BACULOVIRUS-MEDIATED EXPRESSION OF A MANDUCA-SEXTA CHITINASE GENE - PROPERTIES OF THE RECOMBINANT PROTEIN

We constructed a recombinant nonoccluded baculovirus, Autographa calif ornica nuclear polyhedrosis virus (AcMNPV), containing a 1.8 kb DNA fr agment from a Manduca sexta (tobacco hornworm) chitinase cDNA under th e control of the polyhedrin gene promoter. When Spodoptera frugiperda (fall armyworm) cells (SF9) were infected with this recombinant virus, a protein with an apparent molecular weight of 85 kDa was secreted in to the culture medium. This protein hydrolyzed chitin and cross-reacte d with a polyclonal antibody to M. sexta molting fluid chitinase. Tuni camycin treatment of infected SF9 cells and subsequent western blot an alysis indicated that the secreted enzyme was a glycoprotein. GC-MS an alysis revealed that carbohydrate accounted for approximately 25% of t he mass of glycoprotein. The recombinant chitinase and the molting flu id enzyme were indistinguishable by N-terminal sequencing, polyacrylam ide gel electrophoresis and carbohydrate analysis, indicating that the recombinant protein was similar, if not identical, to the molting flu id enzyme. Analysis of the expression level of recombinant chitinase i n SF9, SF21 and Trichoplusia al (Hi-5) cell lines showed that the yiel ds were in the order Hi-5 > SF21 > SF9. Chitinase accumulated in hemol ymph after injection of fourth instar M. sexta and S. frugiperda larva e with recombinant virus. The median time for mortality of S. frugiper da fourth instar larvae infected with the recombinant virus was approx imately 20 h shorter than that for insects infected with a wild type v irus. The results support the hypothesis that insect chitinase has pot ential to enhance the insecticidal activity of entomopathogens.