Giardins, unique component of the cytoskeleton of Giardia, have been identified as four major classes: alpha ([alpha]), beta ([beta]), gamma ([gamma]), and delta ([delta]) .
The localization experiments showed that a few [alpha]-giardins ([alpha]-3, [alpha]-5, and [alpha]-17) are localized in the adhesive disc; [alpha]-15 and [alpha]-16 giardins had a patchy distribution along the plasma membrane, while the others ([alpha]-1, [alpha]-2, [alpha]-6, [alpha]-7.2, [alpha]-7.3, [alpha]- 9, [alpha]-10, and [alpha]-14) were mainly distributed in plasma membrane, adhesive disc, flagella, or cytoplasm.
For purification of soluble [alpha]-13 giardins, BL21 (DE3) cells harboring pET-28a(+)-g-[alpha]13 were cultured in 1000 mL of LB medium for 5h at 25[degrees]C with 50 [micro]g/mL kanamycin.
Four classes of giardins, [alpha], [beta], [gamma], and [delta], have been identified as cytoskeleton components of the Giardia.
Different giardins have different subcellular localizations in G.
Svard, "Annexin-like alpha giardins: a new cytoskeletal gene family in Giardia lamblia," International Journal for Parasitology, vol.
In this research work, we proceed to provide new reports on prokaryotic expression of [alpha]-13 giardin gene and its intracellular localization in G.
Two primers specific to [alpha]-13 giardin gene, A13E (5'-CGG GAT CCA TGC CTG TTC TGA CCC C37) with Bam HI restriction site (underlined) and A13F (57CCC AAG CTT CTA ATC CAC ATC CCA GAG CC-3') with Hind III restriction site (underlined), were designed based on the G.
Expression of [alpha]-13 Giardin. The recombinant plasmids were transformed into E.
The [alpha]-13 giardin proteins were detected using the ECL Plus Western blotting detection system (Tiannon, Shanghai, China) by rabbit anti-His tag monoclonal antibody (diluted 1: 2000) and horseradish peroxidase- (HRP-) labeled goat anti-rabbit IgG antibody (Transgen, Beijing, China) at dilution of 1: 3000.
Total trophozoite extract and the purified [alpha]-13 giardin fusion protein were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane.
Primary antibody was diluted in dilution buffer (at 1: 500), after reacting with the rabbit anti-[alpha]13- giardin antiserum for 1 h; the samples were washed three times with PBS and then incubated for 1 h in the dark with Alexa Fluor 488 anti-rabbit IgG (H+L) from goat (1:200) (Beyotime, Jiangsu, China) at room temperature.