ALV-J (avian leukosis virus-J subgroup) pol geneconserved sequence based siRNA (small interfering ribonucleic acid) recombinant interference vector as well as preparation method and application thereof
A technology of avian leukosis virus and interference carrier, applied in the field of genetic engineering, can solve problems such as accelerated virus mutation and evolution, persistent damage to immune response, unscientific, etc., and achieve the effect of reducing economic losses
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Embodiment 1
[0037] The preparation of embodiment 1 double-stranded DNA
[0038] Targeting the conserved region of the pol gene, the start site is 4614, the end site is 4633, and the siRNA is designed and synthesized, its base sequence is ACAGATATCAGACAGACTT, and its gene sequence is shown in the sequence table SEQ ID NO: 1;
[0039] Designing and synthesizing the hairpin structure containing the above-mentioned target fragment, wherein the gene sequence of the top strand DNA is shown in the sequence table SEQ ID NO: 2; and the gene sequence of the corresponding bottom strand DNA is shown in the sequence table SEQ ID NO: 3; with ddH 2 O was dissolved to 100 μM, 5-10 μl of each complementary single strand was mixed in pairs, and annealed according to the system given in Table 1. The mixture was heated at 95°C for 5-10 minutes, then left at room temperature for 20 minutes to form double-stranded DNA.
[0040] Table 1. oligo DNA annealing system
[0041] 100μM top strand oligo
Embodiment 2
[0042] The construction of embodiment 2 recombinant interference vector:
[0043] The annealed double-stranded DNA was sterilized with ddH 2 O was further diluted to a concentration of 10 nM, and the system was connected at room temperature for 30-45 minutes according to Table 3.
[0044] Table 2. Enzyme Ligation System
[0045] 5×ligation buffer
Embodiment 3
[0046] Embodiment 3 conversion test
[0047] Take 10 μl of the ligation product to transform 100 μl of competent cells DH5α, spread on LB plates (containing 50 μg / ml spectinomycin), and incubate at 37°C.
[0048] Pick 3 clones from the transformation plate, shake the bacteria to extract the plasmid, and then sequence it to verify whether the sequence of the inserted fragment in the recombinant clone is consistent with the designed oligomeric single-stranded DNA, that is, the sequence of top strand DNA and bottom strand DNA;
[0049] The recombinant vector obtained by cloning was sent to a sequencing company for sequencing. It was found that the recombinant vector was obtained by the present invention. The sequence of the inserted fragment in the recombinant clone was consistent with the designed oligomeric single-stranded DNA sequence. It can be seen that the target fragment has been successfully inserted into the cloning vector. .
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