Cloning and Prokaryotic Expression of <i>Apple Stem Pitting Virus</i> CP Gene in Ya Pear in <i>E. coli</i>

Abstract

Abstract In order to clarify evolutionary relationship and genetic diversity of apple stem pitting virus (ASPV), and preparation the corresponding special antiserum. In this study, total RNA was extracted from phloem tissue of Ya (Y) pear which infected by ASPV and used as template for cDNA synthesis. The coat protein (CP) gene of ASPV was cloned and sequenced. The CP gene of Ya pear was cloned into expression vector PET-28a, and transformed into E . coli BL21 (DE3), and SDS-PAGE electrophoresis analysis. The results showed that the complete CP gene consisted of 1 194 nucleotides and encodes a polypeptide of 397 amino acid (aa). Comparison of the amino acid sequences of Ya pear CP gene with other ASPV isolates showed approximately 70% similarity. Phylogenetic tree showed that all isolates of the coat protein (CP) genes of ASPV isolates at AA sequence revealed three groups. All ASPV isolates from apple were clustered to group I, whereas pear were clustered to groups â…i (except NC_003462) and the Ya pear were clustered into group â…¢. Results of SDS-PAGE showed that specific expression of a 42 kD fusion protein was achieved by the inducing of 1 mmol/L IPTG. The complete CP gene and prokaryotic expression vector of Ya pear provided additional baseline data for preparation recombinant polyclonal antibody of ASPV and further study of ASPV molecular biology.