The Baculovirus Expression System: A laboratory guide

ISBN-13: 9789401050470 / Angielski / Miękka / 2012 / 229 str.

Linda King

Opis

The decision to write a book about the practical aspects of the baculovirus expression system stems from the numerous phone calls for help we have had, and from the many visitors to our labora- tories requiring assistance to find the elusive polyhedrin-negative virus containing their favourite gene. We have also organized two expression system workshops and from the manuals we wrote for these, it seemed a logical progression to extend them into book form. We appreciate that those who are 'old-hands' at the baculovirus expression system may have differing views on some of our procedures, but the methods in this book are presented in the light of our own experiences in the laboratory and from our practical workshops, and we hope that the book will be especially useful to those new to the system. The first three chapters give the background information to the baculovirus expression system, and includes advice on how to choose the right transfer vector and discusses the various methods that are available to select recombinant viruses. The practical chapters concentrate on those aspects which are novel to the baculovirus system (insect cell culture, virus amplification and titration, etc. ) and, in general, leave the standard molecular biological techniques to the other excellent laboratory manuals that are available. However, for completeness sake and to avoid constant reference to other manuals, we have included brief details of some standard techniques where they are integral to the success of the baculovirus protocols.

Spis treści

1 The baculoviruses.- 1.1 Introduction.- 1.2 Isolation and host range.- 1.3 Structure and classification.- 1.4 Baculovirus replication in vivo.- 1.5 Baculovirus replication in vitro.- 1.5.1 Baculovirus gene expression and replication.- 1.5.2 Baculovirus gene promoters.- 1.6 Genetic engineering of baculovirus insecticides.- 2 The development of baculovirus expression vectors.- 2.1 Introduction and historical perspective.- 2.2 The merits of the baculovirus expression system.- 2.2.1 Advantages.- 2.2.2 Disadvantages.- 2.3 General principles for inserting foreign genes into the baculovirus genome.- 2.4 Baculovirus transfer vectors.- 2.4.1 Polyhedrin promoter-based expression vectors.- 2.4.2 p10 promoter-based transfer vectors.- 2.4.3 Multiple expression vectors.- 2.4.4 Transfer vectors utilizing other baculovirus gene promoters.- 2.5 Selection of recombinant viruses.- 2.5.1 Selection of a polyhedrin-negative phenotype.- 2.5.2 Selection of f3-galactosidase-negative viruses.- 2.5.3 Recombinant virus selection using dot-blot hybridization.- 2.5.4 Screening for a positive phenotype.- 2.5.5 Enhancing the numbers of recombinant viruses.- 3 Processing of foreign proteins synthesized using baculovirus vectors in insect cells.- 3.1 Introduction.- 3.2 Glycosylation.- 3.3 Phosphorylation, acylation and amidation.- 3.4 Proteolytic processing.- 3.5 Cellular targeting and secretion.- 3.6 Tertiary and quaternary structure formation.- 3.7 Expression of viral genes.- 3.8 Expression of bacterial and fungal genes.- 3.9 Post-transcriptional processing.- 4 Construction of transfer vectors containing the foreign gene.- 4.1 Introduction.- 4.2 Isolation of foreign gene coding sequences.- 4.2.1 Some general guidelines.- 4.2.2 Isolation of DNA fragments from agarose gels.- 4.3 Modifying the ends of DNA molecules.- 4.3.1 Mung bean nuclease.- 4.3.2 Klenow fill-in.- 4.4 Preparation of the transfer vector.- 4.5 DNA ligations.- 4.6 Transformation of bacteria.- 4.7 Screening for recombinant baculovirus transfer vectors.- 4.7.1 Colony hybridization.- 4.7.2 Rapid isolation of bacterial plasmid DNA (mini-preps).- 4.8 Analysis of recombinant transfer vectors.- 4.9 Isolation of highly purified plasmid DNA (maxi-preps).- 5 Insect cell culture media and maintenance of insect cell lines.- 5.1 Introduction.- 5.2 Cell lines.- 5.3 Culture media.- 5.4 Preparation of culture media.- 5.4.1 Preparation of TC100/FCS growth medium.- 5.4.2 Preparation of Grace’s (TNM-FH) growth medium.- 5.4.3 Preparation of 4.5 1 TC100 medium from powdered formula.- 5.4.4. Preparation of TC100 medium from individual ingredients.- 5.4.5 Specialized TC100 media.- 5.4.6 Alternative insect cell culture media.- 5.5 Glassware and disposable plasticware.- 5.5.1 Suggested cleaning regime for tissue culture glassware.- 5.6 Insect cell culture.- 5.6.1 Routine sub-culturing of Sf cell lines (monolayer cultures).- 5.6.2 Routine sub-culturing of Sf cells maintained in spinner cultures.- 5.7 A guide to Sf cell seeding densities for experimental work.- 5.8 Freezing, storage and recovery of insect cells in liquid nitrogen.- 5.8.1 Freezing and storage of cells in liquid nitrogen.