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Prokaryotic Expression of α-13 Giardin Gene and Its Intracellular Localization in Giardia lamblia.
- Source :
-
BioMed Research International . 2/14/2017, Vol. 2017, p1-7. 7p. - Publication Year :
- 2017
-
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-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. [ABSTRACT FROM AUTHOR]
- Subjects :
- *PROTEIN analysis
*ANIMAL experimentation
*BIOLOGICAL assay
*CELL culture
*CYTOSKELETAL proteins
*ELECTROPHORESIS
*ENZYME-linked immunosorbent assay
*ESCHERICHIA coli
*FLUORESCENT antibody technique
*GENE expression
*GENES
*GENETIC techniques
*GIARDIA lamblia
*IMMUNOGLOBULINS
*PEPTIDES
*POLYMERASE chain reaction
*RABBITS
*RECOMBINANT proteins
*RESEARCH funding
*WESTERN immunoblotting
*PHENOMENOLOGICAL biology
Subjects
Details
- Language :
- English
- ISSN :
- 23146133
- Volume :
- 2017
- Database :
- Academic Search Index
- Journal :
- BioMed Research International
- Publication Type :
- Academic Journal
- Accession number :
- 121269732
- Full Text :
- https://doi.org/10.1155/2017/1603264