cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

摘要: Abstract A mRNA transcript that codes for a phospholipase (PLA2) was isolated from single venom gland of the Bothrops ammodytoides viper. The PLA2 cloned onto pCR®2.1-TOPO vector and subsequently expressed heterologously in E. coli strain M15, using pQE30 vector. recombinant named rBamPLA2_1, is composed an N-terminal fusion protein 16 residues, along with 122 residues mature includes 14 cysteines form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 obtained inclusion bodies extracted chaotropic agent. had experimental molecular mass 15,692.5 Da concurred its theoretical mass. refolded vitro conditions after refolding, three main fractions similar masses, were identified. Although, considered to represent different oxidized cystine isoforms, their secondary structures comparable. All isoforms active on egg-yolk phospholipid recognized cell membrane phospholipids be native PLA2s, B. venom. mixture used immunize horse order produce serum antibodies (anti-rBamPLA2_1), which partially inhibited indirect hemolytic activity anti-rBamPLA2_1 not able recognize crotoxin, related but viper genus, Crotalus durissus terrificus, they PLA2s other venoms regional species Bothrops.