Immunolocalization of [alpha]18- and [alpha]12-giardin in Giardia lamblia trophozoites

• Published in: Parasitology research (1987) 2016-11, Vol.115 (11), p.4183
• Main Authors: Wu, Sheng, Pan, Weida, Shi, Xianli, Abdullahi, Auwalu Yusuf, Wang, Zhen, Yu, Xingang, Jiang, Biao, Li, Kangxin, Xu, Chang, Li, Guoqing
• Format: Article
• Language: English
• Subjects: Gene expression; Genetic aspects; Genetic vectors; Giardia; Health aspects; Observations
• ISSN: 0932-0113
• DOI: 10.1007/s00436-016-5194-z

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.