Recombinant adenovirus carrier for expressing African swine fever virus B646L gene, construction method and preparation method of recombinant adenovirus
An African swine fever virus, B646L technology, applied in the field of B646L gene overexpression adenovirus vector and its construction
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Embodiment 1
[0067] African swine fever virus B646L gene Gene ID: KJ195685.1, the African swine fever virus B646L gene was synthesized by Shanghai Sangong Co., Ltd. and added restriction sites XhoI and AsisI, and the synthesized B646L gene was connected to the T vector to obtain T vector of the B646L fragment.
Embodiment 2
[0069] The gene synthesis sequence B646L (that is, the T vector with the B646L fragment) and the adenovirus shuttle plasmid vector pKO-FH ( Figure 4 ) to obtain the linearized B646L gene fragment and the pKO vector fragment respectively. The enzyme digestion reaction system of the double enzyme digestion reaction is as follows in Table 1:
[0070] Table 1 Double enzyme digestion system
[0071] B646L gene fragment or pKO-FH vector
20uL
10×Buffer
3μL
AsisⅠ
1μL
MLuⅠ
1μL
wxya 2 o
5μL
total
30μL
[0072] The temperature of the double enzyme digestion is 37° C., and the time of the double enzyme digestion is 1 hour.
[0073] After the reaction, use 1% agarose gel electrophoresis to detect the size of the digested band, and use a gel recovery kit to recover the target fragment. The vector pKO-FH was digested in the same way, and the vector sequence was recovered from the gel.
Embodiment 3
[0075] The linearized B646L gene fragment and the pKO vector fragment were ligated with T4 ligase to obtain pKO-B646L. The connection system is shown in Table 2:
[0076] Table 2 connection system
[0077] Target gene B646L fragment
2~6μL
pKO vector fragment
2~4μL
10×T4Buffer
1μL
T4 DNA ligase (10U / μL)
1μL
total
10 μL
[0078] After mixing, microcentrifuge and connect at 22°C for 2h.
[0079] The ligation product was transformed into Escherichia coli DH5α competent cells. Spread on the LB plate containing kanamycin resistance for screening, pick a single colony, extract the plasmid after expanding the culture, Asis Ⅰ and MLu Ⅰ double enzyme digestion to identify positive clones, and sequence verification to obtain the correct pKO-B646L clone, Sequencing was performed using CMV-F (SEQ No. 2: 5'CAATGGGAGTTTGTTTTGGCACCA-3') and FH-R (SEQ No. 3: 5'CTTATTAGTGGTGGTGGTGGTGGTGCTCG-3').
[0080] The conversion process is...
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