Recombinant Newcastle disease virus vector containing novel coronavirus double-antigen target sequence combination and corresponding vaccine strain and vaccine
A technology of Newcastle disease virus and coronavirus, which is applied in the field of recombinant Newcastle disease virus vectors and corresponding vaccine strains and vaccines, can solve the problems of restriction, limited immunogenicity, decreased viral load in the lungs of immunized mice, etc., and achieve enhanced immunity The effect of exerting originality and reducing toxic and side effects
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Embodiment 1
[0054] Candidate vaccine strains NDV-S2P-VH-RBD-Fc, NDV-S2P-HSA-deltaRBD-Fc, NDV-S2P-HSA-RBD-Folden, NDV-S2P-M-HSA-RBD-TBSV-F, NDV-S2P -M-HSA-deltaRBD-Fc-F, NDV-S2P-M-HSA-deltaRBD-Ferritin-F, NDV-S2P / GSAS-HSA-deltaRBD-Fc, NDV-S2P / GSAS-HSA-deltaRBD-Folden, NDV -S6P-M-HSA-deltaRBD-Fc-F, NDV-S2P-HAS-scRBD, NDV-S2P-M-scRBD-F, NDV-deltaS2P / F87-M-HSA-deltaRBD-Fc-F, NDV-deltaRBD -NTD-Bann-M-HSA-deltaRBD-Fc-F, NDV-deltaRBD-NTD-Folden-M-HSA-deltaRBD-Fc-F and comparative example vaccine strains NDV-S2P, NDV-deltaS2P / F87, and NDV- Design and construction of VH-RBD-Fc viral vector
[0055] The design of the S protein variant is as follows: the K986P and V987P mutations are performed on the S protein of the original novel coronavirus antigen strain to form the S2P coding sequence, and the R682~R685 amino acids of S2P are mutated to GSAS to form the S2P / GSAS coding sequence, and the S2P is further mutated to F817P , A892P, A899P and A942P mutations to form the S6P coding sequence. At the...
Embodiment 2
[0068] 1. Candidate Vaccine Strain Virus Rescue
[0069] All the recombinant NDV viral vectors constructed in Example 1, and the virus rescue helper plasmids pCI-NP, pCI-P and pCI-L respectively, were transfected into BHK21-T7 cells after the plasmid concentration was measured by Nano drop (disclosed in CN112011521A ). The specific transfection process is as follows: spread BHK21-T7 cells on a six-well plate, and when the cell density reaches 70-80%, take 500 μL Opti-MEM into a 1.5 mL centrifuge tube according to the instructions of Lipofectamin LTX, and then follow the 1:1:1: The ratio of 1 was added to the recombinant NDV virus vector in Example 1, and the pCI-NP, pCI-P and pCI-L helper plasmids, and the total amount of plasmids in each tube was 4 μg, which was used for 1 well. Add 3 μL Plus reagent to each of the above solutions, add 9 μl Lipofectamine LTX after 5 min at room temperature, mix gently, and place at room temperature for 30 min; then take out the BHK21-T7 cell...
Embodiment 3
[0075] In Vitro Evaluation of Recombinant NDV Vector Virus Vaccine Strains
[0076] 1. Determination of HA titer of recombinant vaccine candidate strain
[0077] Dilute each virus vaccine candidate strain of NDV collected to 2 1 Finally, multiple samples of the same virus vaccine strain were inoculated with the allantoic fluid of 9-11-day-old chicken embryos, and the inoculated chicken embryos were sealed with medical tape and cultured at 34 °C. After 72 hours, 100 μl of the allantoic fluid was taken and placed in 96 wells. For V-type hemagglutination plate, dilute the allantoic fluid in 10 gradient series with PBS, and take 50 μl of PBS to set up a negative control at the same time to read the HA titer.
[0078] The results showed that most of the HA titers of initially rescued viruses were 4-8log2, and after passage and adaptation, the vaccine candidate strains all produced higher HA titers, up to 10log2 or more (Table 3).
[0079]
[0080] 2. Antigen expression of reco...
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