Eukaryotic Expression Of Two Baculovirus Enhancin Genes And In-depth Proteomic Study Of Peritrophic Membrane In Heliothis Armigera

Compared with chemical pesticide,biological pesticide has many advantages,no environmental pollution,no harm to natural enemy,harmless to hu-man and other higher animals,continuous insecticidal effect.However,it defects in relatively slow insecticidal speed and increasingly serious pest resistance.Researches have shown that insect peritrophic membrane is the most important organism against pesticides.Insect baculovirus protein ENHANCIN has metal prot-ease activity,which can destroy the the structural integrity of host peritrophic memb-rane or mediate fusion of virus and insect mid-gut epithelial cell,then accelerate the invasion of virus particles and improve the infectivity of viruses and other pathogens.Hence,ENHANCIN shows a broad application prospect in the aspect of improving the insecticidal efficiency.This study adopts AcMNPV Bac-to-Bac system to reveal the molecular mecha-nism of AsGV ENHANCIN fuctions.AsGV enhancin and AsGV enhancing-2679 gene obtained by our previous work were transposed into shuttle plasmid AcMNPV bacmid by donor plasmid pFastBacHT,then transfected into Sf9 insect cells for eukaryotic expression.Now we have constructed the eukaryotic expression vector Bacmid-AsGV-En/2679 and successfully transfected to Sf9;In order to obtain sufficient TnGV ENHANCIN protein to study the mechanism of protease function,recombinant baculovirus eukaryotic expression plasmid Bacmid-TnEn and its peer plasmid with a EGFP reporter gene,Bacmid-TnEn-GFP,both cons-tructed previously,were transfected into insect cell Sf9 for mass suspension expressi-on and purification.3 days after transfection,cell pathological changes were obser-ved by Microscopic examination,and green fluorescence by fluorescence microscopy.Then cells were collected for electrophoresis examination.Results showed that the ex-ogenous gene was successfully expressed;In the past,in the study of host-pathogen interactions,scholars tend to focus more on pathogen research.However,in recent years,a growing understanding is that proteins on the peritrophic membrane of insect relate to researches in the function way,the orientation transformation methods of pathogens and the receptors involved in ins-ect resistant mechanism,which can't be ignored.Therefore,this research focuses on Helicoverpa armigera.It's proteins on peritrophic membrane were analyzed by liquid chromatography-mass spectrometry techniques and 77 proteins successfully identif-ied.Further analysis found that these proteins were mainly involved in digestion,im-mune responses,detoxifcation metabolism and so on.This explains the peritrophic membrane not only assists the mid-gut for food digestion and absorption by enzymo-lysis,but also is involved in immune defense process to protect the insect mid-gut.Our research will provide a theoretical basis for explicit physiological function of proteins on peritrophic membrane,looking for new targets of pest control and dev-elopment of new effective biological insecticide.