Structure of targeted hepatitis C virus (HCV)F gene and application of targeted HCVF gene
A technology of application and small molecule interference, which can be used in antiviral agents, gene therapy, drug combinations, etc., and can solve the problem of low homology between types
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0034] Chemical synthesis of siRNA
[0035] F-siRNA was designed and sent to Shanghai Gemma Pharmaceutical Technology Co., Ltd. for synthesis. The synthesis method adopted is a common method, and the siRNA molecule is deprotected at the 2-position, desalted, purified and annealed to form a double strand, and then dissolved in DEPC-treated distilled water.
[0036] 1. Synthesize the sense RNA fragment and antisense RNA fragment of siRNA respectively
[0037] 2. Annealing to form double-stranded siRNAs
[0038] 1) Dissolve the synthetic siRNA sense and antisense RNA fragments in diacetic acid pyrophosphate (DEPC)-treated water.
[0039] 2) Prepare annealing buffer (2×): 200mM potassium acetate, 4mM magnesium acetate, 60mM Hepes-KOH (pH7.4)
[0040] 3) Prepare 20 μM siRNA stock solution:
[0041] 2×Annealing buffer 100μl
[0042] siRNA sense RNA fragment Aμl
[0043] siRNA antisense RNA fragment Bμl
[0044] DEPC treated water (100-A-B)μl
[0045] The above ingredients we...
Embodiment 2
[0053] Construction of siRNA expression vector
[0054] Use the vector pRNAi-U6H1 / Neo (Biomics Company) with H1 promoter and U6 promoter (see the plasmid map figure 1 ), were respectively connected to the DNA oligonucleotide chains of the above-designed F-siRNA target site sequence (21 bases), to form a plasmid vector capable of expressing F-siRNA.
[0055] DNA oligonucleotide strands are:
[0056] Sense DNA fragment (corresponding to siRNA sense RNA fragment):
[0057] 5′-AAACCUCGUGGAAGGCGACAACdTdT-3′
[0058] Antisense DNA fragment (corresponds to siRNA antisense RNA fragment)
[0059] 5′-AAAGUUGUCGCCUUCCACGAGGdTdT-3′
[0060] The two DNA oligonucleotide strands are annealed to form double-stranded DNA oligos.
[0061] 1) DNA single strand synthesis
[0062] It can be synthesized by many DNA synthesis companies.
[0063] 2) DNA single strand anneals to form a double strand
[0064] The annealing reaction system is as follows: sense DNA fragment oligonucleotide (100 μ...
Embodiment 3
[0081] Construction of Luciferase Expression Vector and Construction of Stable Transfection Cell Line
[0082] Use biological software to analyze the HCV subtype 1b virus sequence published in GenBank, design primers on the HCV C gene fragment, first amplify the sequence of 42-144aa in the +1 frame, and then use two pairs of primers, plus the front of the 0 frame 41 aa sequences, the following HCV 1b subtype F gene sequence is obtained (the F gene is terminated at the 42nd codon + 1 frame shift and the 144th codon TAG):
[0083] ATG AGC ACG AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC ACC AAC CGC
[0084]CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT GGT CAG ATC GTT GGT GGA GTT
[0085] TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG CGC GCG ACT AGG AAG
[0086] ACT TCC GAG CGG TCG CAA CCT CGT GGA AGG CGA CAA CCT ATC CCA AAG GCT
[0087] CGC CAA CCC GAG GGC AGG GCC TGG GCT CAG CCC GGG TAC CCT TGG CCC CTC
[0088] TAT GGC AAT GAG GGC TTG GGG TGG GCA GGA TGG CTC CTG TCA CCC CGC G...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More