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. 2021 Dec 23;9(12):e002843. doi: 10.1136/jitc-2021-002843

Figure 1.

Figure 1

Generation and characterization of VV-scFv-TIGIT. (A) A schematic diagram of homologous recombination. To generate VV-scFv-TIGIT, the shuttle plasmid pVV-scFv-TIGIT was used for homologous recombination with a western reserve (WR) strain of VV by using the left (L) and right (R) flanking sequences of thymidine kinase (TK). T2A, thoseaasigna virus 2A. (B) The virus plaque formed in Hela-S3 cells infected with VV-scFv-TIGIT. (C) Western blot analysis of HA-tagged scFv-TIGIT in the supernatants of Hela-S3 cells, a Multiple Tag was used as the positive control. (D) Western blot analysis of scFv-TIGIT in four murine tumor cell lines. (E) ELISA was used to detect the binding of the secreted scFv-TIGIT to the recombinant TIGIT (r-TIGIT). (F) Luciferase-linked immunosorbent assay was used to test the blocking effect of scFv-TIGIT on the binding of PVR and r-TIGIT. (G, H) Crystal violet staining was used to detect the oncolytic ability of VVs against murine tumor cells. (I) TCID50 method was used to detect viral replication in murine tumor cells. ****p<0.0001. scFv, single-chain variable fragment; TIGIT, T-cell immunoglobulin and ITIM domain; VV, vaccinia virus.