A kind of foot-and-mouth disease virus-like particle vaccine and preparation method thereof
A foot-and-mouth disease virus and particle technology, applied in the field of genetic engineering, can solve the problem of unable to express multiple capsid proteins at the same time, and achieve the effects of complete protective immunity, long immune protection period and lower production cost.
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Embodiment 1
[0049] The gene sequence optimization of the P12A and 3C proteins of embodiment 1 encoding foot-and-mouth disease
[0050] According to GenBank serial number AET43040, (2332 aa linear VRL 15-FEB-2012; DEFINITIONpolyprotein[Foot-and-mouth disease virus-type O] ACCESSION AET43040, the main virus strain (serum type O) and Its gene sequence. On this basis, according to the preference of codons in the translation process of insect cells, the coding gene fragment required for FMD VLP was designed. The packaging of FMD VLP requires P12A and 3C proteins, so the chemically synthesized sequences correspond to P12A respectively Genes and 3C genes.
[0051] The nucleotide sequence of the P12A gene is shown in SEQ ID NO.1, and the sequence structure is: RsrII-Kozaksequence-initiation codon-HBS signal peptide-VP4-VP2-VP3-VP1-2A-stop codon-SV40PolyA Signal - EcoRI.
[0052] The nucleotide sequence of the 3C gene is shown in SEQ ID NO.2, and its sequence structure is: SphI-Polyhedrin promot...
Embodiment 2
[0053] Example 2 Construction of recombinant expression vector pFastBac HT-B-P12A-3C
[0054] 1. The P12A gene and 3C gene of foot-and-mouth disease virus were inserted in series into the transfer vector pFastBac HT-B
[0055] The 3C gene fragment and pFastBac HT-B were double digested with Sph I and Hind III, and the 3C gene fragment was ligated and transformed with pFastBac HT-B after digestion to obtain pFastBac HT-B-3C; The P12A gene fragment and pFastBac HT-B-3C were subjected to double enzyme digestion, followed by ligation transformation. For enzyme digestion results, see figure 1 and figure 2 .
[0056] The above reaction mixture was mixed and centrifuged, then ligated overnight at 16°C. Take out the E.coli DH5α competent cells frozen at -80°C and put them on ice until they melt, add the ligation product, flick the tube wall to mix, ice bath for 30min, heat shock in 42°C water bath for 90sec, immediately ice bath for 1min, add 800μL In LB medium without antibioti...
Embodiment 3
[0066] Example 3 Expression Identification of Recombinant Baculovirus Target Protein
[0067] 1. Sf9 cells transfected with rBacmid DNA
[0068] 1) Cultivation of Sf9 cells Sf9 suspension cells were cultured with SF900 III SFM, and cultured with shaking at 27° C. and 110 rpm. One day before transfection, Sf9 cells were subcultured to 6-well plates with a density of 8×10 per well. 5 .
[0069] 2) Transfection Take two Eppendorf tubes, add solution A and solution B respectively, and the system is as follows: Solution A: Mix 10 μL of recombinant Bacmid DNA and 90 μL of serum-free and antibiotic-free SF900 III medium.
[0070] Solution B: Mix 6 μL of liposome Cellfectin II transfection reagent (mix well by inverting 5-10 times before use) with 94 μL of serum-free and antibiotic-free SF900 III medium.
[0071] After liquid A and liquid B were allowed to stand at room temperature for 5 min, mix liquid A and liquid B, flick the tube wall to mix well, and place at room temperature ...
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