Cloning And Expression Of BmNPV Chitinase In E.coli & Insect Cells And The Synergistic Effect Of The Expressed Products With Bt On Killing Insect Larvae

Baculoviruses are pathogentic to insects of the order Lepidoptera and are attractive biological agents for the control of agriculturally important insect pests. The baculovirus-insects expression system is one of the promising eucaryotic systems in which a lot of foreign genes of economic value have been efficiently expressed. Progress in the field of baculovirus molecular biology have made with more and more concerns taken to study the baculoviruses in the nearing years. This dissertation contains two aspects concerning BmNPV chitinase gene expressed in E. coli and insect cells and the synergistic effect of the expressed products with Bt on killing the insect larvae.1. The complete chitinase gene (chiA) and the putative signal peptide-deficient encoding region (chis-) were amplified from BmNPVDNA by using PCR method. The target gene (chiA) was inserted into the expression vector pET28a, under the control of phage T7 promoter. Then the recombinant vector pETchiA was transformed into ?coli (DE3) for expression. An expressed band of about 64 kD was identified by SDS-PAGE and further confirmed by Western blot. The expressed target proteins accumulated up to about 22% of the total cellular proteins in the bacterial cells.2. The putative signal peptide-deficient encoding region of chitinase gene (chis-) was amplified from BmNPV DNA by using PCR method. The target gene (chis-) was inserted into donor plasmid pFASTBACHTb under the control of polyhedin promoter and flanked by the left and right ends of Tn7. The target gene was transposed to the Bacmid in E. coli (DH10), by Tn7 transposition function. Recombinant Bacmid DNA was transfected into Tn-5Bl-4 cell mediated by Lipofectin. An expressed protein band of 63 kD was determined by SDS-PAGE and Western blot. The expressed target protein accumulated up to about 12% of the total cellular proteins in the insect cells.3. The expression products of those two fragments above from BmNPV mixed with Bt were fed to the second instar larvae of Bombyx mori. After treatment all the lethal time and weights of the killed were recorded, respectively, and the LT50 as well as the LT90 were calculated. The results indicated that lethal time of the larvae of Bombyx mori fed with the Bt added with chitianses from BmNPV expressed in E. coli and Tn cells advanced for 24. 5 and 24. 6 hours, respectively, and the weights augmented were less against the controls. With these results conducted by TMD there was synergistic effect of the expressed products with Bt on killing the insect larvae.