Strain dpv CHv-gEΔCT without trace deletion of duck plague virus ge gene ct region and its construction method
A technology of duck plague virus and construction method, which is applied in the field of genetic engineering and can solve problems such as residual bases and residual MiniF elements
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Embodiment 1
[0051] Example 1 Preparation of duck plague virus gE gene CT region seamless deletion strain DPV CHv-gEΔCT
[0052] Duck plague virus gE gene CT region traceless deletion strain DPV CHv-gEΔCT, its construction method comprises the following steps:
[0053] 1. Prepare GS1783 electroporation competent, electrotransform pBAC-DPV plasmid
[0054] (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;
[0055] (2) Extract the pBAC-DPV plasmid according to the operating instructions of the QIAGEN Plasmid Midi Kit;
[0056] (3) Resuscitate GS1783 frozen-preserved bacteria in LB solid medium, culture overnight at 30°C;
[0057] (4) Pick a single colony of GS1783 and inoculate it in 5 mL of LB liquid medium, and culture it overnight at 30°C to obtain a seed solution;
[0058] (5) Add 5mL seed liqu...
Embodiment 2
[0160] Determination of the Growth Curve of DPV CHv-gEΔCT of Example 2 Scarless Deletion Strain
[0161] 1. Determination of one-step growth curve
[0162] The parental virus DPV CHv and gE gene CT region traceless deletion strain DPV CHv-gEΔCT were inoculated into DEF cells at 2 MOI respectively, and the supernatant and cells were collected 6h, 12h, 18h, and 24h after inoculation, and each time point was repeated three times. After the collection was complete, the freeze-thaw was repeated twice, and the virus titer was detected in a 96-well plate. The results showed that the deletion of the CT region of the gE gene affected the replication of the DPV CHv virus.
[0163] 2. Determination of multi-step growth curve
[0164] The parental virus DPV CHv and gE gene CT region traceless deletion strain DPV CHv-gEΔCT were inoculated into DEF cells at 0.01 MOI respectively, and the supernatant and cells were collected 12h, 24h, 48h, and 72h after inoculation, and each time point was ...
Embodiment 3
[0165] Example 3 Plaque formation experiment of DPV CHv-gEΔCT strain without trace deletion
[0166] The parental virus DPV CHv and gE gene CT region traceless deletion strain DPV CHv-gEΔCT were respectively inoculated at 0.01 MOI in a 6-well plate covered with DEF cells, 37 ° C, 5% CO 2 After absorbing for 2 hours, remove the supernatant, add 2 mL of 1% methylcellulose, 37 ° C, 5% CO 2 After culturing for 48 h, remove 1% methylcellulose, wash 3 times with PBS, fix overnight at 4°C with 4% paraformaldehyde, wash 3 times with PBS, add H 2 o 2 Mix the mixture with methanol at a volume ratio of 1:50, incubate at room temperature for 30 minutes, wash with distilled water three times, add 5% BSA blocking solution, incubate at room temperature for 30 minutes, add rabbit anti-DPV, incubate overnight at 4°C, wash with PBS three times, add Biotinylated goat anti-rabbit IgG, incubated at 37°C for 30 minutes, washed 3 times with PBS, added dropwise SABC substrate, incubated at 37°C for...
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