Ravilov Rustam, Galeeva Antonina, Frolov Gennadiy, Efimova Marina, Zakirova Elena, Rizvanov Albert, Hisamutdinov Almaz, Garipov Lenar, Mingaleev Danil
Kazan State Academy of Veterinary Medicine named after N.E. Bauman, Kazan, Russia.
Federal Center for Toxicological, Radiation and Biological Safety, Kazan, Russia.
Vet World. 2023 Dec;16(12):2425-2430. doi: 10.14202/vetworld.2023.2425-2430. Epub 2023 Dec 6.
Adeno-associated virus serotype 2 (AAV2) represents a promising basis for developing a virus-vector vaccine against African swine fever (ASF). This study aimed to create genetic constructs based on AAV2 to deliver the immunodominant genes of ASF virus (ASFV) and to evaluate their functionality . The efficiency and specificity of transgene expression, as well as their non-toxicity in cells of target animals, were evaluated.
Bioinformatics analysis methods were used to identify the immunodominant genes of ASFV. The target genes , , , and were identified and subsequently cloned into the pAAV-MCS vector. Assembly of recombinant AAV2 (rAAV2) was performed by cotransfection of AAV293 cells with the following plasmids: pAAV-MCS with the gene of interest, envelope, and packaging. Quantitative polymerase chain reaction was used to determine the AAV2 titer. The functionality of the constructs was evaluated in HEK293 and SPEV cells by determining the presence of mature proteins in the cell lysate and the expression levels of messenger RNA. The specificity of the target proteins in cell lysates was confirmed by Western blotting.
The proposed AAV2 assembly protocol makes it possible to achieve a concentration of mature viral particles of at least 280 billion/mL of virus-containing material. The rAAV2 could effectively transduce host SPEV cells. The expression of both cistrons was detectable during the transduction of cells; therefore, the combined expression of immunogens in the cells of target animals should be possible using this method.
This study demonstrated the potential of using genetic constructs based on AAV2 for the delivery of ASFV genes .
2型腺相关病毒（AAV2）是开发抗非洲猪瘟（ASF）病毒载体疫苗的理想基础。本研究旨在构建基于AAV2的基因载体，用于递送非洲猪瘟病毒（ASFV）的免疫显性基因，并评估其功能。评估了转基因表达的效率和特异性，以及它们在靶动物细胞中的无毒性。
采用生物信息学分析方法鉴定ASFV的免疫显性基因。鉴定出目标基因，并随后将其克隆到pAAV-MCS载体中。通过将AAV293细胞与以下质粒共转染来进行重组AAV2（rAAV2）的组装：携带目的基因的pAAV-MCS、包膜和包装质粒。采用定量聚合酶链反应测定AAV2滴度。通过测定细胞裂解物中成熟蛋白的存在情况和信使RNA的表达水平，在HEK293和SPEV细胞中评估构建体的功能。通过蛋白质免疫印迹法确认细胞裂解物中目标蛋白的特异性。
所提出的AAV2组装方案能够使成熟病毒颗粒的浓度达到至少2800亿/mL含病毒材料。rAAV2能够有效地转导宿主SPEV细胞。在细胞转导过程中可检测到两个顺反子的表达；因此，使用该方法应该能够在靶动物细胞中联合表达免疫原。
本研究证明了使用基于AAV2的基因构建体递送ASFV基因的潜力。
