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Baculovirus-mediated expression of a Manduca sexta chitinase gene: Properties of the recombinant protein

1995; Elsevier BV; Volume: 25; Issue: 2 Linguagem: Inglês

10.1016/0965-1748(94)00070-x

1879-0240

Bhuvana Gopalakrishnan, Subbaratnam Muthukrishnan, Karl J. Kramer,

Entomopathogenic Microorganisms in Pest Control

We constructed a recombinant nonoccluded baculovirus, Autographa californica nuclear polyhedrosis virus (AcMNPV), containing a 1.8 kb DNA fragment from a Manduca sexta (tobacco hornworm) chitinase cDNA under the 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 into the culture medium. This protein hydrolyzed chitin and cross-reacted with a polyclonal antibody to M. sexta molting fluid chitinase. Tunicamycin treatment of infected SF9 cells and subsequent western blot analysis indicated that the secreted enzyme was a glycoprotein. GC-MS analysis revealed that carbohydrate accounted for approximately 25% of the mass of glycoprotein. The recombinant chitinase and the molting fluid enzyme were indistinguishable by N-terminal sequencing, polyacrylamide gel electrophoresis and carbohydrate analysis, indicating that the recombinant protein was similar, if not identical, to the molting fluid enzyme. Analysis of the expression level of recombinant chitinase in SF9, SF21 and Trichoplusia ni (Hi-5) cell lines showed that the yields were in the order Hi-5 > SF21 > SF9. Chitinase accumulated in hemolymph after injection of fourth instar M. sexta and S. frugiperda larvae with recombinant virus. The median time for mortality of S. frugiperda fourth instar larvae infected with the recombinant virus was approximately 20 h shorter than that for insects infected with a wild type virus. The results support the hypothesis that insect chitinase has potential to enhance the insecticidal activity of entomopathogens.