Vaccine against african horse sickness virus
a technology of african horses and vaccines, applied in the field of vaccination of subjects against african horses and diseases, can solve problems such as difficulty in immunizing young animals
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example 1
Construction of the Canarypox Recombinant Viral Vectors
[0187]Synthetic genes encoding the VP2 and VP5 proteins of African Horse Sickness Virus were used in the construction of a recombinant canarypox virus vector. Briefly, the L2 and M5 gene segments that respectively encode VP2 and VP5 of African Horse Sickness Virus serotypes 4, 5 and 9 were amplified by reverse-transcriptase polymerase chain reaction (RT-PCR) and sequenced using a protocol previously described by Bonneau K R, Mullens B A, (2001) Bonneau K R, et al. (1999).
[0188]The sequences of the L2 / VP2 (SEQ ID NO:48) and M5 / VP5 (SEQ ID NO:50) genes of a virulent field isolate of AHSV-4 (hereinafter referred to as “the AHSV4 Jane Strain”) were compared to the published sequences of the same genes of other strains of AHS serotype 4 available at GenBank, and optimized synthetic sequences were then derived using GeneOptimizer® software (Geneart GmbH) for chemical synthesis of an array of oligonucleotides that encompass each indivi...
example 2
Construction of the pLHD3460.4 Donor Plasmid Expressing the H6 Promoter-Driven Synthetic AHSV-4-VP2 and the 42K Promoter-Driven Synthetic AHSV-4-VP5
[0191]FIG. 1 shows the construction scheme for pLHD3460.4 (SEQ ID NO:6), the C3 donor plasmid for generation of the ALVAC recombinant expressing AHSV-4-VP2 and AHSV-4-VP5 viral proteins. The genes encoding AHSV-4-VP2 (SEQ ID NO:4) and AHSV-4-VP5 (SEQ ID NO:5) are synthetic with codon optimization for expression in mammalian cells. The synthetic AHSV-4-VP2 (SEQ ID NO:4) gene was placed under the control of vaccinia pC3H6p promoter and the synthetic AHSV-4-VP5 (SEQ ID NO:5) gene was placed under the control of vaccinia 42K promoter. The plasmid contains also a gene conferring ampicillin resistance.
[0192]The plasmid containing synthetic AHSV-4-VP2 gene was digested with BamHI and NruI. The resulting 3.2 Kb AHSV-4-VP2 insert was isolated and cloned into the BamHI / NruI sites of a shuttle vector prepared from pJY1107.5 (pF8 AIV H7N2 HA) to cre...
example 3
Construction of Recombinant Viral Vector vCP2377 (ALVAC C3 H6p-Synthetic AHSV-4-VP2 / 42 Kp-Synthetic AHSV-4-VP5)
[0197]To produce the vCP2377 recombinant viral vector, the donor plasmid, pLHD3460.4 (SEQ ID NO:6), and the parental virus, ALVAC (4.4×1010 pfu / mL), were recombined in vitro using primary chicken embryo fibroblast (primary CEF, or CEF) cells. FIG. 3 outlines this procedure. Plaque hybridization by AHSV-4-VP5 specific probe was used to confirm recombinant viral vector.
[0198]The in vitro recombination (IVR) was performed by transfection of primary CEF cells with NotI-linearized donor plasmid pLHD3460.4 (15 μg) using Fugene reagent (Roche, Palo Alto, Calif. 94304-1353). The transfected cells were subsequently infected with ALVAC (4.4×1010 pfu / mL) as the rescue virus at a multiplicity of infection (MOI) of 10. After 24 hours, the transfected-infected cells were harvested, sonicated and used for recombinant virus screening.
[0199]The recombinant plaques were screened based on the...
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