Type 1 bovine viral diarrhea virus-like particle as well as preparation and application thereof
A virus-like, particle-based technology, applied in applications, viruses, antiviral agents, etc., can solve the problems of short immune duration and low immune activity, and achieve good immune effect, good compatibility, and improved efficiency
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Embodiment 1
[0033] Embodiment 1: Construction of a kind of BVDV-1 structural protein gene recombinant baculovirus
[0034] 1. Construction of pFBD-BVDV-1 recombinant baculovirus transfer vector
[0035] According to the sequence of the structural protein coding gene of type 1 BVDV strain GS4 (partially submitted to GenBank, accession number: KC700344), an upstream primer (SEQ ID No.3) 5'-GCC with a restriction site was designed GGATCC BamHI ATG TCCGACACAAATACAGAAGG-3' and downstream primer (SEQ ID No.4) 5'-CTC AAGCTT HindIII CTAs ACCCGAGGTCATTTGTTCTG-3', with start codon and stop codon (italics) added to the upstream primer and downstream primer, respectively, for type 1 BVDV structural protein C-E rns - Amplification of genes encoding E1-E2. By reverse transcription polymerase chain reaction (RT-PCR), using type 1 BVDV viral RNA as a template, using upstream primer (SEQ ID No.3) and downstream primer (SEQ ID No.4) to amplify C-E rns -E1-E2 coding genes, recovery of target frag...
Embodiment 2
[0041] Example 2: Expression and preparation of type 1 BVDV VLPs in insect cells
[0042] Seed sf-9 cells in 5 cm 2 Cell culture flasks were inoculated with 1 MOI of F3stock seed virus when the cells grew into a 90% monolayer, and the supernatant was collected and the cells were ultrasonically lysed at 72 hours, and the cell debris was removed by centrifugation at 4000g for 30 minutes at 4°C, and the Amicon Ultra- Concentrate the supernatant to 2 mL in 15 ultrafiltration centrifuge tubes, then add 10-60% sucrose density gradient and centrifuge at 4°C and 35,000 rpm for 2 hours in a Beckman ultracentrifuge (SW41 rotor), take 1 mL and label the order to prepare E2 protein Western detection was performed. For the detection of samples containing E2 protein, take 10 μL and drop it on a 200-mesh copper grid covered with a support film and a carbon film, absorb at room temperature for 3 minutes, stain with 3% phosphotungstic acid after drying, and use FEI Tecnai G 2 F20 transmissi...
Embodiment 3
[0043] Example 3: Preparation of type 1 bovine viral diarrhea virus-like particle vaccine and assay of immune efficacy
[0044] The F3stock seed virus in Example 1 was used to inoculate sf9 cells at an MOI of 1, and the diseased cells were collected 72 hours after inoculation, ultrasonically disrupted and centrifuged at 4°C, 6000g for 10 min to remove cell debris, and the BCA protein concentration assay kit (Beiyuntian Biology) was used to inoculate sf9 cells. technology company) to measure the protein concentration of the crude antigen, then inactivate it with 10mM binary ethyleneimine (BEI) for 48h, add a final concentration of 15mM sodium thiosulfate for neutralization, and fully emulsify with Merckinade SDA-201 biphasic oil adjuvant for at least Drop in cold water in the form of oil droplets without spreading. The prepared type 1 bovine viral diarrhea virus-like particle vaccine is aseptically dispensed quantitatively and stored at 4°C.
[0045] Two BVDV antibody-negative...
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