Baculovirus

Baculovirus P10 Structures and Role in Insect Cells

A blog on baculovirus P10 structures and the role in insect cells might appear a slightly odd topic for a blog from a company based on expression of recombinant proteins. However, a recently published paper that is the product of a collaboration between the Insect Virus Research Group at Oxford Brookes University and OET Ltd offers some insight to the role of P10 structures in infected insect cells and why our flashBAC ULTRA vector is so successful (Graves et al., 2019). flashBAC ULTRA, like all other versions of this expression vector, offers a one-step process for making recombinant viruses but lacks several of the baculovirus genes, including p10. (more…)

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Baculovirus Transfer Vector Plasmid Compatibility

A frequent customer query concerns baculovirus transfer vector plasmid compatibility with either flashBACTM or other systems for making recombinant viruses.  Obviously, if you are buying flashBACTM and our pOET range of transfer vectors you won’t have a problem.  However, given that baculovirus expression vectors have been around since 1983 and many labs have produced variations around a theme, it is not surprising that confusion can arise. (more…)

baculoQUANT Titration of Recombinant Baculoviruses

There are many methods available for the titration of recombinant baculoviruses.  One of the most convenient is the use of the quantitative polymerase chain reaction (QPCR), which assesses how much virus DNA is present in a freshly-amplified stock.  This is compared with a standard curve derived from a control virus stock to give an infectious titre expressed as Q plaque forming units (pfu) per millilitre.  OET Ltd market the baculoQUANT™ all-in-one virus extraction and titration kit for this purpose. (more…)

Transduction of Whole Porcine Kidneys with BacMams

One of the unexpected offshoots of the baculovirus expression system has been its development as a vehicle for the delivery of genes to mammalian cells.  These vectors are otherwise known as BacMams.  Although baculoviruses are unable to replicate in mammalian cells, they can enter them via receptor-mediated endocytosis.  In Frederick Boyce’s original paper the Rous sarcoma virus promoter was used to express beta-galactosidase in the HepG2 human liver cell line.  Further work showed that over 70% of cells in primary cultures of rat hepatocytes displayed expression of beta-galactosidase after exposure to the virus.  Although in this study other cell lines did not appear to be able to express the reporter gene, the subsequent use of other promoters, such as that from cytomegalovirus, seems to have overcome this problem.  (more…)

Baculovirus Gene Mutations and Protein Expression

Have you ever wondered why the baculovirus we use for most protein expression purposes is called AcMNPV?  This is short for Autographa californica nucleopolyhedrovirus, which derives from the Latin name of the alfalfa looper, a pest of alfalfa crops.  The convention for naming baculoviruses is to use the host insect from which they were isolated.  This does mean that some viruses can effectively be named twice, if they are isolated from different insect species.  (more…)

Producing Virus-Like Particles (VLPs) in Insect Cells

Using baculovirus expression vectors to produce virus-like particles (VLPs) is not new.  Intact, non-infectious poliovirus1 and bluetongue virus2 VLPs were synthesised using some of the earliest baculovirus vectors with considerable success.  In many of the projects we undertake for customers we have also generated VLPs for a wide range of different viruses.  They are ideal candidates for use as sub unit vaccines.  The immunogenic parts of the virus can be replicated without assembling an infectious entity.  Although we can’t give specific details of these projects owing to client confidentiality we can offer some general guidelines about how best to produce different types of VLPs, based on their predicted structures. (more…)

Longevity of Infectious Budded Baculovirus Stocks

A question that comes up repeatedly is the long term stability of recombinant budded baculovirus stocks.  This is a particular issue now that most of them are generated using insect cells grown in serum-free medium.  It has long been a mantra in virology that you should always have a little bit of protein in your virus suspension to help stabilise the virion structure or prevent non-specific binding to glass or plastic vessels.  (more…)

Rapid Virus Titration by QPCR

Knowing the infectious titre of a recombinant virus stock is really important prior to testing for protein production in insect cells.  It enables you to add the optimal quantity of virus to initiate virus infection.  Too much virus and you are wasting your stock.  Too little virus and you won’t establish a synchronous infection.  For some proteins this may not matter, as the virus produced by the initial round of replication will infect all other cells and result in protein production.  However, if your protein target is particularly unstable, you may lose some of the product via its degradation in the population of cells initially infected.  (more…)