Optimization of the protocol for the assembly of recombinant adenoassociated serotype 2 viruses for the delivery of African swine fever virus genes into mammalian cells
Year: 2024
Type: article
Abstract: African swine fever (ASF) is a highly contagious viral disease of theSuidaefamily representatives, the mortality rate in primary foci of which reaches 100 %. To date, no specific means of preventing ASF have been developed. Despite the fact that researchers have proposed various methods for creating candidate vaccines against ASF, the issue of developing alternative antigenic variants with low reactogenicity and high immunogenicity is still relevant. It is known that the production of recombinant adeno-associated virus, a potential tool for delivering ASF virus target genes into mammalian cells, is influenced by many factors, in particular, the cell line, expression system, cell culture conditions after transfection, and the quality of the initial plasmid DNA. This work presents the results of optimization of the assembly protocol for recombinant AAV2 carrying the major capsid protein gene of the ASFVB646Las a model cargo. During the research, it was established that the protocol used allows to achieve a veritable virus titer of (2.45 ± 0.17) × 107 viral particles per μl, while the share of fully assembled viral capsids accounts for up to (79.3 ± 2.3) % of all genomic copies. When assessing the potential cytopathogenic effect of recombinant AAV2 on target cells (SPEV, porcine MSCs), it was found that high MOI (up to 10,000 viral particles per cell) does not lead to an increase in the proportion of apoptotic cells. The functionality of the developed AAV2-based construct was confirmed: in the lysates of transduced cells, the mature p72 protein with a molecular weight of 73 kDa was detected, specifically reacting in a western blot with hyperimmune pig serum. Our data confirm the potential of AAV2 as a tool for delivering ASF virus genes into porcine cells, which makes it a promising basis for the design of candidate vaccines.
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: hybrid
OpenAlex ID: W4396574530