Construction of the recombinant baculovirus AcMNPV with cathepsin B-like proteinase and its insecticidal activity against Helicoverpa armigera

The Helicoverpa armigera cathepsin B-like proteinase (HCB) has been shown to have a wide spectrum of substrates. It has been involved in the degradation of yolk protein during embryonic development and also in the decomposition of the adult fat body. To study the possibility of using HCB to improve the insecticidal activity of bioinsecticides, it was inserted into the pFASTBACDUAL-green fluorescent protein (GFP) donor plasmid under the strong polyhedrin promoter, and the polyhedrin gene was retained behind the HCB gene so that the virus could orally infect the host and survive in the natural environment. After the recombinant plasmid transfected the Sf21 cells, the recombinant baculovirus Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV)-GFP-HCB-Polh+ was produced. A 37-kDa recombinant procathepsin B was expressed in AcMNPV-GFP-HCB-Polh+-infected Sf21 cells and processed into 29/25-kDa mature forms. Gelatin zymography revealed that the proteolytic activities of the recombinant HCB and other proteases were activated or enhanced by HCB in AcMNPV-GFP-HCB-Polh+-infected larvae. Green fluorescence was observed earlier and was more intense in AcMNPV-GFP-HCB-Polh+-infected larvae than in HCB-free AcMNPV-GFP-Polh+-infected ones that were infected at the same time; this indicated that the AcMNPV-GFP-HCB-Polh+ virus spreaded faster in larvae than the AcMNPV-GFP-Polh+ virus. A bioassay revealed that infection with the AcMNPV-GFP-HCB-Polh+ virus shortened the median survival time of H. armigera larvae by 12 h as compared with those infected with the AcMNPV-GFP-Polh+ or the wild-type AcMNPV virus. These results suggest that HCB may possibly play a role in the recombinant virus in improving the rate of killing larvae.