Prokaryotic Expression And Intracellular Localization Of ?-13 And EF-1? Of Giardia Lamblia Trophozoite

Giardia lamblia is a common zoonotic parasitic protozoa that parasitize in the small intestines of humans and other animals, which can lead to serious abdominal cramps, acute or chronic diarrhea and malabsorption. The trophozoites of G. lamblia has abundant and complex cytoskeleton system(microtubules, microfilaments and cytoskeletal protein), and often release some secretory proteins when co-culture with intestinal epithelial cells. Both the cytoskeleton and the secretory protein are closely related to the pathogenesis of G. lamblia. Therefore, G. lamblia assemblage A was taken as the research object in this study, the DNA encoding ?-13 giardin and EF-1? were amplified by PCR and the bioinformatic analysis of their genes were conducted respectively. The target protein were expressed by induction after their recombinant plasmid p ET-28a(+)-g-?13 and p ET-28a(+)-EF-1? transformed into E. coli BL21(DE3). The target proteins were purified by Ni-NTA Resin and were used to immunize laboratory animals to produce polyclonal antibodies. Finally, high titer polyclonal serums were obtained respectively and specific antiserum were selected out to determine the intracellular location of ?-13 giardin and EF-1? with fluorescent microscope. The present study may lay a foundation for functional research on ?-13 giardin and EF-1? protein of G. lamblia.Firstly, prokaryotic expression and intracellular localization of ?-13 giardin gene of G. lamblia trophozoite were conducted. The DNA encoding ?-13 giardin was amplified by PCR and cloned into prokaryotic expression vector p ET-28a(+). The positive recombinant plasmid p ET-28a(+)-g-?13 was transformed into E. coli BL21(DE3). The target protein was expressed by using conventional Isopropyl ?-D-Thiogalactoside(IPTG) induction and auto-induction expression system(ZYM-5052), validated by SDS-PAGE and Western blot analysis, and purified by Ni-NTA Resin. The purified protein that emulsified with equal amounts of Freund's adjuvant was used to immunize Kunming mice to produce polyclonal antibodies. Specific antiserum was verified and selected out by Western blot, and then was used to determine the intracellular location of ?-13 giardin with fluorescent microscope. The results showed that the length of ?-13 gene was 1038 bp, encoding a polypeptide of 345 amino acids. The SDS-PAGE and Western blot analysis showed that the expressed product was a fusion protein with about 40 k Da largely present in the soluble form. The antibody possessed good antigenic specificity as well as excellent binding activities with antigens. Immunofluorescence assays revealed that ?-13 giardin proteins mainly localized to the plasma membranes and the flagella.Secondly, prokaryotic expression of EF-1? gene and intracellular localization of EF-1? of G. lamblia trophozoite were carried out. The DNA encoding EF-1? was amplified by PCR and cloned into prokaryotic expression vector p ET-28a(+). The positive recombinant plasmid p ET-28a(+)-EF-1? was transformed into E. coli BL21(DE3). The target protein was expressed by using conventional IPTG induction, validated by SDS-PAGE and Western blot analysis, and purified by Ni-NTA Resin. Synthetic antigenic peptides designed using bioinformatics were coupled to KLH(an immunogen) and then administered to rabbits. The obtained antibodies were purified using protein A resin. The affinity and specificity of antibodies were determined in Western blot using recombinant EF-1? protein. The anti-FL1192 antibody was used to localize EF-1? in trophozoites by immunofluorescence. The results showed that the length of EF-1? gene was 1329 bp, encoding a polypeptide of 432 amino acids. The SDS-PAGE and Western blot analysis showed that the expressed product was a fusion protein with about 50 k Da. The fusion protein with high purity was obtained. The anti-FL1192 antibody possessed good antigenic specificity as well as excellent binding activities with fusion protein. The EF-1? protein mainly localized to the cytoplasm around the two nucleus.