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. 2010 Feb 10;84(8):4083–4088. doi: 10.1128/JVI.02117-09

FIG. 1.

FIG. 1.

CMV gB expression on surfaces of infected cells and construction of chimeric immunoreceptors. (A, left) Time course of gB surface expression on CMV-infected HFF cells as determined by fluorescence-activated cell sorting (FACS) analysis using the 27-287 antibody and a secondary Cy5-labeled anti-mouse antibody compared to that for an isotype control. (Right) Release of viral particles from infected cells into the supernatant determined by quantitative real-time PCR at the indicated time points. Small amounts of human CMV (HCMV) genomes detected within the first 48 h postinfection are due to residual input virus. (B) Construction of an scFv. RNA was isolated from the hybridoma cell line 27-287, which produces an antibody against CMV gB. cDNA was generated, and the antibodies' variable regions were amplified by PCR, cloned, and sequenced. This was followed by fusion with specific primers in an overlap extension PCR. PCR products were cloned into the vector pBullet #607 (14), which already contained the cIR signaling domains, and sequenced. Vh, variable region of immunoglobulin heavy chain; Vl, variable region of immunoglobulin light chain. (C) Specific binding of 27-287-scFv-myc (gB-scFv-myc) protein to CMV gB was monitored by Western blotting. Protein lysates of uninfected and CMV-infected HFF cells were prepared in radioimmunoprecipitation assay (RIPA) buffer. Twenty micrograms of protein was used for SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, blocked, and incubated (1 h) with the 27-287 hybridoma supernatant or the pCDNA4-27-287-scFv-MH-transfected 293T cell supernatant. gB-scFv-myc protein was detected with anti-myc antibody and anti-mouse horseradish peroxidase (HRP) antibody; 27-287 monoclonal antibody (MAb) was detected with anti-mouse HRP antibody by enhanced chemiluminescence and documented with a Fuji LAS-1000 system. (D) Schematic representation of a chimeric immunoreceptor. The extracellular gB scFv was fused to an Ig hinge region and intracellular signaling domains of the CD28 and CD3ζ cDNAs.