Abstract
Bunchy top disease is one of the most destructive viral diseases caused by the Banana Bunchy Top Virus (BBTV). Using of virus particles as an antigen for polyclonal and monoclonal antibodies production by common serological techniques has several disadvantages concerning the purity and concentration of the viral particles and by extension the produced antibodies. In this study, the coat protein gene (CP) of BBTV was expressed using baculovirus expression vector system (BEVS) under the control of the Polyhedrin promoter (Polh). Accordingly, the generated cassette consisting of the CP gene and the improved Green Florescent Protein (GFP) gene. The generated recombinant virus (vAc-CPpolh-GFPp10) was proved using PCR analysis. Spreading of the recombinant virus in Sf9 insect cells was successfully detected using GFP protein florescence under inverted fluorescent microscopy. The generated recombinant virus was amplified in Sf9 cells and the expression of CP was tested using the infected cell lysate by Dot blot analysis. The anti-BBTV polyclonal antibodies serologically reacted with the recombinant CP which was expressed in Sf9 cells as well as to the BBTV infected plants. This result suggested the possibility of using the expressed CP of BBTV for further development of antisera that can be implemented for BBTV detection in infected plants.
Highlights
By the Banana Bunchy Top Virus (BBTV)
The amplified 513 bp coat protein gene (CP) gene fragment was subsequently inserted into pFastBac-Dual vector downstream of the Polh promoter generating the modified plasmid pFBD-CPpolh
The generated cassette consisting of the CP gene and the improved Green Florescent Protein (GFP) gene
Summary
By the Banana Bunchy Top Virus (BBTV). Using of virus particles as an antigen for polyclonal and monoclonal antibodies production by common serological techniques has several disadvantages concerning the purity and concentration of the viral particles and by extension the produced antibodies. The generated recombinant virus (vAc-CPpolhGFPp10) was proved using PCR analysis. Spreading of the recombinant virus in Sf9 insect cells was successfully detected using GFP protein florescence under inverted fluorescent microscopy. The generated recombinant virus was amplified in Sf9 cells and the expression of CP was tested using the infected cell lysate by Dot blot analysis. The anti-BBTV polyclonal antibodies serologically reacted with the recombinant CP which was expressed in Sf9 cells as well as to the BBTV infected plants. This result suggested the possibility of using the expressed CP of BBTV for further development of antisera that can be implemented for BBTV detection in infected plant
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