Cloning And Expression Of Bombyx Mandarina Carboxylesterase Gene In E.coli

According to the predicted carboxylesterase sequence(GenBank:DQ443360) from thegenome of Bombyx mori, a pair of primers were designed, and Bombyx mandarina Carewas successfully cloned from its midgut by RT-PCR with GenBank accession numberEF157830. Sequence analysis results showed that it contained a 1923 bp open readingframe, which encode 641 amino acid residues (m.w. 71.7 kD) with pI 4.82. Thecomparison of Care nucleotide sequences and amino acid sequences between Bombyx moriand Bombyx mandarina showed that they shared highest identity 98.3% and 98.6%respectively, but lower identity with Myzus persicae,Drosophila pseudoobscura,Helicoverpa armigera et al. However, they had common properties in highly conservedamino acid sequences were esterases active central site and disulfide bond. So we predictthat Care from Bombyx mandarina might be related with insect resistence.The Care gene was cloned into prokaryotic expression pET-28a, transformed into Ecoli. BL21, and then expressed by the induction of 1 mmol/L IPTG. SDS-PAGE analysisindicated that Bombyx mandarina showed a specific approximately 75.7 kD bandcompared with the control groups of empty BL21 and BL21 transformed by emptypET-28a. So we predicted that carboxylesterase protein from Bombyx mandarina wassuccessfully expressed. Western blot analysis also showed that Bombyx mandarina showeda specific approximately 75.7 kD band compared with the control groups. The successfulexpressions of carboxylesterase protein from Bombyx mandarina lay the foundation forconstructing in vitro recombinant enzymatic systems to study carboxylesterase-mediatedxenobiotics metabolism and finally confirm their functions.