Biochemical Characteristics Of Chitin Binding Protein SeCBP66 From Spodoptera Exigua

The peritrophic matrix(or peritrophic membrane , PM) is a semi-permeable extracellular matrix that often lines the midgut of insects separating the contents of the gut lumen from the digestive epithelial cells lining the midgut.It is composed of a network of chitin and proteins to which enzymes and other components associate.Isolation and identification of intestinal peritrophic membrane protein provides the prerequisite and basis for the further study of pest biocontrol mechanism,looking for biological control target,and the molecular mechanism of the interaction between the membrane and pathogenic microorganisms.In this study, Spodoptera exigua peritrophic membrane chitin binding protein SeCBP66 for study, based on the beet armyworm midgut cDNA library built and screened secbp66 gene codes for SeCBP66 protein in the past,we try to express SeCBP66 in vitro and study its biochemical characteristics, and the tissue localization in the beet armyworm larvae.In order to get SeCBP66,Pichia pastoris GS115 expression system was used.The nucleotide acids codes for singal of SeCBP66 was removed and the gene fragment codes for mature SeCBP66 was be amplified by PCR and cloned into pPIC9k which has aα-factor signal.The recombinant plasmid was transformed into Pichia pastoris GS115 and the aox gene in the plasmid would be exchanged with the aox1 gene in the genomes and the△s-secbp66 would be integrated into the genome.SeCBP66 was be induced by methanol and detected by Western blot.The results showed a dispersion of unequal size.This may be caused by the existence of a large number of N-linked glycosylation sites, as well as yeast cells in different forms of N-linked glycosylation modification of the protein.Then SeCBP66 was designed to express in insect cell lines, the secbp66 that add a 6×His Tag in the end was amplified by PCR and cloned into pFastBacTM .The recombinant plasmid was transformed into E. coli DH10Bac,and the target gene inserted into Bacmid with Mini-attTn7 transposon.The recombinant Bacmid transfected insect cell lines Sf9 and the virus which was named VSeCBP-1 contain secbp66 was harvested in the media of cell . For amplifying the virus, VSeCBP-1 infected Sf9 for a time and we got VSeCBP-2 . Then SeCBP66 was expressed in the media when VSeCBP-2 infect BTI-Tn-5B1-4(HighFive).SeCBP66 was detected by Western blot with antibodies to His-Tag.The results showed the protein had a most expression at 72h after infection.After binded with regenerated chitin,SeCBP66 was only partially dissociated with 6M Urea and 2%SDS.However,it was solubilized from the bound chitin by 1% Calcoflour or by 2% SDS in the presence of 5%β-mercaptoethanol.It indicates SeCBP66 can strongly bind chitin in the network of peritrophic membrane.SeCBP66 was purified with regenerated chitin and the specific antibody was be prepared from the antiserum to peritrophic membrane.The immunolocalization of SeCBP66 showed it present in gut and peritrophic membarne.Interestingly,it showed lower than the apparent molecular weights of the recombinant SeCBP66.indicated that the SeCBP66 protein in the process of formation of the peritrophic membrane may be partially degraded, but still retain their chitin binding activity of fragments to form a peritrophic membrane.