Molecular characterization of Tuberose mild mosaic virus and preparation of its antiserum to the coat protein expressed in bacteria

A 2-kb DNA product was amplified from purified Tuberose mild mosaic virus (TMMV) virions as well as from infected tissues of tuberose by the use of degenerate primers for potyvirus. The PCR product was subsequently cloned and its sequence analyzed. It was found comprised of 1947 nucleotides (nts) corresponding to the 3´-terminal region of potyviruses. The deduced amino acid sequence contained 598 residues encoding part of the 3´-terminal region of NIb gene (319 residues) and the complete sequence of coat protein (CP) gene (279 residues). A 136 nts of non-coding region (NCR) was found located at the 3´-terminal region of the DNA. A genetic code for aphid transmissibility of potyviruses, DAG triplet, was found at the 19-21 residues from the N-terminus of CP gene. Compared to the known sequences of potyviruses, the percent of nucleotide identities of the CP gene and the NCR were less than 62% and 39%, respectively. Similarly, percent identities of TMMV's CP amino acid sequence to those of other known potyviruses were all below 58%, confirming our previous finding that TMMV is a new species of Potyvirus. Using directional cloning technology, a 39-kDa fusion protein containing a complete CP sequence of TMMV and a partial sequence encoded by the expression vector plasmid (pET-30b, Novagene) was highly expressed and purified from E. coli cell cultures. The antigenicity of the fusion protein was determined to be indistinguishable from the viral CP. Antiserum prepared against this fusion protein showed comparable reactivities in the serological detection of TMMV with the conventional antibodies against purified virus particles