Molecular Cloning And Expression Of Insect Intestinal Mucin From Helicoverpa Armigera

Peritrophic membrane (PM) is a semi-permeable membrane secreted by midgut epithelial cells in most insects and plays a very important role in the midgut digestion process and protecting the midgut epithelium from microbial and pathogens infections.Peritrophic membrane is a natural defense system of insect midgut,it is mainly comprised of chitin and protein.Wang&Granados were the first to identify a highly glycosylated insect gut protein specifically a PM protein in Trichoplusia ni,for which they coined the term Insect/Invertebrate intestinal mucin(IIM).It has chitin binding activity.A highly glycosylated mucin can protect epithelial cells from physical damage.In this study,we researched Helicoverpa armigera larvae,which are world widely important insect in agriculture.1) Using the total RNA of midgut as template, IIM3,IIM4,IIM86were amplified by PCR method.Sequence analysis showed that IIM3open reading frame (ORF) is669bp in length,encoding a polypeptide of223amino acid residues with the predicted" molecular mass of24.8kDa.IIM3contained two chitin binding domains,one signal peptide;IIM4ORF is909bp in length,encoding a polypeptide of303amino acid residues with the predicted molecular mass of33kDa.IIM4contained two chitin binding domains,one mucin domain and one signal peptide; IIM86ORF is2553bp in length,encoding a polypeptide of851amino acid residues with the predicted molecular mass of86.8kDa.IIM86contained five chitin binding domains,one mucin domain,one signal peptide and two glycine-aspartic acid (D-G) rich region.IIM3,IIM4,IIM86gene were cloned into pGEM-T vectors,then constructed recombinant baculoviruses,the recombinant baculoviruses were transfected into Sf9 cells.2)Using the plasmid conferred to IIM86gene as template,Mucin gene was amplified by PCR method.Then,Mucin was recombined to pET28a vector and expressed.Western Blot and Purification showed Mucin protein could be expressed in E.coli.cell BL21. Sequence analysis showed that Mucin is475bp in length,encoding a polypeptide of158amino acid residues with the predicted molecular mass of15.5kDa.Cloning and identification of insect intestinal mucin were provided the prerequisite and basis for this new strategy to break the host insect defense mechanisms to take advantage of the virus through mucin degrading enzyme.The study on the structure compositions and function of insect intestinal mucin will determine the molecular mechanism of interaction between the insects and pathogenic microorganisms,provide new avenues for pest biocontrol in practice and provide a new gene sources for the transgenic plants.