Research Of Neuropeptides Inhibitting Ecdysteroidogenesis In The Prothoracic Gland

Bombyx mori prothoracicostatic peptide(Bom-PTSP)the primarystructure is AWQDLNASAW–NH2 and molecular mass 1089.78 Da wassynthesized by artificial method, and coupled to different carriers(BSA orOVA) by glutaraldehyde one-step method to get immunogen BSA-PTSP,which was used to immunize BALB/c mice by general method, and checkingantigen OVA-PTSP. Through Dot blot and indirect ELISA methods weobtained mice with highly immunizing response against PTSP whosespleenocytes were fused to Sp2/0 by 50% PEG. Hybridomas were cloned bythrice single-cell cloning and one stable monoclonal cell line was generatedfinally which can produce and secretes monoclonal antibodies against PTSPstably and can expand indefinitely in vitro and in vivo. Monoclonal antibodiesin the ascites fluids of mice were typed as IgM. Western Blotting bypolyclonal and monoclonal antibody anti-PTSP showed as follows: polyclonalantibody recognized BSA, BSA-PTSP and OVA-PTSP, but did not recognizeOVA, and more ,a protein which molecular mass is 80KD and roots in brains ofSamia cynthia ricini larvae on day 2 of the fifth instar was rencognized by polyclonalantibody too ;monoclonal antibody recognized BSA-PTSP, OVA-PTSP and aprotein derived from hemolymph of Bombyx mori larvae on early days of thefifth instar, which molecular mass is about 75KD. The result suggested thatthis protein is similar to PTSP in structure or maybe is the original protein ofPTSP. Moreover, the Brains' cDNA library of Samia cynthia ricini larvae on day 2 ofAbstractthe fifth instar was constructed accoriding to Clontech's method of SMART cDNASynthesis by Primer Extension (SSPE), and immunoscreening method hasbeen used to screen the cDNA library by polyclonal antibody against PTSP. Severalclones rencognized by polyclonal antibody have been isolated from the cDNA library, andsecondary-cloning is being performed to the primary positive clones . To continuefurther studies of gene cloning of PTSP in Samia cynthia ricini through PCR and RACEtechnologys will be advantageous to discover more information about PTSP. Further research such as : the function mode of PTSP, the distributing ofanalog peptides of PTSP in different insects and the relationship with PTTH,is not only important for study on the complexity of hormonal regulatingmechanism on ecdysteriodogensis, but also crucial to deep understand thebiological phenomenon and the rules of insect life activities, which has,moreover, very significant value in basic science and practice meanings toprovide new way of pest management and cure for peple.