期刊
RNA BIOLOGY
卷 18, 期 12, 页码 2330-2341出版社
TAYLOR & FRANCIS INC
DOI: 10.1080/15476286.2021.1907055
关键词
Amber codon; genetic code expansion; premature termination codon (ptc); foot-and-mouth disease virus; genetic stability
资金
- Chinese Academy of Agricultural Science and Technology Innovation Project [CAAS-ASTIP-2020-LVRI]
- Frontier Exploration Project of Chinese Academy of Agricultural Sciences [1610312017003]
- National Key R&D Program of China [2018YFD0500103]
- National Natural Sciences Foundation of China [31672585]
The foot-and-mouth disease virus (FMDV) is the causative agent of FMD, a highly infectious disease that affects domestic and wild cloven-hoofed animals, leading to severe economic losses and social consequences. Genetic code expansion technology for generating PTC-FMD vaccines needs further improvement, as amber codons showed a high mutation rate during replication of PTC-FMDV, raising concerns about genetic stability.
The foot-and-mouth disease virus (FMDV) is the causative agent of FMD, a highly infectious and devastating viral disease of domestic and wild cloven-hoofed animals. FMD affects livestock and animal products' national and international trade, causing severe economic losses and social consequences. Currently, inactivated vaccines play a vital role in FMD control, but they have several limitations. The genetic code expansion technology provides powerful strategies for generating premature termination codon (PTC)-harbouring virus as a live but replication-incompetent viral vaccine. However, this technology has not been explored for the design and development of new FMD vaccines. In this study, we first expanded the genetic code of the FMDV genome via a transgenic cell line containing an orthogonal translation machinery. We demonstrated that the transgenic cells stably integrated the orthogonal pyltRNA/pylRS pair into the genome and enabled efficient, homogeneous incorporation of unnatural amino acids into target proteins in mammalian cells. Next, we constructed 129 single-PTC FMDV mutants and four dual-PTC FMDV mutants after considering the tolerance, location, and potential functions of those mutated sites. Amber stop codons individually substituted the selected amino acid codons in four viral proteins (3D, L, VP1, and VP4) of FMDV. We successfully rescued PTC-FMDV mutants, but the amber codon unexpectedly showed a highly degree of mutation rate during PTC-FMDV packaging and replication. Our findings highlight that the genetic code expansion technology for the generation of PTC-FMD vaccines needs to be further improved and that the genetic stability of amber codons during the packaging and replication of FMDV is a concern.
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