Cloning, Expression And Subcellular Localization Of Giardia Lamblia Alpha-18 And-12 Giardin Genes

Giardia lamblia is a zoonotic protozoan that parasitizes the upper small intestine of human and many mammals, which causes giardiasis typically characterized by diarrhea. The parasite has a highly developed cytoskeleton system composed of microtubules, microfilaments and cytoskeletal proteins. Motility and attachment to the intestinal wall are mediated by cytoskeletal structures, so the pathogenesis of Giardia depends directly on the cytoskeleton. Among a number of cytoskeletal proteins involving in the composition, giardins are one of unique ingredients of the Giardia cytoskeleton. The study of the giardins will provide an empirical basis not only for investigating the pathogenic mechanisms of Giardia but also for new drug targets. So far, there are no reports about ?-18 and-12 giardin, therefore the study aimed at cloning, expression and subcellular localization of G. lamblia ?-18 and-12 giardin genes.Firstly, to study the genetic variation, prokaryotic expression and subcellular localization of G. lamblia ?-18 giardin gene, the primers of ?-18 giardin gene were designed based on reference sequences from Gen BankTM. ?-18 giardin genes were amplified from G. lamblia assemblages A and F by PCR and were cloned into p MD-18 T vector. The sequenced ?-18 giardin genes were analyzed by bioinformatics software. According to theoretical value obtained by the bioinformatics tool, the p ET-28 a vector was screened as the prokaryotic vector, and then the recombinant plasmids p ET28a-A?human?-AG-18 and p ET28a-F?cat?-AG-18 were transformed respectively in Escherichia coli Rosseta?DE3? strain for the expression. The expressed ?-18 giardin fusion protein was validated by SDS-PAGE and Western-blot analysis, and purified by Ni-Agarose resin. Mice was immunized with purified fusion protein?human-derived? for preparation of polyclonal antibody, and then to determine the subcellular localization of ?-18 giardin by fluorescence immunoassay. Results showed that the ?-18 giardin gene was 861 bp in length, encoding 286 amino acids; it was 100% homologous between human-derived and dog-derived G. lamblia assemblage A, but it was 86.8% homologous with G. lamblia assemblage F?cat-derived?. ?-18 giardin fusion protein was about 36 k Da in molecular weight, with good reactivity. Prediction based on in silico analyses: it had hydrophobicity, without signal peptide and transmembrane domain, and contained 11 alpha regions, 13 beta sheets, 1 beta turn and 7 random coils in secondary structure. The concentrations of purified ?-18 giardin were 1.18 mg/ml and 1.5 mg/ml for human- and cat-derived ?-18 giardins, respectively. The titer of ant-?18 giardin polyclonal antibody was as high as 1:25600 dilutions. The localization showed that ?-18 giardin was mainly observed at four pairs of flagella of G. lamblia trophozoites, suggesting that ?-18 giardin is flagella associated protein. The above information would lay the foundation for research about the biological function of ?-18 giardin in G. lamblia.Secondly, in order to carry out the prokaryotic expression and subcellular localization of G. lamblia ?-12 giardin, the specific primer of ?-12 giardin gene was designed based on reference sequence from Gen BankTM. ?-12 giardin gene was amplified from G. lamblia assemblages A by PCR and was cloned into p MD-18 T vector. The sequenced ?-12 giardin gene was analyzed by bioinformatics software. According to theoretical value obtained by the bioinformatics tool, the p ET-28 a vector was screened as the prokaryotic vector, and then the recombinant plasmid p ET28a-AG-12 was transformed in E. coli Rosseta?DE3? strain for the expression. The expressed ?-12 giardin fusion protein was validated by SDS-PAGE and Western-blot analysis, and purified by Ni-Agarose resin. Mice was immunized with purified fusion protein for preparation of polyclonal antibody, and then to determine the subcellular localization of ?-12 giardin by fluorescence immunoassay. Results showed that the ?-12 giardin gene was 972 bp in length, encoding 323 amino acids; it was 100% homologous with reference sequence?XM₀01708393?. ?-12 giardin fusion protein was about 40 k Da in molecular weight, with good reactivity. Bioinformatics analysis revealed that it had hydrophily, without signal peptide and transmembrane domain, and contained 5 alpha regions, 1 beta sheets, 4 beta turn and 4 random coils in secondary structure. The concentration of purified ?-12 giardin was 0.86 mg/ml. The titer of ant-?12 giardin polyclonal antibody was as high as 1:25600 dilutions. The localization showed that ?-12 giardin was mainly observed at the cytoplasm of G. lamblia trophozoites, suggesting that ?-12 giardin is a cytoplasm associated protein. The above information would lay the foundation for research about the biological function of ?-12 giardin in G. lamblia.