Goose astrovirus-like particle novel genetic engineering subunit vaccine
An astrovirus, amino acid technology, applied in genetic engineering, viruses, viral peptides, etc., can solve the problems of weak immune protection, strong strain virulence, incomplete inactivation, etc., to achieve strong immunogenicity, protein Good immunogenicity and the effect of reducing production costs
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Embodiment 1
[0109] Example 1 Construction and Identification of Transfer Vector pF-Cap
[0110] 1. Cap protein gene amplification and purification The codon-optimized GAstV Cap protein gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript Co., Ltd. and cloned into the pUC17 plasmid to obtain the pUC-Cap plasmid vector. Use the pUC-Cap plasmid as a template, and Cap-F and Cap-R as upstream and downstream primers for PCR amplification (the gene sequences of Cap-F and Cap-R are shown in SEQ ID NO.3 and 4), and the amplification system is shown in the table 1.
[0111] Table 1 Cap protein gene amplification system
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[0114] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.
[0115] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as figure 1 As shown, the target band appea...
Embodiment 2
[0127] Embodiment 2 recombinant baculovirus genome Bac-Cap construction
[0128] 1. Transformation of DH10Bac bacteria Take 1 μl of the pF-Cap plasmid in Example 1 and add it to 100 μl of DH10Bac competent cells, mix well, bathe in ice for 30 minutes, heat shock in a water bath at 42°C for 90 seconds, and then bathe in ice for 2 minutes, add 900 μl without Amp LB liquid medium, cultured at 37°C for 5 hours. After 100 μl of bacterial solution was diluted 81 times, 100 μl of diluted bacterial solution was applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and incubated at 37°C for 48 hours.
[0129] 2. Select a single clone Use an inoculation needle to pick up large white colonies, then streak on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and culture at 37°C for 48 hours. Then pick a single colony and inoculate it into LB liquid medium containing gentamicin, kanamycin and tetracycline for culture, pres...
Embodiment 3
[0130] Example 3 Recombinant Baculovirus Transfection
[0131] Inoculate 0.8×10 per well in a six-well plate 6 Sf9 cells, the cell confluence is 50-70%. Prepare the following complexes for each well: dilute 4 μl of Cellfectin transfection reagent with 100 μl of transfection medium T1, and briefly vortex; dilute 3 μg of the recombinant Bacmid-Cap plasmid in Example 2 with 100 μl of transfection medium T1, Prepare the transfection mixture by mixing the diluted transfection reagent and plasmid separately and blowing gently. Add the above-mentioned transfection complex after the cells adhere to the wall, incubate at 27°C for 5 hours, remove the supernatant, add 2ml of SF-SFM fresh medium, culture at 27°C for 4-5 days, and harvest the supernatant. The recombinant baculovirus rBac-Cap was obtained, and the virus titer was detected by the MTT relative potency method for the harvested P1 generation recombinant baculovirus, and the rBac-Cap P1 virus titer was 3.4×10 7 pfu / mL. The a...
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