- 5.8.2 Recovery of cells from liquid nitrogen.- 5.9 A guide to adapting cells to serum-free media.- 6 Propagation, titration and purification of AcMNPV in cell culture.- 6.1 Introduction.- 6.1.1 Safety considerations: general rules for working with baculoviruses.- 6.2 Infection of cells with virus for experimental work.- 6.2.1 Infection of Sf cells in monolayer culture.- 6.2.2 Infection of Sf cells in suspension culture.- 6.3 Titration of virus by plaque-assay.- 6.3.1 Standard plaque-assay.- 6.3.2 Plaque-assay of lacZ-positive viruses.- 6.4 Plaque-picking and plaque-purification.- 6.5 Amplification of virus stocks.- 6.5.1 To prepare a seed stock of virus from a plaque-pick.- 6.5.2 Preparation of an intermediate stock of virus.- 6.5.3 Preparation of a high-titre working stock of virus.- 6.6 Large-scale production of virus for the purification of virus particles.- 6.7 Purification of infectious virus DNA.- 6.8 Titration of virus by TCID50.- 7 Production and selection of recombinant virus.- 7.1 Introduction.- 7.2 Preparation of linear AcMNPV.lacZ (or AcMNPV.SC) DNA.- 7.3 Co-transfection of insect cells.- 7.3.1 Co-transfection by lipofection.- 7.3.2 Co-transfection by calcium phosphate co-precipitation.- 7.4 Separation of parental and recombinant viruses by plaque-assay.- 7.5 Plaque-purification and amplification of recombinant virus stocks.- 7.6 Amplification and detection of recombinant viruses by limiting dilution and dot-blot hybridization.- 8 Characterization of recombinant viruses.- 8.1 Introduction.- 8.2 Analysis of recombinant virus genomes.- 8.2.1 Extraction of DNA from virus-infected cells.- 8.2.2 Analysis of virus DNA with restriction endonucleases.- 8.2.3 Southern hybridization analysis of virus DNA.- 8.3 Analysis of foreign gene expression by polyacrylamide gel electophoresis, using unlabelled or radiolabelled cell proteins.- 8.3.1 Radiolabelling proteins in virus-infected insect cells.- 8.3.2 Polyacrylamide gel electrophoresis of infected cell extracts.- 8.4 Analysis of recombinant protein synthesis in insect cells using immunological techniques.- 8.4.1 Immunofluorescence.- 8.4.2 Western blot analysis of virus-infected cell proteins.- 8.4.3 Immunoprecipitation of virus-infected cell proteins.- 8.5 Analysis of post-translational processing events in insect cells.- 8.5.1 Glycosylation.- 8.5.2 Phosphorylation.- 8.5.3 Palmitylation and myristylation.- 8.6 Analysis of transcription in recombinant virus-infected cells.- 8.6.1 Extraction of RNA from insect cells.- 8.6.2 Analysis of RNA using Northern blot hybridization.- 9 Scaling up the production of recombinant protein in insect cells; laboratory bench level.- 9.1 Introduction.- 9.2 Large-scale culture of insect cells.- 9.2.1 Large-scale culture of insect cells in monolayer cultures.- 9.2.2 Large-scale culture of insect cells in suspension cultures.- 9.3 The importance of highly infectious virus stocks.- 9.4 Multiplicity of infection.- 9.5 The optimum time to harvest virus-infected cells.- 9.6 Purification of recombinant protein from infected cell cultures.- 10 Propagation of baculoviruses in insect larvae.- 10.1 Introduction.- 10.2 Rearing insects in the laboratory.- 10.3 Infection of insect larvae with polyhedra from cell culture.- 10.3.1 Preparation of virus polyhedra from infected cells in rulture.- 10.3.2 Propagating the virus in insect larvae.- 10.4 Purification of polyhedra from infected larvae.- 10.5 Bioassays of polyhedra.- 10.5.1 LD50 assays.- 10.5.2 LT50 assays.- 10.6 Purification of virus particles and DNA from polyhedra.- 10.7 Isolation of virus particles from infected larvae to establish infections in cell culture.- 10.7.1 Purification of virus particles from polyhedra for the infection of cells in culture.- 10.7.2 Purification of virus particles from haemolymph for the infection of cells in culture.- 10.8 Preparation of semi-synthetic insect diet.- 11 Trouble-shooting guide.- 11.1 Introduction.- 11.2 Insertion of foreign gene coding sequences into transfer vectors.- 11.2.1 The transfer vector.- 11.2.2 DNA sequences for insertion into transfer vectors.- 11.2.3 Ligations.- 11.3 Cell culture.- 11.3.1 Cells fail to thrive and attach to glass/plastic surfaces.- 11.3.2 Cells are contaminated with virus.- 11.3.3 Cells are contaminated with yeast, fungi or bacteria.- 11.3.4 Crystals of precipitate in the medium.- 11.4 Preparation of virus stocks and infectious DNA.- 11.4.1 Virus stocks.- 11.4.2 Infectious virus DNA purification.- 11.5 Co-transfections.- 11.6 Baculovirus plaque-assays.- 11.6.1 Condition of the cells.- 11.6.2 Plaque-assay manipulations.- 11.6.3 General problems.- 11.7 Screening for recombinant viruses.- 11.8 Instability of recombinant viruses.- 11.9 Poor yields of recombinant protein.- Appendix A list of selected suppliers.- References.