Structure and usage of antisense oligonucleotide inhibiting ABHD2 gene expression
A technology of antisense oligonucleotides and uses, which is applied in the field of bioengineering drugs to achieve important social and economic benefits
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Embodiment 1
[0029] Materials and Methods
[0030] 1. Drug preparation
[0031] Lamivudine was dissolved in PBS to a final concentration of 10 mmol / L.
[0032] 2. Cell Culture
[0033] The cells used were the HepG2 cell line of the liver cancer cell line and the HepG2.2.15 cell line transfected with HBV DNA. HepG2.2.15 cells are derived from HepG2 cells, contain integrated HBV DNA, and can continuously and stably secrete Dane's particles, HBsAg, HBV DNA, etc. into the culture medium during cell culture. HepG2 cells were cultured with DMEM cell culture medium containing 10% fetal bovine serum (FBS, Gibco), and HepG2.2.15 cells were cultured with MEM cell culture medium containing 10% fetal bovine serum, 380 μg / ml G418 (Promega).
[0034] 3. Cell Dosing
[0035] HepG2.2.15 cells were passaged at a ratio of 1:3 after they became confluent, and 48 hours later, they were replaced with MEM medium containing 2% FBS, and lamivudine was added. The final concentration of lamivudine was 25 μmol / ...
Embodiment 2
[0048] Materials and Methods
[0049] 1. Design and synthesis of S-ASODN
[0050] Search the nucleic acid sequence database in GeneBank, select the ABHD2 mRNA reference sequence NM 007011 published by NCBI, and use the artificial neural network prediction algorithm patented by our laboratory based on the multi-level prediction structure of target genes and the antisense online design designed by our laboratory. The software AODesigner (software copyright number: 2005SR12155) designed five ASODN sequences targeting ABHD2. By aligning with the GeneBank online blast sequence, the selected target sequences have good specificity and will not interfere with the expression of other normal human genes (see sequence table 1-5). All oligonucleotides were synthesized by ABI8909 automatic DNA synthesizer and thio-modified during synthesis. The process was as follows: The thio-reagent (Beaucage regent, Transgenomic) was dissolved in anhydrous acetonitrile to make the final concentration 1...
Embodiment 3
[0072] Materials and Methods
[0073] 1. Design and synthesis of ASODN
[0074] According to the results of Example 2, the antisense oligonucleotide sequence AB3 with better effect was selected, and its sense oligonucleotide was synthesized (see sequence table 6). The synthesis of all thiooligonucleotides is the same as that in Example 2.
[0075] 2. Cytotoxicity assay of thioantisense oligonucleotide sequence AB3
[0076] Hep2.2.15 cells were cultured in MEM medium containing 10% fetal bovine serum (Gibco) and 380 μg / ml in a 37° C., 5% CO2 incubator. Observe that the cells grow well, and after culturing to the logarithmic growth phase, inoculate a 96-well plate, 0.75×10 5 Cells / well, incubated at 37°C, 5% CO2 for 48-72 hours, after reaching 40-60% cell confluence, in a serum-free state, use liposomal Lipofectin (Invitrogen, 1mg / ml) reagent and operate according to the instructions Transfection was performed. The concentrations of antisense oligonucleotide AB3 were 0.2 μM...
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