Immunolocalization Of The Celangulin V Receptor And The Construction Of CDNA Library Of The Midgut Of Oriental Armyworm, Mythimna Separata Walker (Lepidoptera: Notuidae)

There are two routes for the new pesticides discovery. One is the innovation of thechemical structure, i. e., computer-aided design (CAD), chemical/biosynthesis and new leadcompounds from the nature products. The other is the innovation of the pesticidal targets, i. e.utilizing the knowledge of bioinformatics, molecular biology and pharmacology to find newpesticidal targets and active mechanisms and thus instruct the discovery of the lead chemicalstructures. To find a new target, the localization of receptor on the tissular and cellular levelare required. Immunoelectron microscopy (IEM), however, is the main technique fordetecting the distribution of complicated and activated nature components in the bio-tissues.Celangulin V, a nature product from insecticidal plant, Chinese bittersweet, Celastrusangulatus Max., had been discovered as a new insect digestive poison for its serious damagein the epithelium of the midgut of the sensitive pests. For the illustration of its mechanism ofaction, the artificial antigens of Celangulin V were synthesized to elicit monoclonalantibodies, with which the distribution of the poison in the epithelium of the midgut of thearmyworm was performed. Meanwhile, for the purposes to isolate the receptor and thus toanlysis its structure, the cDNA library of the oriental armyworm was constructed. The resultswere as follow:1. The hapten was synthesized by a condensation reaction of succinic anhydride withCelangulin V and the structure was characterized by NMR, IR, Optical rotation and HRMSanalysis. All the analysis data give a common results that the product was1β,2β-diacetyloxy-8α,13-diisobutanoyloxy-6α-succinoyl-9β-benzoyloxy-4α-hydroxy-β-di-hydroagarofuran(called hapten),introducing a carboxyl on the skeleton of Celangulin V,and with which three carrier proteins,bovine serum albumin(BSA),keyhole limpethemocyanin(KLH)and ovalbumin(OVA),were coupled to obtain corresponding artificialantigens:hapten-BSA,hapten-KLH and hapten-OVA．Successful conjugates information wasgave by the scanning of UV(200～400nm)and IR(400-4000cm^-1).2．After intraperitoneal injection using hapten-KLH as the immunoantigen,thesplenocyte were fused with SP2／0 myeloma cells．And then colonization and screening of the positive clones by ELISA with hapten-BSA and hapten-OVA as coating-antigens werefollowed. Three hybridoma cell lines, C₆-E₉-B₁1,C₆-E₉-C₅ and C₆-E₉-H₃, were selected fortheir capability of production of monoclonal antibodies specifically against Celangulin V.The cell lines were inoculated in mice abdomen and the titre of anti-Celangulin Vantibodies in the ascites were measured to be about 7.5×10⁴, much enough for the studydemand.3.The microstructure of the midgut of intoxicant oriental armyworm larvae wasobserved. The electron microscopy photos indicted that Celangulin V could result in aserious damage on the microvilli, mitochondria and rough endoplasmic reticulum of thecolumnar and goblet epithelium of the pest midgut, microvilli were fragmented, lodged,disordered, malformed and bifurcated, mitochondria swelled irregularly, the inner and outermembranes uncoupled, and the ridges deletion, the cisternae lumen of endoplasmic reticulumdilation and vacuolization, nucleolus apoptosis, nucleolus and chromatin condensed andelectron density increased. And amount of vesicles budded from intramembranes to intercellur,implying that this pathological appearance maybe relate with the mechanism of dehydration.4. The ultrathin sections of the midgut from the oriental armyworm were prepared for theimmunoelectron microscopy. This was followed by incubations with anti-Celangulin Vmonoclonal antibody and colloidal gold-labeled goat-anti-mouse immunoglobulin, in orderly.The immunoelectron microscopy revealed that Celangulin V could bind on the microvilli,mitochondria and rough endoplasmic reticulum of the epithelium of the midgut. And thebinding quantity, as well as the damage degree, was increased according to the insect'ssuffering time. From above, it can be confirmed that there are receptors in the epithelium ofthe midgut of sensitive insects, which can be enacted specifically by Celangulin V.5. With the techniques of Trizol and oligo (dT) chromatography, the total RNA andmRNA extracted from the midgut of the oriental armyworm. And the cDNA library, using thephage ofλTriplEX as express vector, was firstly constructed with a qualified original titre of1.07×10⁷ and recombination fraction of 95.8%. The library was screened using CelangulinV, anti-Celangulin V monoclonal antibody and HRP-labeled goat-anti-mouse immuno-globulin as probe, the first aitibody and second antibody, respectively, and then developedwith the ECL kit. The results showed that no Celangulin V receptor gene was found in thelibrary. But it gave a presume that the receptor maybe a homo- or hetero-polymer whichcomposed by several subunits, and even couple with cell membranes, which made thetraditional method of screening cDNA library inactivated for the receptor separating.