells in 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 (the MOI of H1299 and U251 cells was 10, and the MOI of SGC7901 was 20), an appropriate amount of PAK7shRNA virus was added, and the culture medium was replaced after 24 hours of cultivation. After the infection time reached 5 days, the cells in logarithmic growth phase were collected. Experimental group of cells. 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 ...
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