Production of polyclonal antisera to a recombinant coat protein of Potato virus Y expressed in Escherichia coli and its application for immunodiagnosis.

摘要

A polyclonal antiserum to a recombinant Coat Protein (CP) of Potato virus Y (PVY) was developed and its effectiveness was measured with double antibody sandwich immunosorbent assay (DAS-ELISA), indirect ELISA (I-ELISA), Indirect Plate Trapped Antigen (IPTA) ELISA, Western Blotting (WB) and dot blotting immuno binding assay (DBIA). The CP gene of PVY was amplified with the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using primers, designed from a recombinant CP sequence for PVYO (common strain) and PVYN (the necrotic strain), to amplify the full CP from a mixture of different PVY isolates (PVYO, PVYN, PVYNTN and PVYN:O). The full 800 bp-CP amplicon gene was cloned and expressed into pBAD-C terminal 6xHis Tag TOPO expression vector in Escherichia coli BL21. The CP fraction from bacterial lysates was purified, under native and denatured conditions, by nickel-nitrilotriacetic acid (Ni-NTA) batch chromatography; yielding 0.6 mg mL-1. Antigenicity of the purified CP fraction was measured with Western Blotting (WB) analysis. For immunization, the CP fusion protein was injected into rabbits. The recombinant PVY-CP antiserum reacted with PVY-infected potatoes using IPTA-ELISA and DBIA and with a wide spectrum of local and foreign strains of PVY including PVYO, PVYN and PVYNTN in DAS-ELISA and DBIA. The data indicated that the produced recombinant antiserum was efficient and accurate in determination of negative and positive results in ELISA tests. Therefore, this antiserum is suitable for certification programs of potatoes due to its low cost, high specificity, feasibility and its endless supply from recombinant bacterial clones carrying the CP genes for this virus.