IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine
A subunit vaccine, bursal disease technology, applied in the direction of viral peptides, antiviral agents, viral antigen components, etc., can solve the complex procedures of recombinant silkworm baculovirus, expensive insect cell culture medium, and unfavorable farm acceptance, etc. problems, to achieve the effect of intuitive and convenient screening, easy operation, and simplification of tedious steps
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Embodiment 1
[0030] This embodiment describes the method for obtaining the infectious bursal disease virus (IBDV) VP2 gene provided by the present invention. The cloning method is nested RT-PCR, comprising the following steps:
[0031] (1) Extraction of Genomic RNA of Infectious Bursal Disease Virus (IBDV) LYZS Strain:
[0032] The IBDV LYZS strain virus was extracted from the bursal disease material clinically diagnosed as chicken infectious bursal disease (IBD) from Luoyang area. After homogenization, it was extracted according to the TIANamp virus RNA of Tiangen Biochemical Technology (Beijing) Co., Ltd. Kit instructions for the extraction of IBDV genomic RNA.
[0033] (2) Cloning of VP2 gene of IBDV LYZS strain:
[0034]1. Design a pair of primers located in the peripheral region of the VP2 gene sequence according to the sequence of the IBDV strain registered in GenBank to select the conserved region for amplifying a large fragment containing the VP2 gene, with the purpose of determin...
Embodiment 2
[0039] The present embodiment has described the method for producing Infectious Bursal Disease Virus (IBDV) VP2 albumen, and it expresses above-mentioned gene protein with silkworm or chrysalis, and comprises the steps:
[0040] (1) Transform the recombinant vector pFastBac-VP2 obtained above into the Escherichia coli competent cell DH10Bac (Invitrogen, Catalog: 10361012) containing the full-length baculovirus genome shuttle vector, recombine in the Escherichia coli cell, and pass the cyanobacteria Bacmid was screened for the recombinant shuttle vector, and the recombinant shuttle vector was named Bacmid-VP2.
[0041] (2) Transfect insect sf9 cells (Invtrogen, Catalog: 11496015) with the recombinant shuttle vector Bacmid-VP2 described in step (1), and produce recombinant baculovirus in the cells, named vBac-VP2: prepare 5 × 10 5 cells / ml of sf9 cell suspension, insert 2ml / well of sf9 cells into a 6-well cell culture plate, and let stand at 27°C for more than 1 hour to allow th...
Embodiment 3
[0045] This example describes the IBDV genetically engineered subunit vaccine provided by the present invention. It is an injection vaccine: including the above-mentioned dilution of hemolymph of silkworm larvae infected with the recombinant virus, immune adjuvants, preservatives and stabilizers, etc., the above-mentioned silkworm hemolymph is diluted with PBS (0.01M, pH=7.4) When the concentration of agar expansion antigen of VP2 protein reaches 1:16, the obtained dilution is mixed with immune adjuvant at 1:1~1:3 (V:V), and the immune adjuvant includes mineral oil, Siben-80, spit Wen-80, aluminum stearate and ISCOM etc. The ratio of hemolymph diluent and immune adjuvant can be 1:2. The production method is as follows: collect the above silkworm hemolymph, process it with a high-pressure homogenizer, treat it twice at 4°C and 800 bar pressure, centrifuge at 12,000 rpm for 30 minutes, collect the supernatant, and dilute the above-mentioned blood by 200 times with PBS (0.01M, p...
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