Nucleic acid transferring vector and transferring system and uses
A nucleic acid and dilute acid technology, applied in nucleic acid transport system and its application fields, can solve the problems of low efficiency, inorganic nanoparticles cannot be degraded in vivo, and cannot effectively protect nucleic acid efficiency, achieving high efficiency, mild conditions, and low equipment requirements Effect
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Embodiment 1
[0053] Preparation of chitosan-oligonucleotide polymer
[0054] (1) Preparation of chitosan solution
[0055] 1. Dissolve chitosan in 1% acetic acid solution to obtain a solution with 1% chitosan content, and stir for 24 hours to completely dissolve it.
[0056] 2. Add an equal volume of 1% ammonia water to the resulting solution to precipitate the dissolved chitosan and filter it; pre-cool the precipitate at -80°C overnight, then vacuum freeze-dry the precipitate; redissolve in 1% acetic acid solution (this step Optional, if possible, it is best to do it, mainly to remove impurities such as protein in chitosan).
[0057] 3. Adjust the pH to 5.5 with sodium hydroxide.
[0058] 4. Dilute with 1% acetic acid solution to a concentration of 0.02%, and then adjust the pH to 5.5 again.
[0059] 5. Sterilize by filtration in a sterile room and refrigerate at 4°C.
[0060] (2) Preparation of oligonucleotide solution: oligonucleotide 200 μg / ml solution is mixed with an equal volume...
Embodiment 2
[0065] Preparation of chitosan-RNA polymer
[0066] (1) Preparation of chitosan solution
[0067] 1. Dissolve chitosan in 1% acetic acid solution to obtain a solution with 1% chitosan content, and stir for 24 hours to completely dissolve it.
[0068] 2. Add an equal volume of 1% ammonia water to the resulting solution to precipitate the dissolved chitosan and filter it; pre-cool the precipitate at -80°C overnight, then vacuum freeze-dry the precipitate; redissolve in 1% acetic acid solution (this step Optional, if possible, it is best to do it, mainly to remove impurities such as protein in chitosan).
[0069] 3. Adjust the pH to 5.5 with sodium hydroxide.
[0070] 4. Dilute with 1% acetic acid solution to a concentration of 0.25%, and then adjust the pH to 5.5 again.
[0071] 5. Sterilize by filtration in a sterile room and refrigerate at 4°C.
[0072] (2) Preparation of RNA solution: RNA was dissolved in 20% sodium sulfate solution, and the final concentration of RNA was...
Embodiment 3
[0075] Application of chitosan-oligonucleotide polymer in inhibiting Mycobacterium tuberculosis.
[0076] 1. The ino1 gene is a gene that plays an important role in the growth and virulence of Mycobacterium tuberculosis, and the ino1 gene of Mycobacterium tuberculosis is selected as the gene to be suppressed.
[0077] 2. Check the full sequence of the ino1 gene on Genebank. Four antisense oligonucleotides were designed according to OLIGO-5 SECONDARY STRUCTURE ANALYSIS software. The sequence is as follows: 5'-CGACGGGCTCCGCGTCGGAG-3', 5'-TCGGTGAACTTCTTGGCCCA-3', 5'-CTTGGAGATCTTCTTGGACT-3', 5'-TTGGCGATCTTGGCCGCCCG-3'.
[0078] 3. Chitosan and antisense oligonucleotides are prepared into chitosan-oligonucleotide polymers according to method one.
[0079] 4. Dissolve the polymer in PBS to 0.5 mg / ml and add it into the culture medium in which Mycobacterium tuberculosis is cultured, and co-cultivate for 2-4 weeks.
[0080] 5. Take a small amount of bacterial solution and sufficien...
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