Additional file 3: Figure S3.
TgCyp18 mutants, namely 17GEH19 to 17AAA19 and 149RP150 to 149YV150, which are located in the N and C termini of the protein, respectively, had reduced interactions with CCR5 . To generate TgCyp18 mutants, primers containing an EcoRV site (boldface) (5'-CAT GGA TAT CGA CAT CGA CGC AGC AGC TGC-3') and a NruI restriction site (boldface) (5'-CCG TGA TTT TCG CGA CCT TAG ACA CGT AGC-3') were used. Amplicons were digested with EcoRV and NruI and then ligated into pCR4-TOPO-TgCyp18, which had been treated with EcoRV and NruI to give pCR4-TOPO-MTgCyp18. pCR4-TOPO-MTgCyp18 was digested with NcoI and NheI and the resulting products ligated into pHXNTPHA, resulting in the plasmid, pHXNTP-MTgCyp18HA. The coding sequence corresponding to the full-length TgCyp18 mutant fused to HA (MTgCyp18-HA) was obtained from pHXNTP-MTgCyp18HA by NcoI and BglII digestion. Liberated fragments were treated with the Klenow fragment and inserted into the EcoRV site of pDMG. The pDMG-MTgCyp18HA vector contained expression cassettes for GFP, DHFR-TS and MTgCyp18-HA. The resultant recombinant T. gondii clones of pDMG-MTgCyp18HA were designated RH-DN. Western blot analysis of T. gondii tachyzoite of RH-DN clones (C1, C2, C3) including RH-WT and RH-OE clones (C1, C2 and C3) was performed. Because the RH-DN C3 clone expressed high levels of MTgCyp18-HA it was selected for further study.
Format: TIFF Size: 684KB Download file
Ibrahim et al. BMC Microbiology 2014 14:76 doi:10.1186/1471-2180-14-76