Inactivation Analysis Of BmNPV Cysteine Protease Gene And Chitinase Gene And Expression Of HGM-CSF

Insect baculovirus expression vector system (BEVS) is a super high-level eukaryotic expression system developed in 1980s. Bombyx mori BEVS has many additional advantages than normal BEVS.In this thesis, BEVS was further reformed to expreted to improve the expressing level of foreign protease .first of all, we coloned the BmNPVsu Cysteine protease gene and Chitinase gene and also analyzed the sequence of them. Then by cloning the two gene separately into pBluescript II SK+ and inserting the fragment of BmNPV poly gene between them, Recombinant transfer vector pBm ChiA-polh-CP was constructed.By cotransfection of the pBm ChiA-polh-CP and BmNPV-GMCSF DNA, which contained granulocyte macrophage colony stimulating factors gene (GMCSF) into BmN cells, and substituting the promoter of CP and ChiA gene with the polyhedrin gene , the recombinant virus BmNPVpolh+ CP ChiA'GMCSF^was produced. The recombinant virus , in which CP gene and ChiA gene were inactive, could express GMCSF gene and form polyhedrin BmNPV polh+ CP ChiA' GMCSF*. with the recombinant virus infecting BmN cells revealed that CP and ChiA gene were not essential for viral replication and have no significant effect on polyhedron formation, and the infected cells survived 2~3 days more than those infected with BmNPV and BmNPV-GMCSF. which was 10 times as high as that expressed in BmNPV-GMCSF.The expression of recombinant virus infecting Bombyx mori larva indicates that the Bombyx mori larva infected with the recombinant virus survived 0.5-1 day more then those infected with BmNPV, and its epidermis was not easy to damage. The above result showed: Chia and CP gene were not essential for virus replication, and by inactivating ChiA and CP gene, the expressing level of foreign protease of BEVS was improved. At the same time, Because polyhedrosis was formed and fed to Bombyx mori lavra. Many difficulties of virus inoculation in practical production can be resolved.