Optimized porcine circovirus type 2 cap protein gene and its recombinant plasmid and application
A technology of porcine circovirus and recombinant plasmid, applied in the field of molecular biology, can solve problems such as low efficiency
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[0058] 3. Preparation and transformation of Escherichia coli DH5α competent cells
[0059] Preparation of competent Escherichia coli DH5α: Streak inoculated frozen DH5α strains on LB plates without antibiotics, and streak on resistant medium as a control, culture overnight at 37°C. The next day, a single colony was picked and inoculated in 3mL LB liquid medium, and cultured overnight at 37°C with shaking at 200rpm. Take 10 μL of the bacterial liquid and inoculate it into a new 3 mL LB liquid medium, and culture it with shaking at 37°C for about 2 hours until the OD600 reaches 0.3-0.4. Under sterile conditions, the culture was poured into ice-precooled sterile Eppendorf tubes, centrifuged at 12,000 rpm at 4°C for 30 s, the supernatant was discarded and inverted to dry. Use 800 μl ice-cold 0.1mol / LCaCl 2 Resuspend the bacterial pellet in the solution, put it in an ice bath for 30 minutes, centrifuge at 12000rpm at 4°C for 30s, discard the supernatant, invert and dry it, and fi...
Embodiment 3
[0065] In vitro expression and identification of embodiment 3 recombinant plasmids
[0066] 1. Purification of Plasmids
[0067] The method refers to the instructions of Promega's plasmid mini-purification kit. The specific operation is as follows: pick DH5α single colonies containing recombinant eukaryotic plasmids pVAX-Cap, pVAX-SynCap, pVAX-SynCap(m) and pVAX-SynCap-2A-SynCap respectively, and inoculate them in 3.0mL In the LB medium of penicillin, shake culture overnight at 200rpm. Take 5×1.5mL bacterial solution and centrifuge at 12000rpm for 30s to precipitate the bacterial cells; discard the supernatant completely and add 250μL 4℃ pre-cooled solution I (20μg / mL RNaseA) to resuspend the bacteria; add 250μL solution II and gently invert the centrifuge tube After repeated lysis is complete, place the centrifuge tube on ice (not more than 5 minutes); add 350 μL of solution III, cap the tube tightly, invert the tube and gently invert until the protein denatures into a whit...
Embodiment 4
[0077] The preparation of embodiment 4 recombinant plasmid and carrying recombinant plasmid attenuated Salmonella and animal immunity test
[0078] 1. Mass Preparation of Plasmids
[0079] Method with reference to embodiment 3. Inoculate 10 μL of the strain into 3 mL of LB medium containing 50 μg / mL kanapenicillin, and shake at 200 rpm at 37°C for 6 hours; pour all 3 mL of the bacterial culture into 200 mL of LB medium containing 50 μg / mL kanapenicillin, at 37°C Shake culture overnight at 200rpm; the next day, collect the bacterial solution and centrifuge at 7500rpm for 10min, discard the supernatant completely, add 100mL 4℃ pre-cooled STE (10mM Tris-HCl, 0.1MNaCl, 1mMEDTA, pH8.0), resuspend the pellet; centrifuge at 7500rpm for 10min; After discarding the supernatant, add 2 mL of 4°C pre-cooled solution I (50 mM glucose; 25 mM Tris-Cl, pH 8.0; 10 mM EDTA) to fully resuspend the bacteria, and distribute them into EP tubes, 200 μL / tube; add 400 μL of newly prepared solution II...
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