Duck plague virus US1 gene maker-free deletion virus strain and building method thereof
A duck plague virus and deletion virus technology, applied in the field of genetic engineering
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Embodiment 1
[0067] Example 1 Construction of US1 gene traceless deletion strain GS1783-pBAC-DPV-ΔUS1 strain
[0068] The US1 single-copy gene scarless deletion strain GS1783-pBAC-DPV-ΔUS1 was constructed on Escherichia coli GS1783 by Red / ET modification technology, and the construction method included the following steps:
[0069] 1. Prepare GS1783 electroporation competent, electrotransform pBAC-DPV plasmid
[0070] (1) Escherichia coli with pBAC-DPV plasmid was revived in LB solid medium containing chloramphenicol, cultured overnight at 37°C; single colonies were inoculated in LB liquid medium containing chloramphenicol, and cultivated overnight at 37°C;
[0071] (2) Extract the pBAC-DPV plasmid according to the operating instructions of the QIAGEN Plasmid Midi Kit;
[0072] (3) Resuscitate GS1783 frozen-preserved bacteria in LB solid medium, culture overnight at 30°C;
[0073] (4) Pick a single colony of GS1783 and inoculate it in 5 mL of LB liquid medium, and culture it overnight at...
Embodiment 2
[0114] Example 2 Construction of US1 gene traceless deletion strain GS1783-pBAC-DPV-2ΔUS1 strain
[0115] The US1 double-copy gene scarless deletion strain CHv-BAC-G-2ΔUS1 was constructed on Escherichia coli GS1783 by Red / ET modification technology, and the construction method included the following steps:
[0116] 1. Amplify the I_sceI-Kana-US1 targeting fragment
[0117] (1) Escherichia coli carrying the pEPkan-S plasmid was cultured overnight at 37°C in LB solid medium containing kanamycin; single colonies were inoculated in LB liquid medium containing kanamycin at 37°C Cultivate overnight, and extract the pEPkan-S plasmid using the plasmid mini-extraction kit (see pEPkan-S plasmid map figure 1 );
[0118] (2) Using the pEPkan-S plasmid as a template and 1808F and 1808R as primers, amplify the base fragment containing the I-SceI restriction site and the Kana element and the 40bp homology arms of the upper and lower reaches of the US1 gene by PCR Targeting fragment 1808, ...
Embodiment 3
[0147] Example 3 Rescue CHv-BAC-G-ΔUS1, CHv-BAC-G-2ΔUS1 virus
[0148] 1. Thaw frozen GS1783-pBAC-DPV-ΔUS1 and GS1783-pBAC-DPV-2ΔUS1 bacteria in LB solid medium containing chloramphenicol, and culture overnight at 30°C.
[0149] 2. Extract pBAC-DPV-ΔUS1 and pBAC-DPV-2ΔUS1 plasmids according to the instructions of QIAGEN Plasmid Midi Kit:
[0150] (1) Pick a single colony of GS1783-pBAC-DPV-ΔUS1 and GS1783-pBAC-DPV-2ΔUS1 and inoculate it in 100 mL of LB liquid medium containing chloramphenicol, and cultivate overnight at 30°C;
[0151] (2) Harvest the bacterial cells: centrifuge at 4°C for 15 minutes with a centrifugal force of 6000×g.
[0152] (3) Resuspend the bacteria with 4mL Buffer P1 (add RNase A to Buffer P1 before use).
[0153] (4) Add 4mL buffer P2, quickly invert the tube 4-6 times, and let stand at room temperature (15-25°C) for 5min.
[0154] (5) Add 4 mL of pre-cooled buffer P3, mix immediately and vigorously turn over 4-6 times, and incubate on ice for 15 min....
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