Application of human PSMD7 gene and related product
A gene and application technology, applied in the application of human PSMD7 gene and related products, can solve the problems of PSMD7 gene and other problems
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Embodiment 1
[0119] Example 1 Preparation of RNAi lentivirus against human PSMD7 gene
[0120] 1. Screening for effective siRNA targets against the human PSMD7 gene
[0121] Retrieve PSMD7 (NM_002811) gene information from Genbank; design effective siRNA targets for PSMD7 gene. Table 1-1 lists the screened effective siRNA target sequences against the PSMD7 gene.
[0122] Table 1-1 is targeted at the siRNA target sequence of human PSMD7 gene
[0123] SEQ ID NO TargetSeq(5'-3') 1 TGAGGAAGTTGGAGTTGAA
[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 Medical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragments.
[0126] Table 1-2 Double-stranded DNA Oligo with sticky ends ...
Embodiment 2
[0145] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0146] Human pancreatic cancer ASPC-1 cells and PANC-1 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 value (MOI, ASPC-1:20, PANC-1:20), add an appropriate amount of the lentivirus prepared in Example 1, replace the medium after 24 hours of cultivation, and collect the cells after the infection time reaches 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 ...
Embodiment 3
[0153] Example 3 Western Blot detection target reduces PSMD7 gene protein level expression
[0154] 1. Extraction of total cell protein
[0155] (1) Infect the target cells (human pancreatic cancer ASPC-1 cells, pancreatic cancer PANC-1 cells, MOI, ASPC-1:20, PANC-1) with the control virus and the RNAi virus targeting PSMD7 interference target respectively :20). Cell samples were received and washed twice with PBS. Take an appropriate amount of RIPA lysate, add PMSF within a few minutes before use, so that the final concentration of PMSF is 1mM. (Use RIPA lysate, link to instructions: http: / / www.beyotime.com / ripa-lysis-bufferm.htm)
[0156] (2) Add an appropriate amount of RIPA lysate, and lyse on ice for 10-15 minutes. Scrape the cells and transfer them into a new EP tube, and then ultrasonically disrupt the cells (20 times at 40W, 1 s each time, 2 s apart).
[0157] (3) 4°C, 12000g, centrifuge for 15min, take the supernatant and measure the protein concentration by BCA ...
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