What is p10 promoter?
The ubiquitous polyhedrin gene promoter, from which most baculovirus expression vectors are developed, has an oft forgotten rival – the p10 gene promoter. These sequences remain under the control of the polyhedrin gene promoter to obtain high levels of protein production.
What is polyhedrin gene promoter?
The polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus was analysed with respect to which sequences are required upstream of the mRNA transcription initiation (CAP) site for efficient promoter activity. Removal of the TATA motif did not affect lacZ gene expression.
What is baculovirus expression?
The baculovirus expression system (BEVS) has become a vital gene expression “tool” in the production and characterization of numerous recombinant proteins. Polyhedrin is a protein produced by wild-type baculovirus to protect the virus from environmental pressures such as temperature and osmolarity.
What is polyhedral occlusion bodies?
Polyhedrins are a type of viral protein that form occlusion bodies (also called polyhedra), large structures that protect the virus particles from the outside environment for extended periods until they are ingested by susceptible insects.
Why is CMV promoter used?
The CMV promoter is a commonly used promoter for the production of high level recombinant protein in mammalian cells17. However, the expression level of the transgene driven by CMV promoter decreases with extended culture times because of transcriptional silencing, which is associated with DNA methylation18, 19.
What is a Bacmid?
The bacmid is essentially a very large plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking site inserted in the lacZ coding region. The most commonly used cell line for expressing recombinant proteins from baculovirus vectors is Sf9.
What is occlusion body?
Occlusion bodies are protein lattices that protect infectious particles after release into the environment. This strategy is mostly used by insect viruses to keep long-term infectivity of tissues after the host death. Poxviridae viruses produce a kind of occlusion bodies called “A type inclusions” (ATI).
How do you amplify baculovirus?
The Quick Amplification Guide suggests the best way to amplify, should the baculovirus yield be lower than average….
- Combine and mix the following: 0.5ml 0.4% trypan blue + 0.3 ml 1x PBS + 0.2 ml Sf9 cells.
- Dampen the haemocytometer, either side of the trough, and press coverslip firmly into place.
