Cloning And Prokaryotic Expression Of The Wax Synthase Gene Of The Chinese White Wax Scale

The Chinese white wax scale,Ericerus pela Chavannes,is a unique resource insect of China,which has a specialized characteristic of wax seceretion.The wax produced by white wax scale has been widespread used in machinery,light industry,pharmaceucial,food and other industries.Wax synthase(WS)is one of the key enzymes in the synthesis process of wax ester.At present,WS has not yet been identified in insects.In this study,we aim to obtain the full length cDNA sequence of the wax synthase gene of the white wax insect,analysis the amino acid sequence of Ericerus pela WS and heterologously express the enzyme in Escherichia coli.The result of this study lays an important foundation of the research on antibody preparation,organizational and subcellular localization,which also ensures the accomplishment of the successive functional research and enzyme activity assays and provides references for the research on wax ester synthesis in other insects.1.Cloning and sequence analysis of the wax synthase gene of the Chinese white wax scaleIn this study,we obtained the full-length cDNA sequence of the ws gene by using RACE based on the partial sequence obtained previously.The full-length cDNA sequence of the ws gene was 1,518 bp,which included the 5'-UTR(untranslated region)with 94 bp,3'-UTR with68 bp,and the open reading frame with 1359 bp.Amino acid sequence analysis showed that,the gene was deduced to encode 452 amino acid residues with the putative protein molecular weight of 51.8 kDa,and the theoretical isoelectric point of 6.35.It has a secretory signal peptide and a transmembrane region containing 22 amino acid residues in the N-terminal.The phylogenetic tree of WS amino acid sequence showed that,Ericerus pela WS and the WS of other known species do not get together.Research results showed that,the structure,transmembrane,conserved regions of Ericerus pela WS sequence are different from with the well-known WS from other species.Further researchs are needed for identification of Ericerus pela WS.2.Prokaryotic expression of the Ericerus pela WSThrough the analysis of the sequence and structure of Ericerus pela WS,we amplified a partial amino acid sequence of Ericerus pela WS,which performs better specificity and higher hydrophilic properties.We added a His tag at the end of the sequence and enzyme cleavage site at both ends.We constructed a prokaryotic expression vector named pET-22b/EpelWS and transferred into the Escherichia coli BL21(DE3).Then we induced the Escherichia coli to express the interest proteins abundantly under the condition of 28? and 1mM concentration of IPTG.The band of the interest proteins was found in the result of SDS-PAGE analysis.Western Blot result showed that,The interest proteins with His tag was combined with the primary antibodies“mouse polyclonal antibody to His tag” and also the secondary antibodies “Goat polyantibody to IgG(HRP)” specially,which confirms the fact of large expression of the Ericerus pela WS in Escherichia coli.