Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors

Abstract

Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location.

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection

“…Though designed as baculovirus shuttle vectors, pBAC-5-based plasmids are excellent for transient gene expression in insect cell culture (Ogay et al, 2006), due to the presence of an AcMNPV gp64 early/late promoter (Whitford et al, 1989;Blissard & Rohrmann, 1991). Homologous recombination of plasmids such as pBAC-5 with circularized viral DNA is an infrequent event (less than 1 %) (Kitts et al, 1990) and thus would not be significant for these transient experiments. Also, pBAC-5 plasmids are designed to eliminate the polh gene after recombination and any incidental recombinant viruses would not produce the polyhedra that were used to perform experiments.…”

Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

“…Recombinant viruses were obtained through homologous recombination by co-transfection of Sf21 cells with Bsu36I-linearized AcRP23.lacZ viral DNA (Kitts, Ayres, and Possee, 1990) using calcium phosphate precipitation (O'Reilly, Miller, and Luckow, 1992). Recombinant viruses were isolated by four rounds of plaque assay (Summers and Smith, 1987) …”

Development of an Enzyme-Linked Immunosorbent Assay Using Recombinant Chicken Anemia Virus Proteins Expressed in a Baculovirus Vector System.

“…Lipofectin (4 µl) was added dropwise to 12 µl of a mixture containing 1 µg of each transfer vector DNA and 20 ng of linearized BacPAK6 (Clontech Laboratories, Inc., CA) baculovirus DNA [7], and then incubated for 15 min at room temperature. The mixture was added to 1.0 × 10 6 Sf cells in serum-free Grace's medium.…”