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

• Published in: Parasitology research (1987) 2016-11, Vol.115 (11), p.4183-4187
• 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: Animals; Biomedical and Life Sciences; Biomedicine; Crystal structure; Cytoplasm; Cytoplasm - chemistry; Cytoplasm - genetics; Cytoplasm - metabolism; Cytoskeletal Proteins - analysis; Cytoskeletal Proteins - genetics; Cytoskeletal Proteins - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Female; Flagella; Flagella - chemistry; Flagella - genetics; Flagella - metabolism; Fusion protein; Giardia lamblia - chemistry; Giardia lamblia - genetics; Giardia lamblia - metabolism; Giardiasis - parasitology; Humans; Immunoassay - methods; Immunology; Localization; Medical Microbiology; Mice; Microbiology; Original Paper; Plasmids; Polyclonal antibodies; Polymerase chain reaction; Protein Transport; Proteins; Protozoa; Protozoan Proteins - analysis; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Recombinant Proteins - analysis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Structure-function relationships; Trophozoites; Trophozoites - chemistry; Trophozoites - metabolism
• ISSN: 0932-0113; 1432-1955
• DOI: 10.1007/s00436-016-5194-z

To study subcellular localization of α18- and α12-giardin in Giardia lamblia trophozoites, the α18- and α12-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 α18- and α12-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 α18- and α12-giardin was determined by fluorescence immunoassay. Results showed that the concentrations of purified α18- and α12-giardin fusion proteins were 1.20 and 0.86 mg/ml, respectively. The titers of anti-α18- and anti-α12-giardin polyclonal antibody were both as high as 1:25600 dilutions. Immunofluorescent analysis showed that α18- and α12-giardin proteins were mainly localized at four pairs of flagella and the cytoplasm of G. lamblia trophozoites, suggesting that α18- and α12-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 α18- and α12-giardin.