Prokaryotic Expression of α-13 Giardin Gene and Its Intracellular Localization in Giardia lamblia. (14th February 2017)

• Record Type: Journal Article
• Authors: Yu, Xingang; Abdullahi, Auwalu Yusuf; Wu, Sheng; Pan, Weida; Shi, Xianli; Hu, Wei; Tan, Liping; Li, Kangxin; Wang, Zhen; Li, Guoqing
• Other Names: Benchimol Marlene Academic Editor.
• Is Part Of: BioMed research international. Volume 2017(2017)
• Publication Date: 2017-02-14
• Subjects: Medicine -- Periodicals; Biology -- Periodicals; Biotechnology -- Periodicals; Life sciences -- Periodicals; 610.5
• DOI: 10.1155/2017/1603264
• Languages: English
• ISSNs: 2314-6133

Abstract

Abstract : To study prokaryotic expression and subcellular localization of α -13 giardin in Giardia lamblia trophozoites, α -13 giardin gene was amplified and cloned into prokaryotic expression vector pET-28a(+). The positive recombinant plasmid was transformed into E. coli BL21(DE3) for expression by using IPTG and autoinduction expression system (ZYM-5052). The target protein was validated by SDS-PAGE and Western blotting and purified by Ni-NTA Resin. Rabbits were immunized with purified fusion proteins for preparation of polyclonal antibody; then the intracellular location of α -13 giardin was determined by fluorescence immunoassay. The results showed that the length of α -13 giardin gene was 1038 bp, encoding a polypeptide of 345 amino acids. The expressed product was a fusion protein with about 40 kDa largely present in soluble form. The target protein accounted for 21.0% of total proteins after being induced with IPTG, while it accounted for 28.8% with ZYM-5052. The anti- α 13-giardin polyclonal antibody possessed good antigenic specificity as well as excellent binding activity with recombinant α -13 giardin. Immunofluorescence assays revealed that α -13 giardin was localized in the cytoplasm of G. lamblia trophozoite, suggesting that it is a cytoplasm-associated protein. The present study may lay a foundation for further functional research on α -13 giardin of G. lamblia .