Small interfering RNA for silencing CD317, recombinant vector and medicine, and applications thereof
A technology of small molecule interference and recombinant vectors, which is applied in the fields of genetic engineering technology, biomedicine, and molecular biology, can solve the problems of dependence on immune function, weak ADCC effect, and rapid antibody clearance, so as to promote tumor cell apoptosis and apoptosis. death effect
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Embodiment 1
[0047] Embodiment 1siRNA design
[0048] According to the basic principle of siRNA target sequence, a siRNA sequence of 21 nucleotides was designed for the human CD317 gene transcript (NM_004335.3), that is, si-CD317, including the sense strand and the antisense strand, and its base sequence is as follows:
[0049] Justice chain (underlined as SEQUENCE NO.1):
[0050] 5'- CCAGGUCUUAAGCGUGAGA dTdT-3';
[0051] Antisense strand (underlined as SEQUENCE NO.2):
[0052] 5'- UCUCACGCUUAAGACCUGG dTdT-3'.
[0053] The base sequence of the negative control (NC) siRNA selected in this embodiment is as follows:
[0054] Sense strand: 5'-UUCUCCGAACGUGUCACGUdTdT-3';
[0055] Antisense strand: 5'-ACGUGACACGUUCGGAGAAdTdT-3'.
[0056] The 3' end of the interference fragment used in the embodiment of the present invention adds two deoxyribonucleotides in a single-stranded suspension structure to enhance the stability of siRNA in vivo and in vitro and prevent degradation.
Embodiment 2
[0057] Example 2 Interference Effect Verification
[0058] The cell lines selected in the present invention are cervical cancer cell Hela, ovarian cancer cell SK-OV-3 and breast cancer cell MCF-7 which highly express human CD317. The transfection method and result verification are as follows:
[0059] 1. Cell transfection
[0060] According to the siRNA synthesis report, add an appropriate amount of DEPC water to prepare a 20 μM stock solution;
[0061] Inoculate the cells in a 12-well plate, and the density should reach 60%-80% after overnight culture;
[0062] Dilute 4 μL Lipofectamine 3000 transfection reagent with 50 μL Opti-MEM medium, mix well, and let stand at room temperature for 5 minutes;
[0063] Dilute 2 μL siRNA with 50 μL Opti-MEM medium, mix well, and let stand at room temperature for 5 minutes;
[0064] Mix the above step 2) with the dilution of step 3), mix well and let stand at room temperature for 10 minutes. At this time, the ratio of siRNA to Lipofect...
Embodiment 3
[0086] Example 3 Analysis of the effect of CD317 silencing on tumor cell apoptosis by flow cytometry
[0087] 1. Cell pretreatment
[0088] 1) 36 hours after the cells were transfected with siRNA, the cells were digested with trypsin-EDTA, resuspended to 3×10 5 cells / mL, and inoculated in a 12-well plate;
[0089] 2) After culturing overnight, change the serum-free medium to continue culturing for 48 hours, and set up a serum group as a control;
[0090] 2. Analysis of cell apoptosis by flow cytometry
[0091] 1) After starving the cells without serum, collect the supernatant, then digest with EDTA-free trypsin, and collect the cell suspension;
[0092] 2) Centrifuge the cell supernatant and cell suspension at 500 g at 4°C for 5 min, collect the supernatant and cells in the cell suspension, and combine them;
[0093] 3) Wash the cells 3 times with pre-cooled PBS, and then resuspend the cells with 100 μL 1×AnnexinV Binding Buffer;
[0094] 4) Add 5 μL Annexin V-FITC and 5 μL ...
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