Prokaryotic Expression of [alpha]-13 Giardin Gene and Its Intracellular Localization in Giardia lamblia

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...

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Bibliographic Details
Published in:BioMed research international 2017-01, Vol.2017
Main Authors: Yu, Xingang, Abdullahi, Auwalu Yusuf, Wu, Sheng, Pan, Weida, Shi, Xianli, Hu, Wei, Tan, Liping, Li, Kangxin, Wang, Zhen, Li, Guoqing
Format: Article
Language:English
Subjects:
Adhesives
Biomedical research
Cloning
Cytoplasm
Cytoskeleton
Deoxyribonucleic acid
DNA
Epidemics
Gene expression
Genes
Genetic aspects
Genetic testing
Giardia
Giardia lamblia
Localization
Microbiological research
Physiological aspects
Plasmids
Proteins
Signal transduction
Zoonoses
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Summary: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-α13-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.
ISSN:2314-6133
2314-6141
DOI:10.1155/2017/1603264