Small interfering RNA aiming at HPV16E6 gene and application of small interfering RNA
A small interference and gene technology, applied in the direction of DNA / RNA fragments, gene therapy, recombinant DNA technology, etc., can solve the problems of large side effects and low specificity, and achieve the effect of low toxicity and side effects and reduced expression
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[0048] The preparation method of the small interfering nucleic acid molecule provided by the invention includes sequence design and sequence synthesis.
[0049] The synthesis of the small interfering nucleic acid molecular sequence can be done by chemical synthesis, or entrusted to a biotechnology company specialized in nucleic acid synthesis, such as entrusting Shanghai Aibosi Biotechnology Co., Ltd. to perform the synthesis.
[0050] Generally speaking, the chemical synthesis method includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalting.
[0051] For example, the specific steps for the chemical synthesis of siRNA with the nucleotide sequence shown in HPV16E6si01 are as follows:
[0052] (1) Synthesis of oligoribonucleotides
[0053] Set to synthesize 1 millimolar RNA on an automatic DNA / RNA synthesizer (for example, AppliedBiosystemsEXPEDITE8909), and set the coupling time of each cycle ...
Embodiment 1
[0062] Embodiment 1: Design HPV16E6 small interfering nucleic acid sequence
[0063] The HPV16E6 mRNA sequence was obtained from the biomedical website NCBI database (http: / / www.ncbi.nlm.nih.gov / ) established by the National Institutes of Health of the United States, and the siRNA sequence specific to this gene was designed online in siDESIGNCenter and DSIR. The principle of selecting the sequence of the small interfering nucleic acid molecule defined by the online software is: mainly based on a linear model combining the characteristics of the sequence of the small interfering nucleic acid molecule and the target sequence. The high stability of the 5' end of the siRNA may block the loading of the sense strand of the siRNA into the RNA-induced silencing complex (RISC); the low stability of the 5' end of the antisense strand of the siRNA may facilitate the loading of the siRNA The antisense strand of the nucleic acid molecule is loaded into RISC; and the low stability of the mi...
Embodiment 2
[0069] Example 2: In vitro verification of the efficiency of the dsRNA sequence inhibiting the expression of HPVE6
[0070] (1) Cell culture
[0071] Human cervical cancer cell lines (SiHa, ATCC) were treated with MEM medium (Gibco) containing 1% penicillin-streptomycin (PS, Gibco) antibiotic mixture and 10% fetal bovine serum (FBS, Gibco), at 37°C, Cultured in a cell incubator with 5% carbon dioxide and 95% air humidity.
[0072] (2) Cell transfection
[0073] (1) One day before transfection, SiHa cells were inoculated in culture medium until the confluence of cells was 30-50% at the time of transfection.
[0074] (2) Prepare transfection samples:
[0075] Separately mix 1 μl of siRNA with a concentration of 20 μM (see Table 1) and 0.5 μl of cell transfection reagent Lipofectamine TM 2000 (Invitrogen) was diluted in 0.05ml Opti-MEM (Invitogen), mixed gently and incubated for 5 minutes. Mix gently and incubate for 5 minutes.
[0076] Use nonsense control siRNA (the sense...
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