Overexpression of Ehcp112 gene in E. histolytica

Ehcp112 complete gene was PCR amplified with specific primers containing BamHI and KpnI restriction sites (forward: 5′-GGGGTACCATGACAGCGATTGTTGTCGCTTTTTT-3′, reverse: 5′-CGGGATCCTTACTTATCGTTCGTCATCCTTGTAATCGATTGTATGATTGTAGAATTGG-3′) under the following conditions: 95°C 15 s, 60°C 30 s, 72°C 90 s during 30 cycles. Then, the PCR product was cloned into pJET1.2 plasmid (Fermentas) according to the manufacturer recommendations. The Ehcp112/pJET construction was digested and the Ehcp112 fragment was subcloned in the pExEhNeo plasmid (Hamann et al., 1995 (link)). This plasmid was amplified in E. coli DH5α, purified by Qiagen Midi Kit (Qiagen) and automatically sequenced. Transfection was performed as described (Avalos-Padilla et al., 2015 (link)). Transfected trophozoites were selected with increasing concentrations of G-418 (a neomycin analog) until stable growth was achieved (20 μg/ml). Overexpression of the Ehcp112 gene was verified by RT-PCR by specific primers and using s2 ribosomal gene as internal control. WB assays were performed using α-EhCP112 (1:5,000) and α-actin (1:3,000) antibodies. Laser confocal microscopy assays were performed using α-EhCP112 (1:100) antibodies in paraformaldehyde fixed and triton X-100 permeabilized trophozoites and nuclei were stained by propidium iodide.