Prokaryotic Expression of -13 Giardin Gene and Its Intracellular Localization in .

To study prokaryotic expression and subcellular localization of -13 giardin in trophozoites, -13 giardin gene was amplified and cloned into prokaryotic expression vector pET-28a(+). The positive recombinant plasmid was transformed into BL21(DE3) for expression by using IPTG and autoinduction expression system (ZYM-5052). The target protein was validated by SDS-PAGE and Western blotting and purified by Ni-NTA Resin. Rabbits were immunized with purified fusion proteins for preparation of polyclonal antibody; then the intracellular location of -13 giardin was determined by fluorescence immunoassay. The results showed that the length of -13 giardin gene was 1038 bp, encoding a polypeptide of 345 amino acids. The expressed product was a fusion protein with about 40 kDa largely present in soluble form. The target protein accounted for 21.0% of total proteins after being induced with IPTG, while it accounted for 28.8% with ZYM-5052. The anti-13-giardin polyclonal antibody possessed good antigenic specificity as well as excellent binding activity with recombinant -13 giardin. Immunofluorescence assays revealed that -13 giardin was localized in the cytoplasm of trophozoite, suggesting that it is a cytoplasm-associated protein. The present study may lay a foundation for further functional research on -13 giardin of .