Background and Aim:
Alkaline phosphatase (AP) is a group of hydrolases that remove the phosphate group from proteins, nucleotides and alkaloids (dephosphorylation). Moreover analytic detection in competitive immunoassays is carried out with primary or secondary antibodies that are labeled with sensitive reporter molecules like AP. An attractive alternative to the chemical coupling of these antibodies is the construction of genetically engineered fusion proteins consisting of an enzyme and a specific antibody (scFv). In other hand due to the extensive damage of fig disease to fig gardens caused by
fig mosaic virus (FMV), identifying and removing infected seedlings and trees is the first step to combat this disease. Accordingly, in this study
recombinant antibody of FMV fused to AP protein was produced for direct detection of mentioned virus in infected samples.Methods: Gene expressing specific FMV
recombinant antibody fused to AP (scFv-AP) was initially cloned in pET۲۶b bacterial expression vector. The construct was transformed to E.coli (BL۲۱ strain) and cells were inoculated in ۲xTY medium containing kanamycin (۵۰µg / ml). Induction was performed by addition of IPTG to a final concentration of ۱mM for overnight at ۳۰°C while shaking. Expression of
(scFv-AP) constructs was investigated by electrophoresis on SDS-PAGE and Western blotting. Finally, specificity of prepared recombinant scFv-AP antibody against naturally infected plants was evaluated through indirect ELISA and dot blot assays.Results: The results of colony PCR, sequencing and SDS-PAGE confirmed to recombinant (scfv-AP) construct was precisely cloned and expressed in the pET۲۶b expression vector in a right reading frame of the histidine sequence. Furthermore direct detection of FMV in infected figs and FMV recombinant protein by ELISA, western and dot blotting indicated to the successful fusion of
recombinant antibody to AP protein.Conclusion: in this study for the first time,
recombinant antibody of FMV which can be used for rapid and direct detection of infected samples and FMV recombinant protein by ELISA, western and dot blotting is produced. Compared to the monoclonal antibodies of mentioned virus, the monoclonal antibody conjugated has a faster detection, accurate, more sensitive and affordable.