WE PROPOSE TO DEVELOP CLONING AND EXPRESSION VECTORS FROM NUCLEAR POLYHEDROSIS VIRUSES (NPV) WITH CAPABILITIES FOR EFFICIENTLY PRODUCING ONE OF SEVERAL GENES SIMULTANEOUSLY IN INFECTED INSECT CELL CULTURES.

WE PROPOSE TO DEVELOP CLONING AND EXPRESSION VECTORS FROM NUCLEAR POLYHEDROSIS VIRUSES (NPV) WITH CAPABILITIES FOR EFFICIENTLY PRODUCING ONE OF SEVERAL GENES SIMULTANEOUSLY IN INFECTED INSECT CELL CULTURES. THE OBJECTIVES IN PHASE I ARE TO CONSTRUCT ALTERNATIVE PLASMID TRANSFER VECTORS WITH INCREASED EXPRESSION CAPABILITIES THROUGH THE ISOLATION AND ENGINEERING OF AT LEAST TWO NEW POLYHEDRIN GENE PROMOTERS, AND TO USE THESE TRANSFER VECTORS IN THE CONSTRUCTION OF VIRAL EXPRESSION VECTORS CAPABLE OF PRODUCING MORE THAN ONE GENE PRODUCT AT ONCE UNDER THE CONTROL OF POLYHEDRIN PROMOTERS. PHASE II WILL CONCENTRATE ON THE EXPRESSION OF SPECIFIED GENES IN THE VECTORS, OPTIMIZATION OF LARGE SCALE CULTURES OF VECTOR INFECTED INSECT CELLS, AND PURIFICATION PROCEDURES FOR THE GENE PRODUCTS. PRELIMINARY STUDIES HAVE DEMONSTRATED THAT THIS EXPRESSION SYSTEM ALLOWS POST-TRANSLATIONAL MODIFICATION, GLYCOSYLATION, AND EFFICIENT SECRETION OF GENE PRODUCTS FROM INFECTED CELLS. DURING THE COURSE OF THIS STUDY WE WILL ALSO COMPLETE A DETAILED COST ANALYSIS COMPARING THIS SYSTEM WITH CLASSICAL BACTERIAL AND YEAST EXPRESSION SYSTEMS. THE NEW VECTORS DEVELOPED IN THIS STUDY WILL ENHANCE THE COMMERCIAL FEASIBILITY OF NPV INFECTED CELL CULTURES AS EFFICIENT EXPRESSION SYSTEMS FOR MANY GENE PRODUCTS WHICH COULD BE MARKETED AS RESEARCH AND CLINICALLY THERAPEUTIC REAGENTS.