Baculovirus

Baculovirus Transfer Vectors Compatible with flashBAC™

A question we are asked from time to time is to identify baculovirus transfer vectors
compatible with our flashBAC™ system. We obviously nominate our own extensive range that is available but frequently someone has an old plasmid from years ago that they want to reuse. (more…)

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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…)

Titrating Stocks of Recombinant Baculoviruses Using the Plaque Assay Technique

We were going to devote this blog to a comprehensive account of how you might determine the infectious titre of your virus stock.  However, when our CEO got wind of this he started ruminating about how things were “back in the day” and how he had to collect seaweed from the seashore to extract agar for his overlays…!  No, we didn’t believe him either, but he is approaching his 7th decade so we cut him some slack!  But if you read on below maybe there is some truth in the rumour.  What this outburst of near senility did prompt was a consideration of the origins of the plaque assay method for determining the infectious titres of virus stocks.  So here goes. (more…)

Production of High Titre Virus Stocks

In our last blog post we talked about the process of transfecting insect cells with virus DNA and how you could recognise success or otherwise.  Transfections are normally done on a fairly modest scale (2-5ml) in culture dishes.  Therefore your P0 virus stock is of low volume and probably modest infectious titre (107 plaque forming units [pfu] per ml or lower; we will discuss how to measure this value in our next blog).  To produce a virus stock with a higher infectious titre and a larger volume you will need to amplify it further in bigger cultures of insect cells – usually Sf9. (more…)