Novel coronavirus pneumonia recombinant human type 5 adenovirus vaccine
A technology of adenovirus and virus strains, applied in the direction of viruses, virus peptides, antiviral agents, etc., can solve problems such as economic losses
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Embodiment 1
[0053] Example 1: Materials and methods
[0054] 1 Materials and methods
[0055] 1.1 Virus strains
[0056] rAd-tPASopti, a recombinant human adenovirus strain rAd-tPASopti expressing SARS-CoV-2S protein optimized by mammalian codon bias, was constructed by the important zoonosis and severe exotic disease innovation team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, save. The SARS-CoV-2 isolate HRB25 and the SARS-CoV-2 mouse-adapted strain HRB26M were isolated, identified and preserved by the important zoonoses and severe exotic diseases innovation team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
[0057] 1.2 cells
[0058] Vero E6 cells (ATCC, No.CRL-1586) were preserved and cultured by the important zoonosis and severe exotic disease innovation team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The 293A cells expressing human adenovirus type 5 E1 protein were...
Embodiment 2
[0084] Embodiment 2: experimental result
[0085] 2.1 Construction and rescue of recombinant adenovirus expressing tPASopti6PB protein gene
[0086] The PCR method was used to amplify the tPASopti6PB protein gene with pBlue-tPASopti6PB as a template and primers pShuttle-CMV-F and pShuttle-CMV-R. The tPASopti6PB protein gene was cloned into the KpnI and XhoI sites of the shuttle vector pShuttle-CMV by enzyme digestion and ligation to construct the recombinant shuttle vector pShuttle-CMV-tPASopti6PB containing the tPASopti6PB protein gene. The results of enzyme digestion and sequence determination showed that the tPASopti6PB protein gene was successfully cloned into the pShuttle-CMV vector. The shuttle vector pShuttle-CMV-tPASopti6PB plasmid linearized by the restriction endonuclease PmeⅠ was transformed into Escherichia coli BJ5183-AD-1 competent cells, the tPASopti6PB protein gene was homologously recombined into the pAdEasy-1 vector, the genomic DNA was extracted, and the sp...
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