Application of human HIST1H2BK gene and related products
A technology of HIST1H2BK and gene, which is applied in the field of application of human HIST1H2BK gene and related products, and can solve the problem of no literature report on the function of HIST1H2BK
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Embodiment 1
[0100] Example 1 Preparation of RNAi lentivirus against human HIST1H2BK gene
[0101] 1. Screening for effective siRNA targets against the human HIST1H2BK gene
[0102] Retrieve HIST1H2BK (NM_080593) gene information from Genbank; design effective siRNA targets for HIST1H2BK gene. Table 1-1 lists the screened effective siRNA target sequences against the HIST1H2BK gene.
[0103] Table 1-1 is targeted at the siRNA target sequence of human HIST1H2BK gene
[0104] SEQ ID NO TargetSeq(5'-3') 1 GAGAAAGAGTATATAAGCT
[0105] 2. Preparation of lentiviral vector
[0106] 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.
[0107] Table 1-2 Double-stranded DNA...
Embodiment 2
[0125] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0126] Human liver cancer SMMC-7721 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, SMMC-7721:10), 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 ...
Embodiment 3
[0133] Example 3 Western Blotting method to detect gene silencing efficiency
[0134] 1. Extraction of total cell protein
[0135] 1) Infect the target cells (SMMC-7721 cells) with the control virus and the RNAi virus targeting the HIST1H2BK interference target according to the multiplicity of infection (MOI: 10).
[0136] 2) After 5 days of infection, collect the cell samples, take an appropriate amount of RIPA lysate (Beiyuntian, P0013C), and add PMSF within a few minutes before use, so that the final concentration of PMSF is 1mM.
[0137] 3) 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).
[0138] 4) Centrifuge at 12000g for 15min at 4°C, take the supernatant and use BCA Protein Assay Kit (manufacturer: Biyuntian, article number: P0010S) to measure the protein concentration.
[0139] 5) Add new lysate t...
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