ORIGINAL ARTICLE Expression of recombinant Chrysanthemum virus B coat protein for raising polyclonal antisera Lakhmir Singh & Vipin Hallan & Raja Ram & Aijaz A. Zaidi Received: 27 March 2010 / Accepted: 15 October 2010 / Published online: 28 January 2011 # Society for Plant Biochemistry and Biotechnology 2011 Abstract Chrysanthemum virus B (CVB genus Carlavirus, family Flexiviridae) is one of the major viral pathogens of chrysanthemum. This virus occurs worldwide, is a potential threat to the floriculture industry and hence is a quarantine pathogen. CVB has a positive sense single-stranded RNA (ssRNA) genome ~8.8 kb. The coat protein gene of CVB was amplified by RT-PCR, cloned and over expressed in E. coli BL21. The protein (CP) was expressed as a fusion protein with Glutathione S-Transferase (GST). Fused protein with GST was purified by GST tagged affinity chromatography and polyclonal (but monospecific) antibodies from rabbits immunized with the fusion protein was used for formulation of ELISA based diagnostic kit for CVB detection. The antisera produced showed specific reaction to CVB from infected chrysanthemums, Nicotiana glutinosa, Nicotiana clevelandii and Chenopodium quinoa at dilution of 1:1000 in ELISA. Results obtained were comparable (some times better) than commercial kit. The IgG against CVB performed favorably in specificity and sensitivity to the virus. Findings present a procedure for production of antibodies for CVB indexing of chrysanthemums propagative and mother stock materials to provide the disease free planting material. Keywords CVB detection . Indexing . Polyclonal antisera . Expression . Diagnostics Abbreviations CVB Chrysanthemum virus B ssRNA Single-stranded RNA GST Glutathione S-Transferase E. coli Escherichia coli ELISA Enzyme-Linked Immunosorbent Assay RT-PCR Reverse transcription-polymerase chain reaction IPTG Isopropyl-β-D-thiogalactopyranoside PBS Phosphate buffer saline TBST Tris-buffered saline Tween 20 DAS-ELISA Double antibody sandwich ELISA Introduction Chrysanthemum is ranked among the top ten most important flower crops in the international cut-flower market (Singh 2000). Several viruses and viroids have been reported in chrysanthemum, including Chrysanthe- mum virus B (Raizada et al. 1989), Tomato aspermy virus (O’Reilly et al. 1991), Tomato spotted wilt virus (Martelik and Mokra 1998), Chrysanthemum stem necrosis virus (Verhoeven et al. 1996), Chrysanthemum chlorotic mottle viroid (Romaine and Horst 1975) and Chrysanthemum stunt viroid (Haselhof and Symons 1981). Of these, CVB is one of the major pathogens of chrysanthemum (Koenig 1982) and with a high incidence level (Indian scenario). CVB is associated with symptoms like leaf mottling, vein clearing, mosaic, malformation and slight to severe necrosis (Hollings and Stone 1972). CVB, a member of Carlavirus group (Wetter and Milne 1981) has slightly flexuous, rod- shaped virus particles with 34 kDa coat protein subunits (Hollings and Stone 1972; Levay and Zavriev 1991). It has been reported to occur worldwide (Kryczynski and Stawiszynska 1996; Suastika et al. 1997; Nam et al. 1999; Bellardi and Bertaccini 2000). During survey of chrysanthe- L. Singh : V. Hallan (*) : R. Ram : A. A. Zaidi Plant Virology Lab, Institute of Himalayan Bioresource Technology, Palampur 176061, HP, India e-mail: rnaivi@gmail.com J. Plant Biochem. Biotechnol. (Jan–June 2011) 20(1):96–101 DOI 10.1007/s13562-010-0033-2