Shrna for inhibiting the replication of African swine fever virus and its use
An African swine fever virus, virus technology, applied in the direction of DNA/RNA fragments, viruses, antiviral agents, etc., can solve the problems of inability to induce protective immunity, limited effect, etc., achieve good inhibition, inhibit replication, and increase specific infection Effect
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[0047] In view of the deficiencies in the prior art, the inventors of the present invention have been able to propose the technical scheme of the present invention through long-term research and extensive practice, which can be summarized as follows: designing shRNA against African swine fever virus, and then using AAV virus as a carrier to construct Recombinant AAV expressing specific shRNA. Preferably, the recombinant AAV is modified for its Cap structural protein, that is, the optimized porcine macrophage C-reactive protein is added to the 3' end of the Cap gene, without affecting the assembly and stability of AAV , the modified AAV virus can bind to the C-reactive protein receptor of porcine macrophages, so that the recombinant AAV can specifically infect target cells, namely porcine macrophages.
[0048] The shRNAs described in the present invention consist of two complementary (sense and antisense) 19-29 base pair sequences separated by a short stem-loop (loop) of 4-11 d...
Embodiment 1
[0053] Example 1 Design and Construction of shRNA Sequences
[0054] 1. Design DNA sequences capable of transcribing shRNA for the A104R gene and I215L gene of African swine fever virus respectively. The DNA sequences are as follows:
[0055] A104R sense strand (SEQ ID NO.3):
[0056] cgcaagagctttactccttagtagcggcagttcaagagactgccgctactaaggagtaaagctcttgctttttta
[0057] A104R antisense strand (SEQ ID NO.4):
[0058] agcttaaaaaagcaagagctttactccttagtagcggcagtctcttgaactgccgctactaaggagtaaagctcttgcggtac
[0059] The above-mentioned sense strand and antisense strand templates include cohesive ends and shRNA sequences, and the sequences of the shRNAs are:
[0060] 5'-gcaagagctttactccttagtagcggcagttcaagagactgccgctactaaggagtaaagctcttgctttttt-3' (SEQ ID NO. 1),
[0061] Among them, uucaagaga is a loop structure, and the enzyme cutting site is Kpn1 (ggtacc).
[0062] I215L sense strand (SEQ ID NO.5):
[0063] cgacacctgatagagaatccctctgagaatttcaagagaattctcagaggggattctctatcaggtgtctttttt...
Embodiment 2
[0072] Example 2 Construction of pAAV-U6-A104R-shRNA plasmid and pAAV-U6-I215L-shRNA plasmid
[0073] Nanjing GenScript synthesized the U6 promoter gene (SEQ ID NO.7) and constructed the pAAV-U6 vector. The specific operations are as follows:
[0074] The pAAV-CAG vector (purchased from Addgene) was transformed, and the pAAV-CAG vector was cut with Nde1 / Kpn1 to remove the CAG promoter, and then the synthetic U6 promoter was connected to the double-digested pAAV-CAG vector, and the The CAG promoter replaced the U6 promoter in adult cells, resulting in pAAV-U6.
[0075] The vector pAAV-U6 was double digested with KpnI and HindIII to recover the target vector.
[0076] 1. The pAAV-U6 plasmid was digested with KpnⅠ and HindⅢ respectively at 37°C for 3 hours. The specific enzyme digestion reaction system is shown in Table 2.
[0077] The digested product was subjected to gel electrophoresis, and the digested pAAV-U6 plasmid was purified using a gel recovery purification kit.
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