Publication Date:
2021-05-19
Description:
In this study lusiferse gene extracted from Vibrio fischeri. V. fischeri strain was kindly provided by Iranian
Research Organization for Science and Technology (IROST). Genomic DNA extraction was carried out by
Phenol-chloroform. A DNA fragment encoding the luxA and LuxB was amplified by PCR using sense and
antisense primers, specific restriction sites for BamH1 and Kpn1 were introduced into 5´ end of forward and
reverse primer, respectively. The PCR product was purified from agarose gel and ligated into cleaved PT257R
cloning vector. Following the confirmation of the cloned Luciferase transformed into E. coli. Recombinant
clones were confirmed by specific PCR and restriction enzyme digestion analysis. The luxA and LuxB fragment
was released subcloned in to the PcDNA3.1\hug and PcDNA3.1\neo expression vectors, respectively.
recombinant plasmid was confirmed through restriction digestion using BamH1 and Kpn1 enzymes and
subsequently, transformation procedure continued into NIH3T3 eukaryote cells by specific kit. Luminescence
ability of recombinant clones was tested by NIH3T3 cells and dechanal (substrate) and Neomysin and
hygromysin. The results showed that luminescence start after 2 hours and then increase after 6 hours. Inaddition,
the protein identity was verified by western blot analysis, the protein bands 76 kD were detected. , which
indicates protein expression of luxB, luxA.
Description:
Iranian Fisheries Science Research Institute
Description:
Published
Keywords:
Lusifrase
;
Vibrio fischeri
;
Transfer gene
;
DNA
;
Lusiferase gene
;
Investihation
;
Genetic
;
Cyprins carpio
;
Rutilus frisii kutum
Repository Name:
AquaDocs
Type:
Report
,
Refereed
Format:
58pp.
