Bovine viral diarrhea-infectious bovine rhinotracheitis bivalent subunit fusion vaccine and identification method thereof
A technology for bovine viral diarrhea and rhinotracheitis virus, which can be applied to vaccines, viruses, and viral peptides, and can solve problems such as difficult differential diagnosis, high content of foreign proteins, and large side effects
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Embodiment 1
[0081] The target genes of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 were synthesized by the biological company, and the primer sequences of SEQ ID NO.5-SEQ ID NO.12 were amplified respectively. Add the above target gene. The result is as figure 1 Shown (where, M: marker, 1: BVDV-E2, 2: BVDV-NS3, 3: IBRV-VP8, 4: control, 5: IBRV-gB).
Embodiment 2
[0083] Connect and Transform
[0084] 1. Plasmid pET-30a (+), BVDV-NS3 gene (SEQ ID NO.1), BVDV-E2 gene (SEQ ID NO.3), IBRV-VP8 gene (SEQ ID NO.2), IBRV-gB gene (SEQ ID NO.4) were subjected to KpnI and BamHI double enzyme digestion respectively;
[0085] 2. Perform 1% agarose gel electrophoresis on the digested product, recover the target band from the gel and purify it;
[0086] 3. T4-DNA ligase connects the plasmid and each gene respectively;
[0087] 4. Kanamycin (100ml / L) solid medium to screen BVDV-NS3-pET-30a (+), BVDV-E2-pET-30a (+), IBRV-VP8-pET-30a (+) and IBRV- gB-pET-30a(+) was transformed into Escherichia coli BL21 positive strain;
[0088] 5. Identification of recombinant prokaryotic expression vector by double enzyme digestion.
[0089] Prokaryotic expression and purification
[0090] 1. Pick a single colony of Escherichia coli BL21 containing the expression vector in LB solid medium (containing 100mg / L kanamycin) and culture it overnight in 20mL of LB liqui...
Embodiment 3
[0098] immune experiment
[0099] 1. Take 300 μg of purified prokaryotically expressed BVDV-NS3 and 500 μg of IBRV-VP8 protein, mix and emulsify with adjuvant ISA206VG, and prepare 10 vaccines for cattle.
[0100] 2. Randomly select 20 cows over 3 months old from cattle farms negative for bovine viral diarrhea and bovine infectious rhinotracheitis, and randomly divide them into an experimental group and a control group, with 10 cattle in each group. The experimental group was inoculated intramuscularly, with 1ml of each head containing 30 μg of BVDV-NS3 and 50 μg of IBRV-VP8 protein; the control group was inoculated with normal saline, and each head was inoculated with 1ml.
[0101] 3. Continue to observe for 14 days after immunization, and monitor body temperature changes, appetite, and mental changes. The results found that compared with the control group, the body temperature of the cattle in the experimental group fluctuated 24 hours after vaccination, with a fluctuatio...
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