ABSTRACT
Background: The advent of recombinant DNA technology has facilitated heterologous expression of proteins from various sources in different host systems including Escherichia coli. If a plant virus coat protein is expressed in the bacterium it can be used as the antigen for antibody preparation. Such a recombinant antigen preparation can be particularly useful where equipment such as ultracentrifuge is unavailable to purify virus particles to use as the antigen for conventional antibody preparation
Objectives: Heterologous protein expression and purification of the full length Potato virus Y [PVY] coat protein [CP] from isolate pot187 [an affiliate of strain N] to be used as an antigen was the aim of the study
Materials and Methods: Reverse transcription Polymerase Chain Reaction [RT-PCR] was carried out to amplify an 801 bp fragment of the CP gene from PVY-infected potato leaves. The amplicon was cloned into pGEM-T Easy. The cloned fragment was restricted by BamHI + SacI and the purified fragment was cloned into the expression vector pET21a[+] which was restricted with the same enzymes. The generated plasmid was introduced into E. coli strain RosettaTM. The expression was induced with isopropyl-beta-D-thiogalactopyranoside [IPTG] and its protein content was subjected to SDSPAGE and western blotting
Results: SDS-PAGE analysis of protein from the induced bacteria showed a tilde 35 KDa protein corresponding to PVY CP. Expression of the recombinant protein was confirmed by anti-His anitibody
Conclusions: The full-length cDNA of PVY-CP was amplified from the infected potato leaves. The cDNA was heterologously expressed in E. coli. The produced recombinant CP can be used as an antigen to generate polyclonal antibody