Baculovirus and Insect Cell Expression Protocols (original) (raw)
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Baculovirus and Insect Cell Expression
The insect cell culture/baculovirus system has three primary applications: (1) recombinant protein synthesis, (2) biopesticide synthesis, and (3) as a model system (e.g., for studying apoptosis). The fundamental techniques involved in these applications are described throughout this book. In this chapter, the most widely techniques are summarized and the reader is directed to detailed information found elsewhere in this book. Furthermore, many useful tips and the author's personal preferences that are rarely published are discussed in this chapter along with quantitative methods to characterize cell growth, baculovirus infection, and metabolism.
Covert Infection of Insects by Baculoviruses
Frontiers in microbiology, 2017
Baculoviruses (Baculoviridae) are occluded DNA viruses that are lethal pathogens of the larval stages of some lepidopterans, mosquitoes, and sawflies (phytophagous Hymenoptera). These viruses have been developed as biological insecticides for control of insect pests and as expression vectors in biotechnological applications. Natural and laboratory populations frequently harbor covert infections by baculoviruses, often at a prevalence exceeding 50%. Covert infection can comprise either non-productive latency or sublethal infection involving low level production of virus progeny. Latency in cell culture systems involves the expression of a small subset of viral genes. In contrast, covert infection in lepidopterans is associated with differential infection of cell types, modulation of virus gene expression and avoidance of immune system clearance. The molecular basis for covert infection may reside in the regulation of host-virus interactions through the action of microRNAs (miRNA). In...
Insect cells–Baculovirus system: Factors affecting growth and low MOI infection
Biochemical Engineering Journal, 2005
The use of low multiplicity of infection (MOI) for the production of baculovirus in insect cell sytems is an attractive alternative for largescale production processes. Such processes may be focused on the production of biological insecticides or on the expression of medically useful foreign genes. In the present study, the effect of MOI, cell inoculum age and inoculation procedure (carry-over of spent medium from the pre-culture) was studied. While some influences of inoculum age and medium were found, the overhelming effect is related to MOI. The data obtained describe the dynamics of the changes of non-occluded virus (NOV) in time, giving a useful insight into the mechanism of the cell-virus system.
Baculovirus studies in new, indigenous lepidopteran cell lines
Indian journal of experimental biology, 2002
Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and ...
Production of selected baculoviruses in newly established lepidopteran cell lines
in Vitro Cellular & Developmental Biology-animal, 2001
One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID50 assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1–47.3×106 TCID50/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3×106 TCID50/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0×106TCID50/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6×106TCID50/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses.
Biotechnology and Bioengineering, 1992
An experimental study has been carried out to investigate the effectiveness of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cells were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2 % fetal bovine serum (FBS), SF-900, and a serumfree medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of 8. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown in 10% serum-supplemented medium. 0 1992 John Wiley & Sons, Inc.