1995) strains of were used for protein expression and cloning, respectively

1995) strains of were used for protein expression and cloning, respectively. Expression of recombinant fusion protein Expression of scFv(FMV-NP)-AP fusion protein in was performed as described (Harper et al. and the native virus presented in infected herb extracts was assessed by ELISA, western blot and dot blot assays. Results revealed that scFv(FMV-NP)-AP is able to detect the presence of FMV in infected fig plants. The novel approach, implementing specific recombinant fusion antibody developed in this research, leads to one-step detection of FMV in plants by avoiding the use of chemical enzyme-labeled secondary antibodies. Keywords: Alkaline phosphatase, Fig mosaic computer virus, Fusion protein, Recombinant antibody, scFv Introduction The recent progress in genetic and protein engineering has led to the development of novel fusion proteins with different functions. Fusion proteins are primarily made by the joining of two or more?distinct genes,?coding separate proteins. Expression of this?new construct leads to create novel polypeptide?having functional properties derived from original peptides.?Recombinant DNA technology is usually recruited for making recombinant fusion proteins?that can be used in biological research and?therapeutics (Hermanson 2013; Khan et al. 2016). Applying conventional polyclonal (pAb) and monoclonal antibodies (mAb) in serological assays suffers from low specificity (Khan SN 2 et al. 2016; Liu SN 2 et al. 2010, 2015), uneven quality of pAb, time consuming process, and high costs. Recently, novel progresses in recombinant antibody engineering has enabled expression of recombinant antibodies with desired affinity and specificity (Fields et al. 2013; Maragos et SN 2 al. 2012; Rajakaruna and Robinson 2016; Ren et al. 2020; Wang et al. 2017b). Single-chain fragments variable (scFv) is made of variable regions of light (VL) and heavy chains (VH) of immunoglobulins along with a flexible and short peptide linker. Generating scFv recombinant antibodies has several advantages over traditional antibodies, including low cost and mass-production by fermentation in microorganisms (He et al. 2017). Furthermore, scFv molecules could be genetically labeled with sensitive reporter molecules such as alkaline phosphatase (AP) (homo-dimeric enzyme) which lead to reduce time consumption and exclude applying conventional secondary conjugated antibodies. These enzyme-labeled antibodies (immune-enzyme conjugate protein) are well known and widely applied in clinical and detection assays. Therefore, immune-enzyme conjugates with fusion protein is the product of linking enzymes and antibodies by genetically designed manipulation. Specific antigen detection, large quantity production in bacterial expression system, and uncomplicated manipulating to prepare genetic applications are the main reasons for using scFv as an antigen detection tools (Muller et al. 2001; Rabbani et al. 1995; Safarnejad et al. 2008, 2013; Shahryari et al. 2013). The common fig ((FMV), causing major disease in fig trees, is a member of genus in family of herb viruses (Elbeaino et al. 2018). FMV is usually a negative-strand RNA computer virus which contains six genomic RNA segments. The RNA1, RNA2, RNA3, and RNA4 segments encode RdRp, GP, NP, and a movement protein, respectively. Main symptoms of infected trees are low fruit yield and distortion, leaf mosaic pattern, defoliation, vein banding, chlorosis of leaves, ringspots, and yellow spotting on fruits. FMV have mainly transmitted actually or by eriophyid mites?through fig plantations (Preising et al. 2021). Planting healthful and certified seedlings which have been evaluated via Immunoassay assessments such as micro plate ELISA, is the most influential way to manage the spread of FMV in fig trees. Recently some poly and monoclonal antibodies against the nucleocapsid protein (NP) of FMV has been generated (Shahmirzaie et al. 2019, 2020). Producing of novel AP-recombinant antibody fusion protein would lead to rapid, sensitive and accurate detection of this computer virus in plants. Hence, we have designed a system for bacterial expression of a fusion protein which consists of antibody fragments and alkaline phosphatase, scFv (FMV-NP)-AP. It is appropriate for producing AP-labeled scFv antibodies (Rau et al. 2002; Wang et al. 2017b, 2017c). Applying this novel recombinant fusion antibody leads to decrease time SN 2 needed for immunoassay analysis by ICAM1 avoiding the use of SN 2 AP-labeled secondary antibodies. Recombinant scFv(FMV-NP)-AP conjugates can be used instead of conventionally generated, enzyme-labeled antibodies in immunoassays. Methods Design.