[In vivo identification of the interaction between var intron and an ApiAP2 transcription factor in Plasmodium...

To investigate the potential interaction between the ApiAP2 protein family member, PF3D7_1107800, and var intron of Plasmodium falciparum in vivo.

Genomic DNA was extracted from Plasmodium falciparum (3D7 strain), 5' end gene fragment of PF3D7_1107800 was amplified by PCR, and cloned into pGEX-4T-1 vector. The constructed pGEX-4T-1-PF3D7_1107800N was transformed into E. Coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was purified through glutathione sepharose. Twenty female BALB/c mice were divided into 2 group. Ten mice in experiment group were immunized with a mixture of the purified protein and Freund's adjuvant by subcutaneous injection. Other 10 mice received PBS injection as control. Sera from mice of 2 group were purified with protein G. The effect of the antibody was testified with Western blotting. DNA products of ChIP assay was analyzed for enrichment of anti-PF3D7_1107800 group in ups C var intron by qPCR.

PCR result of the PF3D7_1107800 gene 5' end segment showed that there was a specific band (about 345 bp), which was consistent with the theoretical value. The constructed vector pGEX-4T-1-PF3D7_1107800N was confirmed by gene sequencing. SDS-PAGE and Western blotting analysis demonstrated that the recombinant protein was about Mr 37,000. The anti-PF3D7_1107800 serum was obtained after the immunization of mice with the purified protein, and reacted with the recombinant protein, the specific band was about Mr 200,000. qPCR result showed that the fold enrichment of anti-PF3D7_1107800 group in var intron was two times higher than that of the reference gene region.

The Plasmodium falciparum ApiAP2 family member PF3D7_1107800 binds to ups C var intron region in vivo.