Background and Aims: Serological assay is considered as one of the best choices for conducting large number of infection tests. Recombinant DNA technology has been used for expression of virus coat protein (CP) gene in prokaryotic bacterial cells such as Escherichia coli and the recombinant CP (rCP) is used as immunogen in antibody production. Heterologous CP protein expression and purification of the full length Prunus necrotic ringspot virus-PNRSV, Ilarvirus genus, from an Iranian isolate as an antigen was the aim of the study.
Materials and Methods: A predominant Iranian PNRSV isolate (PK5) was selected and its CP gene was amplified using specific primers and the nucleotide sequence has been determined. The amplicon was cloned into pET28a(+) expression vector. The amplified CP gene and linearized pET-28a(+) were purified from gel, ligated and transformed into BL21 strain of E. coli. Expression of rCP in transformed BL21 competent cells was tested using SDS-PAGE and Western Blot assays.
Results: RT-PCR on total RNA extracted from the infected leaves resulted in a DNA fragment of approximately 688 bp corresponding to full PNRSV/CP. BLAST analysis of the obtained nucleotide sequence for PNRSV/CP revealed 97% identity to JW isolate (accession no. DQ983491). The size of pET-PNRSV/CP was about 6000 bp. The E. coli BL21 cells harboring recombinant pET-PNRSV/CP successfully expressed the recombinant CP after IPTG induction.
Conclusions: In this study, the recombinant CP gene of a predominant Iranian PNRSV isolates expressed in E. coli. The recombinant CP can be used for producing high quality antibodies against PNRSV.