The Property Study On The BmCPV And BmNPV Polyhedra Embedding Heterologous Protein

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) are two pathogens of the Bombyx mori which can infect and lead the hosts to midgut pus disease and blood pus disease respectively. The polyhedra producted by insect viruses can protect the embedded virions very well. In order to better understand the embedding feature of the BmCPV and BmNPV polyhedra and explore the embedding mechanism. The present study has mainly carried out some studies about the polyhedra of BmCPV and BmNPV incorporated heterologous proteins properties.1. Proteomics study of BmCPV polyhedraThe proteomics study of the BmCPV polyhedra purified from the midgets of infected silkworm was performed by LC-MS/MS technology. Twenty-eight proteins was identified, of which twenty-three proteins belong to Bombyx mori indicating that BmCPV polyhedra could incorporated the host proteins during the polyhedra assembly process. Three dimensional structure analysis of these foreign proteins showed that the structures of part of these proteins have the same things in common with the left-hand structure of the BmCPV polyhedrin or with the N-terminal structure of BmCPV structural proteins VP3.So we speculated that incorporating of the host proteins into BmCPV polyhedra was possibly caused by these structural similarity between these proteins mentioned above.2. Stably transformed cells expressing BmNPV polyhedron and analysis of embedded proteinsThe BmN cells was trasfected with a expression vector pIZT/V5-His including BmCPV polyhedron gene and screened with Zeocin antibiotic to obtain transformed cells that could stably express polyhedrin. Fluorescence observation showed that green fluorescent protein (GFP) crystallization of polyhedron appeared in the cytoplasm of the transformed cells. But the purified polyhedra didn't show significant green fluorescent indicating that BmCPV polyhedrin could assemble into polyhedra by themselves without any virions protein and the GFP didn't been incorporated into the polyhedra. By analysis of the tandem mass spectrometry we found that two proteins that are ubiquitin and ribosomal protein S27a and BTB/POZ domain containing protein included in the purified polyhedra belonged to Bombyx mori. The results of structural analysis showed that the three dimentional structure of the two proteins have the same things in common with the left-hand structure of the BmCPV polyhedron. We speculated that polyhedrin could interact with the proteins which have the similar structures with the polyhedron and then incorporate them into polyhedra. The stable transformed cells were infected with modified linear baculovirus BmPAK6. The results of replied infection assay showed that both the purified polyhedra and its lysate could make the X-gal coloring demonstrating that theβ-galactosidase could be incorporated into the polyhedra.3. Proteomics analysis of BmNPV ODVThe proteins of occlusion-derived virus(ODV) of BmNPV purified from sick silkworms suffering from blood pus disease was detected by LC-MS/MS. Thirty-eight proteins are identified, of which twenty-eight proteins were encoded by BmNPV itself and ten proteins were encoded by Bombyx mori.In addition, some virus nonstructural proteins were identified in the polyhedra. The results indicated that in the assembly process of the BmNPV polyhedra the polyhedrin could incorporate some host proteins. 4. Stably transformed cells expressing BmNPV polyhedronThe BmN cells was trasfected with a expression vector pIZT/V5-His-polh including BmNPV polyhedron gene and screened with antibiotic to obtain transformed cells that could stably express polyhedrin. Observation with fluorescence microscope showed that green fluorescent polyhedra particles appeared in the nucleolus of the transformed cells, and the nucleolus was enlarged which was similar with the BmNPV-infected cells. The results demonstrated that the polyhedron could assembe into polyhedra by themselves in the cells which caused the cells to have pathological changes.The results above provide new methods and clues to further study about the embedding mechanism of the virions and heterologous proteins into the polyhedra, and also provide new technologies and ways to the utility of polyhedra feature that embedding foreign proteins.