Construction Of ORF33 Deleted Bacmid For Helicoverpa Armigera Nucleopolyhedrovirus (HearNPV) And Analysis On HearNPV Virulence Enhancement By Its Chitinase

This thesis reported the rusults of ORF33 deleted Bacmid for Helicoverpa armigera nucleopolyhedrovirus(HearNPV)and analysis on HearNPV virulence enhancement by its chitinase.The main results are as follows:The ORF33 gene was successfully expressed in E.coli BL21 based a constructed expression vector pGEM-4T-2-ORF33. After inducing with IPTG,. The fusing protein GST-Hear33 was purifed by SDS-PAGE and used to generate antibodies in rabbit。Characterized by ELISA and western-blot,the antibody of Hear33 was prepared for the future analysis. The ELISA titer of antiserum against HearNPV ORF33 was about 1:256000.To study the function of HearNPV ORF33 gene, a recombinant vector was structured with PKSE vector which contain an chloramphenicol resistance (Cmr) gene and enhanced green fluorescent protein (GFP) gene. The recommendation system is PKD46, containingλphage recombinant system,and the HearNPV Bacmid which is genetically modified genome of HearNPV. Confirmed by PCR and restriction enzyme digestion, HearNPV ORF33 gene deletion Bacmid recombinant virus genome was successfully constructed.The PCR product of the Chitinase gene was cloned into the prokaryotic expression vector the pMAL-p2x and the PET-32a, and then expressed in E.coli TB1 and DE3, respectively. The Chitinase (ChiA) was expressed with a yield of 15.8% and 26.8% of the total cellular proteins for TB1 and DE3, respectively. And the soluble products were 20.0% and 30.2% to the fusion proteins expressed in TB1 and DE3, respectively. The DE3 product was fed to the third instar larvae of the H. armigera for bioassay of virulence enhancement. The bioassay results show that ,the mortality was 47.0% higher than the control virus at a combination of 2.0μg / ml of soluble ChiA to HearNPV. The virulence of HearNPV could be enhanced by soluble ChiA expressed in E. coli significantly.