II type adenovirus live vector recombinant vaccine of dog for showing lyssa virus protective antigen by using spike
A rabies virus and protective antigen technology, which is applied in the development and application of new animal rabies vaccines, and can solve problems that have not yet been reported.
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Embodiment 1
[0027] Example 1: Cloning of the complete genome of recombinant vaccines displaying rabies virus protective antigens with six neighbor particles
[0028]Eco52I and SnaB I double digested pPOLYII-CAV2, recovered a 12516bp fragment by agarose gel electrophoresis, and cloned it into the pET-28a vector to obtain the recombinant plasmid pET-LH. Then pET-LH was double-digested with Nde I and Mlu I, and the 4325bp fragment was recovered by agarose gel electrophoresis, and cloned into pET-28a vector to obtain the recombinant plasmid pET-SH. The neutralizing epitopes of rabies virus were inserted into four different sites in the hexon by overlapping PCR, and the PCR products were cloned into pMD18-T vector for sequence determination. After the sequencing was correct, the modified hexosome gene was cloned into pET-SH using the two natural restriction sites of hexosome Kpn I and Dra I. Finally, according to the above route, four kinds of recombinant canine adenovirus type II whole genom...
Embodiment 2
[0055] Example 2: Cloning of the complete genome of recombinant vaccines displaying rabies virus protective antigens with fibers
[0056] Using the enzyme cutting sites Sac I and Kpn I in pBluescriptII KS (+ / -), insert the artificially synthesized sequence Linker SK (containing multiple enzyme cutting sites of Sal I, Xma I, Spe I and Pac I in sequence) to obtain recombinant Plasmid pBS-SKI. According to the filament gene sequence, artificially synthesized canine adenovirus type II genome sequence 28050bp ~ 28106bp segment, and inserted it into Linker SK between Sal I ~ Xma I, and obtained the sequence of 30182bp ~ 29358bp in the whole genome of canine adenovirus type II virus by PCR , Insert the two fragments between Spe I~Pac I in Linker SK to obtain the recombinant plasmid pBS-SKII. Then insert the rabies virus neutralizing antigenic epitope between Xma I and Spe I sites in the Linker SK of pBS-SKII according to the transcriptional reading frame of the spike gene to obtain ...
Embodiment 3
[0072] Embodiment 3: the cloning of the complete genome of the recombinant vaccine that displays rabies virus protective antigen with IX protein
[0073]EcoR I and Cla I double-digested pPOLYII-CAV2, recovered a 3610bp fragment by agarose gel electrophoresis, and cloned it into pBluescriptII KS (+ / -) to obtain the recombinant plasmid pBS-LIX. Mlu I and EcoR I double digested pBS-LIX, agarose gel electrophoresis recovered a 340bp fragment, cloned into pEGFP-C1, and obtained the recombinant plasmid pEGFP-SIX. At the stop codon of the IX protein, a single restriction site Ase I was used to insert the rabies virus glycoprotein outer region gene (G') and the SV40 poly A transcription termination signal sequence to realize the fusion expression of the IX protein and the glycoprotein outer region . Finally, according to the above-mentioned route, reverse cloning to obtain the whole genome plasmid pPOLYII-CAV2 / IX-G' of recombinant canine type II adenovirus.
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