关键词: cGAS, PK-15, 稳定细胞系, 慢病毒包装系统

Abstract:

The pathogenic microorganisms can be sensed and recognized by innate immune system once invaded the animals. And the sensing of conserved structural components called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) of the hosts activates a series of signaling events, leading to induction of downstream antiviral effector proteins including type Ⅰ interferons (IFNs) and proinflammatory cytokines to clear the invaders. The nucleotidyltransferase GMP-AMP (cGAMP) synthase (cGAS) is ubiquitously expressed in various types of cells and tissues, and is still the most crucial cytosolic DNA sensor till now. However, the transcription and expression of cGAS are almost undetectable in porcine kidney cell lines PK-15, which play central roles in the study of swine viral disease. To construct the PK-15 cell lines stably expressing cGAS, the cGAS gene was inserted into the genome of the target cells using lentivirus packaging system, following by the identification of gene transcription, expression, and distribution. Finally, the DNA viruses pseudorabies virus (PRV) and porcine parvovirus (PPV) were used as model viruses to confirm the role of cGAS in innate antiviral immunity. The results showed that cGAS is successfully integrated into the genome of the PK-15 cells, and can be detected in the recombinant cell lines PK-15-cGAS as early as generation 3. The infection of PRV and PPV induced markedly higher levels of interferon-related genes, and consistently the replication of PRV and PPV was dramatically inhibited in PK-15-cGAS cells. In summary, the PK-15 cell lines stably expressing cGAS were prepared successfully, which will help to explore the mechanisms of viral infection as well as immune evasion.

Key words: cGAS, PK-15, stable cell line, lentivirus packaging system