Invasive Species Compendium

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Abstract

Influence of deletion of Autographa californica multiple nucleopolyhedrovirus Ac148-150 on viral replication and foreign protein expression.

Abstract

Objective: The effect of Ac148,Ac149 and Ac150(Acc148-150) deletion on AcMNPV replication and expression of foreign proteins was investigated to provide foundation for identification of baculovirus gene functions and shortening baculovirus genome. Method: Ac148-150 genes were knocked out by Red/ET homologous recombination and rpsL-AMP counter-selection systems. The recombinant baculoviruses (wild type virus vAcMNPV/GFP and deletion mutant virus vAcΔ148-150/GFP) were successfully constructed by co-transfection with KOAc148-150 bacmid and wild type bacmid and reporter expression vector in Sf9 cells, and the one-step growth curve of two recombinant baculoviruses was determined. In Sf9 cells infected with recombinant baculoviruses, GFP expression were then observed by fluorescence microscopy, and GFP fluorescence intensity was detected by flow cytometry. The expression of GFP in Ac148-150 deletion mutant was examined by SDS PAGE and Western blot. Result: The knockout of Ac148-150 was verified by PCR. Compared with the wild type virus vAcMNPV/GFP, the one-step growth curve of the deletion mutant virus vAcΔ148-150/GFP was basically the same, indicating that Ac148-150 deletion did not affect viral replication. Fluorescence microscopy and flow cytometry results showed that the deletion of Ac148-150 had no effect on the expression of foreign proteins. As detected by SDS-PAGE and Western blot, there was no significant difference in GFP yield between Ac148-150 deletion mutant and wild-type virus. Conclusion: The deletion of Ac148, Ac149, Ac150 from the AcMNPV genome had no effects on virus replication and the ability of expressing foreign proteins.