Production of Antiserum to Recombinant Coat Protein for Detecting Lily mottle virus in Yunnan, China

Lily mottle virus (LMoV), Potyvirus genus, is very difficult to purify for the preparation of diagnostic antisera. The coat protein (CP) gene of LMoV was amplified by RT-PCR from infected plants and cloned into the prokaryotic pET-30a vector to generate the recombinant plasmid pET-CP. The resulting carboxy-terminal His-tagged CP was over-expressed in Escherichia coli BL 21 cells by Isopropyl-β- d -thiogalactoside (IPTG) induction and purified over Ni-NTA affinity columns. The purified CP was used to elicit a polyclonal antiserum in rabbits. The antiserum had a titre of 1 : 128 in double diffusion tests, and specifically recognized LMoV in Western blots, Enzyme-Linked Immunosorbent Assays and Immuno-Electron Microscopy. The CP antiserum was also used to evaluate LMoV infection of lily bulbs used for commercial production in Yunnan, China. Substantial levels of infection were found in both imported and native bulbs, and provide the basis to implement flexible, rapid and large-scale virus indexing of lily plants for use in propagation and to meet virus-free quarantine regulations.