Molecular Cloning And Characterization Of A Venom Phospholipase A₂ From The Bumblebee Bombus Ignitus

With the development of the study on the life sciences, such as molecular biology and genetic engineering technology, It has become possible to make the research of the insects in medical use in a molecular level. In this study, we propose the molecular cloning, expression and characterization of Bombus ignitus Phospholipase A2 (Bi-PLA2)gene.Phospholipase A2 (PLA2) is one of the main components of bee venom. Here, we identify a venom PLA2 from the bumblebee, Bombus ignitus. Bumblebee venom PLA2 (B1-PLA2) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags (ESTs), encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA2 gene. Both genomic sequence and the cDNA sequence of Bi-PLA2 gene are first reported sequence. Therefore, they have been submitted to Gene Bank, the accession number are FJ768907 and FJ768908, respectively.Bi-PLA2 is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA2 (136 amino acids) possesses features consistent with other bee PLA2S, including ten conserved cysteine residues, as well as a highly conserved Ca2+-binding site and active site. Phylogenetic analysis of bee PLA2S separated the bumblebee and honeybee PLA2 proteins into two groups.The mature Bi-PLA2 purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA2. Immunofluorescence staining of Bi-PLA2-treated insect Sf9 cells revealed that Bi-PLA2 binds at the cell membrane and induces apoptotic cell death.