Immunolocalization of α18-and α12-giardin in Giardia lamblia trophozoites

To study subcellular localization of alpha 18- and alpha 12-giardin in Giardia lamblia trophozoites, the alpha 18- and alpha 12-giardin genes were amplified from G. lamblia assemblage A, respectively. The PCR products were cloned into the prokaryotic expression vector pET-28a(+), and the positive recombinant plasmids were transformed into E. coli Rosetta (DE3) strain for the expression, and expressed alpha 18- and alpha 12-giardin fusion protein were purified by Ni-Agarose resin, respectively. Mice were immunized with purified fusion proteins for preparation of polyclonal antibody, and then the subcellular localization of alpha 18- and alpha 12-giardin was determined by fluorescence immunoassay. Results showed that the concentrations of purified alpha 18- and alpha 12-giardin fusion proteins were 1.20 and 0.86 mg/ml, respectively. The titers of anti-alpha 18- and anti-alpha 12-giardin polyclonal antibody were both as high as 1:25600 dilutions. Immunofluorescent analysis showed that alpha 18- and alpha 12-giardin proteins were mainly localized at four pairs of flagella and the cytoplasm of G. lamblia trophozoites, suggesting that alpha 18- and alpha 12-giardin are the flagella and cytoplasm-associated proteins, respectively. The above information would lay the foundation for research about the crystal structure and biological function of alpha 18- and alpha 12-giardin.