Use of human rhbdd1 gene and related medicines
A gene and drug technology, applied in the use of human RHBDD1 gene and related drug fields
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Embodiment 1
[0069] Example 1: Preparation of RNAi lentivirus for human RHBDD1 gene
[0070] 1. Screening for effective siRNA targets against the human RHBDD1 gene
[0071] The RHBDD1 (NM_032276) gene information was retrieved from Genbank; effective siRNA targets for the RHBDD1 gene were designed using the design software Genechem of Shanghai Jikai Gene Chemical Technology Co., Ltd. In the coding sequence (CDS) region of the RHBDD1 gene, a sequence of 33 bases was obtained starting from every other base, and Table 1 lists 33 effective siRNA target sequences for the RHBDD1 gene.
[0072] Table 1 is targeted at the siRNA target sequence of human RHBDD1 gene
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[0075] Aiming at the siRNA target (taking SEQIDNO: 21 as an example), the double-stranded DNA Oligo sequence (table 2) containing AgeI and EcoRI restriction endonuclease at both ends of synthesis; Act on pGCSIL-GFP carrier with AgeI and EcoRI restriction enzyme Provided by Shanghai Jikai Gene Chemical Technolo...
Embodiment 2
[0093] Example 2: Real-time fluorescent quantitative RT-PCR method to detect the silencing efficiency of RHBDD1 gene
[0094] Human liver cancer HepG2 cells in logarithmic growth phase, human tongue squamous cell carcinoma Tca8113 cells, human breast cancer MCF-7 cells, human pancreatic cancer Panc-1 cells, human gastric cancer SGC7901 cells and human lung cancer 95D cells were trypsinized, Make a 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 (the MOI of Tca8113, HepG2 cells, Panc-1 cells and 95D cells is 10, the MOI of SGC7901 cells and MCF-7 is 20) value, add an appropriate amount of the virus prepared in Example 1, and replace after culturing for 24 hours After the infection time reached 4 days, the cells were collected. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV ...
Embodiment 3
[0102] Example 3: Detection of proliferation ability of tumor cells infected with RHBDD1-shRNA lentivirus
[0103] Human liver cancer HepG2 cells in logarithmic growth phase, human tongue squamous cell carcinoma Tca8113 cells, human breast cancer MCF-7 cells, human pancreatic cancer Panc-1 cells, human gastric cancer SGC7901 cells and human lung cancer 95D cells were trypsinized, Make a 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 (the MOI of Tca8113, HepG2 cells, Panc-1 cells and 95D cells is 10, the MOI of SGC7901 cells and MCF-7 is 20), add an appropriate amount of RHBDD1-shRNA lentivirus, and replace the medium after culturing for 24 hours , after the infection time reached 4 days, the cells of each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2...
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