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Prokaryotic Expression of α-13 Giardin Gene and Its Intracellular Localization in Giardia lamblia
- Source :
- BioMed Research International, BioMed Research International, Vol 2017 (2017)
- Publication Year :
- 2017
- Publisher :
- Hindawi, 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.
- Subjects :
- 0301 basic medicine
Cytoplasm
Article Subject
Protozoan Proteins
lcsh:Medicine
Gene Expression
lac operon
Biology
medicine.disease_cause
General Biochemistry, Genetics and Molecular Biology
law.invention
03 medical and health sciences
law
Gene expression
medicine
Animals
Giardia lamblia
Trophozoites
Gene
General Immunology and Microbiology
lcsh:R
General Medicine
Molecular biology
Fusion protein
Recombinant Proteins
Cytoskeletal Proteins
030104 developmental biology
Polyclonal antibodies
Recombinant DNA
biology.protein
Rabbits
Target protein
Research Article
Subjects
Details
- Language :
- English
- ISSN :
- 23146133
- Database :
- OpenAIRE
- Journal :
- BioMed Research International
- Accession number :
- edsair.doi.dedup.....e6d23e85da37ff9124a7846a431be09b
- Full Text :
- https://doi.org/10.1155/2017/1603264