Purposes and related medicines of human ZNF786 gene
A gene and drug technology, applied in the field of human ZNF786 gene and its related drugs
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Embodiment 1
[0071] Example 1: Preparation of RNAi lentivirus for human ZNF786 gene
[0072] 1. Screening for effective siRNA targets against the human ZNF786 gene
[0073] Retrieve ZNF786 (NM_152411.3) gene information from Genbank; design effective siRNA target for ZNF786 gene by using the design software Genechem of Shanghai Jikai Gene Chemical Technology Co., Ltd. In the coding sequence (CDS) region of the ZNF786 gene, a sequence of 21 bases was obtained starting at every other base, and Table 1 lists 42 effective siRNA target sequences for the ZNF786 gene.
[0074] Table 1 is targeted at the siRNA target sequence of human ZNF786 gene
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[0078] Aim at the siRNA target (taking SEQ ID NO: 19 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 2) containing AgeI and EcoRI restriction sites at both ends; act on pGCSIL- GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and identi...
Embodiment 2
[0097] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of ZNF786 gene
[0098] Human laryngeal cancer Hep 2 cells, lung cancer 95D cells, gastric cancer AGS cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells and glioma U251 cells in logarithmic growth phase were digested with trypsin to make cell suspension ( The number of cells is 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, Hep2: 10, 95D: 2, AGS: 100, SMMC-7721: 20, MCF-7: 20, U251: 10) value, add an appropriate amount of the virus prepared in Example 1 and incubate for 24 hours Afterwards, the medium was replaced, 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 the M-MLV instruction manual of Promega Company, the RNA was reverse-tran...
Embodiment 3
[0106] Example 3: Detection of proliferation ability of tumor cells infected with ZNF786-siRNA lentivirus
[0107] Human laryngeal cancer Hep 2 cells, lung cancer 95D cells, gastric cancer AGS cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells and glioma U251 cells in logarithmic growth phase were digested with trypsin to make cell suspension ( The number of cells is 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, Hep2: 10, 95D: 2, AGS: 100, SMMC-7721: 20, MCF-7: 20, U251: 10), an appropriate amount of virus was added, and the culture medium was replaced after 24 hours. After the infection time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. 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 eac...
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