EXPRESSION OF SWEET POTATO FEATHERY MOTTLE VIRUS COAT PROTEIN AND PRODUCTION OF SPECIFIC ANTISERUM FOR DIAGNOSTIC ASPECTS

SWeetpotato (Ipomoea batatas L.) has a diverse range of positive characteristics including high yield per unit area, nutritional value, and resistance to several production stresses (Kays, 2005). However, yield of sweetpotato is affected by several pathogens (Clark and Moyer, 1988; Moyer and Salazar, 1989). One of these is the sweet potato feathery mottle virus (SPFMV), a major component of the sweet potato virus disease, SPVD (Gibson et al., 1998; Gibson and Aritua, 2000) combined with the sweet potato chlorotic stunt virus, SPCSV (Brunt et al., 1996). SPFMV has long been recognized to occur wherever sweetpotatoes are grown. SPFMV, a type member of the potyviruses, is transmitted by several genera of aphids including the cotton aphid (Aphis gossypii) and the green peach aphid (Myzus persicae) in a non-persistent manner (Brunt et al., 1996). Symptoms vary with regard to the cultivated variety and environmental factors that often makes identification difficult (Brunt et al., 1990). Serological methods are widely used in the detection of viral infection (Portsmann and Kiessig, 1992). Recently, advances in recombinant DNA technology, coupled with its ease to manipulate and its rapid growing rate in a less expensive media had established Escherichia coli (E. coli) as a leading host organism to produce high protein quantities of scientific interest. One of these interests is the expression of proteins as an antigen for antibody production. Several approaches were used for the expression of antigens. One of the aspects to express proteins in E. coli is to clone genes of interest into an expression vector coding for an amino terminus of a highly expressed protein, carrier protein. The carrier sequence is often an E. coli gene, but it can be a gene from any other organism that is strongly expressed in E. coli. The carrier sequence provides the necessary signals for high expression. In such vectors, the portion of the fusion protein encoded by the carrier can be as small as one amino acid (Amann and Brosius, 1985). The carrier sequence can also code for an entire functional protein. s-galactosidase (Ruther and Muller-Hill, 1983), trpE (Koerner et al., 1991), maltose-binding protein (Guan et al., 1987), glutathione-S transferase (GST, Smith and Johnson, 1988), and thioredoxin (LaVallie et al., 1993) are widely used as carrier regions. They also facilitate the purification of the fusion protein by affinity chromatography. The pGEX vectors are designed for foreign polypeptides expression as fusions to the C terminus of GST, a common 26-kDa cytoplasmic protein of eukaryotes. GST gene used in the pGEX vectors was originally cloned from Schistosoma japonicum (Smith et al., 1986). The fusion proteins remain soluble and can be purified from lysed cells via the GST affinity to glutathione immobilized on agarose beads. Recovery of the fusion proteins is achieved by elution with reduced glutathione at neutral pH. The main advantage of this system for expression and recovery of foreign proteins from E. coli is that most fusion proteins remain soluble. Denaturing conditions are not required at any stage during purification. Consequently, foreign polypeptides may retain their functional activities and antigenicity. In this study, SPFMV coat protein gene was expressed in E. coli as a fusion to GST of pGEX4-1 vector. The purified antigen was used to immunize mice and antiserum production. The produced antiserum was evaluated for its utility of SPFMV detection using dot-ELISA.