Small interference RNA sequence of hepatitis B virus gene and preparation method thereof
A technology of ribonucleic acid and hepatitis B virus, applied in the field of genetic engineering, can solve problems such as RNA interference, technical difficulty, and unsuitability for long-term research projects
Examples
Embodiment 1
[0029] Example 1 In vitro transcription and synthesis of interfering RNA inhibits the expression of HBV surface antigen fusion gene in vitro
[0030] Preparation of siRNA
[0031] (1) Design of siRNA According to the design principle of siRNA and the experimental results of direct synthesis of siRNA, the target cDNA target sequences of 2 siRNAs targeting HBV (ayw subtype) S gene were selected. The sequence is as follows:
[0032] (anti-s siRNA1)5'-AAACCTTCGGACGGAAATTGC-3'
[0033] (anti-s siRNA2)5'-AATACCGCAGAGTCTAGACTC-3'
[0034] (2) Design and synthesis of transcription templates for specific siRNAs According to the above cDNA target sequences, two 29-mer DNAs were designed and synthesized as templates, including 8 bases matching the 3' end of the T7 promoter primer (called For the guide sequence, leader aequence) and 21 designed siRNA sequences.
[0035] ① The antisense and sense strands of specific anti-s siRNA1 are as follows:
[0036] Antisense anti-s siRNA1 Oligon...
Embodiment 2
[0074] Example 2 Anti-s siRNA inhibits the expression of green fluorescent protein and HBV S gene expression in HepG2 cells co-transfected with reporter plasmid pGFP-S and siRNA
[0075] (1) Co-transfection of HepG2 cells with reporter plasmid pGFP-S and siRNA Reporter plasmid pGFP-S is a plasmid expressing reporter gene green fluorescent protein and HBsAg fusion protein. HepG2 cells according to 1 × 10 5 Inoculate a 24-well plate per well, cultivate for 24 hours to reach a fusion rate of 80%-90%, and co-transfect pGFP-S and siRNA. For specific steps, refer to the instruction manual of Lipofectamine 2000 from Invitrogen Company. After 6 hours, change to 10% FCS DMEM, and at the same time set up a negative group transfected only with pGFP-S vector, and repeat 3 wells for each group.
[0076] (2) Observation of cell GFP expression by fluorescence microscope After 48 hours of transfection, the expression of GFP in HepG2 cells was observed under an inverted fluorescence microscop...
Embodiment 3
[0079] Example 3 Anti-s siRNA inhibits experiments on the expression of HBsAg and HbeAg in siRNA transfected HepG2.2.15 cells
[0080] (1) siRNA transfected HepG2.2.15 cells HepG2.2.15 cells according to 1×10 4 Inoculate a 24-well plate, cultivate for 24 hours to achieve a fusion rate of 30%-40%, and transfect 0.84 μg of siRNA each. For specific steps, refer to the instruction manual of OLIGOFECTAMINE reagent from Invitrogen Company. Set irrelevant control siGFP group (FEBS Letters 543 (2003) 51-54): sense RNA 5-GGCUACGUCCAGGAGCGCACC-3, anti-sense RNA 5-UGCGCUCCUGGACGUAGCCTT-3. Change to 10% FCS DMEM after 6 hours. Three replicate wells were set up for each group.
[0081] (2) Detection of HBsAg and HBeAg concentration changes by radioimmunoassay At 48 hours after transfection of HepG2.2.15 cells, cell culture fluid was collected, centrifuged to remove cell debris, and the supernatant was detected by radioimmunoassay. For the detection method, refer to the kit instructions. ...
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Description
Claims
Application Information
- IPC
- C07H21/02; C12N15/11; C12N15/10; C12N15/51; A61K31/7088; A61P1/16; C12N15/113
- Inventors
- 朱海红; 陈智