Application of human DUS4L gene and related product of human DUS4L gene
A technology of genes and uses, which can be used in medical preparations containing active ingredients, genetic engineering, plant genetic improvement, etc., and can solve problems such as lack of DUS4L gene
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Embodiment 1
[0103] Example 1 Preparation of RNAi lentivirus against human DUS4L gene
[0104] 1. Screening for effective siRNA targets against the human DUS4L gene
[0105] Retrieve DUS4L (NM_181581) gene information from Genbank; design effective siRNA targets for DUS4L gene. Table 1-1 lists the screened effective siRNA target sequences against the DUS4L gene.
[0106] Table 1-1 is targeted at the siRNA target sequence of human DUS4L gene
[0107] SEQ ID NO TargetSeq(5'-3') 1 ACATCAGCAATCATAGATT
[0108] 2. Preparation of lentiviral vector
[0109] 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.
[0110] Table 1-2 Double-stranded DNA Oligo with sticky ends ...
Embodiment 2
[0129] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0130] Human lung cancer A549 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, A549:10), an appropriate amount of the lentivirus prepared in Example 1 was added, and the culture medium was replaced after 24 hours of culture. 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 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 transcripta...
Embodiment 3
[0137] Embodiment 3Western Blotting method detects the silencing efficiency of gene
[0138] 1. Extraction of total cell protein
[0139] (1) Infect the target cells (A549 cells) with the control virus and the RNAi virus targeting the DUS4L interference target respectively. 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, instruction link: http: / / www.beyotime.com / ripa- analysis-bufferm.htm )
[0140] (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).
[0141] (3) 4°C, 12000g, centrifuge for 15min, take the supernatant to determine the protein concentration by BCA method (link to BCA Protein Assay Kit manual: http: / / www.beyotime.com / p0010s.htm)
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