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This detailed volume provides up-to-date guidance on techniques used to work with baculoviruses and insect cells. The book provides basic methods to create recombinant baculoviruses, to improve productivity, to produce a variety of products, to purify products, to quantify baculovirus stocks or to quantify product produced, and it concludes with alternative uses of either baculovirus or insect cells as tools. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and comprehensive, Baculovirus: Methods and Protocols serves as an ideal guide for researchers looking to overcome some of the limitations associated with the early baculoviral vectors and cell lines.
Table des matières
Expansion and Storage of Insect Cells and Virus Stocks.- Construction of Recombinant Baculoviruses.- A Time and Cost-Effective Pipeline for Expression Screening and Protein Production in Insect Cells Based on the HR-Bac Toolbox to Generate Recombinant Baculoviruses.- Optimized Workflow from Gene to Product.- TOPO Cloning for Efficient Plasmid Construction.- Adaptive Laboratory Evolution to Improve Recombinant Protein Production Using Insect Cells.- RNAi-Mediated Silencing in the Insect Cell-Baculovirus Expression System.- Scarless Baculovirus Genome Editing Using Lambda Red Recombineering in E. coli.- Probing Baculovirus Vector Gene Essentiality for Foreign Gene Expression Using a CRISPR-Cas9 System.- Generation of Complex Protein Structures by Co-Infection with High Quality Recombinant Baculovirus.- Production of Secreted Antibody in Baculovirus Expression Vector System.- Production of Recombinant Viral Antigens Using the Baculovirus-Insect Cell Expression System.- Protein Production at the 100L Scale.- Recombinant AAV Production.- Recombinant AAV Purification.- Extraction of Modified Adeno-Associated Virus-Like Particles Displaying Peptides on Their Surface.- One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.- Purification Process for a Secreted Protein Produced in Insect Cells.- Functional Baculovirus Particle Quantification via Plaque Assay.- TCID50 Assay: A Simple Method to Determine Baculovirus Titer.- Rapid Two-Day Baculovirus Titering Using Flow Cytometry.- A Microsphere Immunoassay for the Quantitative Detection of Antigens in Cell Culture Supernatant.- Non-Viral Platform for Expression of Recombinant Protein in Insect Cells.- Assembly of Baculovirus Vectors for Multiplexed Prime Editing.- Recombinant Protein Production in Suspension Mammalian Cells Using the BacMam Baculovirus Expression System.
