baculovirus recombinant
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Bac-to-Bac Baculovirus Expression System
The Bac-to-Bac™ Baculovirus Expression System provides a rapid and highly effective method to generate recombinant baculoviruses based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) propagated in E coli The major components of the Bac-to-Bac™ Baculovirus Expression System include: |
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Bac-to-Bac Baculovirus Expression System
These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses |
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Guide to Baculovirus Expression Vector Systems (BEVS) and
baculovirus system has become one of the most versatile and powerful eukaryotic vector systems for recombinant protein expression (4) More than 600 recombinant genes have been expressed in baculoviruses to date Since 1985 when the first protein (IL-2) was produced in large scale from a recombinant baculovirus use of BEVS has increased |
Which medium is used for recombinant baculovirus cell culture?
Traditionally, Grace’s Supplemented (TNM-FH) medium has been the medium of choice for insect cell culture. However, other serum/hemolymph-dependent and serum-free formulations have evolved since Grace’s medium was introduced. FIGURE 3. Generation of recombinant baculoviruses and gene expression with the BAC-TO-BAC expression system. TABLE 2.
How does baculovirus infection affect recombinant protein synthesis?
Baculovirus infection of insect cells results in microscopically observable cell lysis within 3–5 d after infection. Cell disruption may lead to increased proteolytic activity and other environmental factors that can result in degradation of recombinant protein.
How many recombinant genes are in a baculovirus?
Since 1983, when BEVS technology was introduced, the baculovirus system has become one of the most versatile and powerful eukaryotic vector systems for recombinant protein expression (4). More than 600 recombinant genes have been expressed in baculoviruses to date.
How do I make a recombinant baculovirus?
Transfect the recombinant bacmid DNA into the insect cell line of choice to generate a recombinant baculovirus. Amplify and titer the baculoviral stock, and use this stock to infect insect cells to express your recombinant protein. This manual is supplied with the products listed below.
IMPORTANT LICENSING INFORMATION
These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. tools.thermofisher.com
IMPORTANT
Changes from previous version Purpose of this manual tools.thermofisher.com
About this guide
Before using this product, read and understand the information in the “Safety” appendix in this document. This manual provides an overview of the Bac-to-Bac® Baculovirus Expression System, and provides instructions and guidelines to: Clone your gene of interest into the pFastBacTM donor plasmid of choice. Transform the pFastBacTM construct into MAX
Types of products
This manual is supplied with the products listed below. For a list of the reagents supplied with each catalog number, see below and the next page. tools.thermofisher.com
Shipping and storage
The Bac-to-Bac® Baculovirus Expression System is shipped in three boxes as described below. Upon receipt, store each box as detailed below. All reagents are guaranteed for six months if stored properly. tools.thermofisher.com
Choosing a pFastBacTM vector
A number of pFastBacTMvectors are available for use with the Bac-to-Bac® Baculovirus Expression System (see table below). Choose the vector that best suits your needs. tools.thermofisher.com
Bac-to-Bac® TOPO® Expression System
The Bac-to-Bac® TOPO® Expression System provides a rapid and highly effective method to generate recombinant baculoviruses by combining the ease of blunt-end TOPO® cloning with the efficiency of site-specific transposition technology of the Bac-to-Bac® System. The Bac-to-Bac® TOPO® Expression System is available separately with a choice of pFastBa
Diagram of the Bac-
The figure below depicts the generation of recombinant baculovirus and the - - tools.thermofisher.com
Cloning considerations
The pFastBacTM1 vector is a non-fusion vector (i.e., no fusion tags are present in the vector). To ensure proper expression of your recombinant protein, your insert must contain: An ATG start codon for initiation of translation A stop codon for termination of the gene tools.thermofisher.com
Note:
Stop codons are included in the multiple cloning site in all three reading frames. Note The production of recombinant proteins requires that your insert contain a translation initiation ATG. Generally, transfer vectors that contain intact polyhedrin (PH) leader sequences (e.g., pFastBacTM vectors) may yield higher levels of expression than vectors
Note
Generally, transfer vectors that contain intact polyhedrin (PH) leader sequences (e.g., pFastBacTM vectors) may yield higher levels of expression than vectors that contain interrupted leader sequences. Protein translation can initiate at the mutated ATG (ATT) upstream of the multiple cloning site; however, initiation from this site is inefficient a
Cloning into pFastBacTM Dual
An ATG start codon for initiation of translation A stop codon for termination of the gene if you don’t use one of the stop codons provided in the multiple cloning site tools.thermofisher.com
Note
The production of recombinant proteins requires that your insert contain a translation initiation ATG. Generally, transfer vectors that contain intact polyhedrin leader sequences (e.g., pFastBacTM vectors) may yield higher levels of expression than vectors that contain interrupted leader sequences. For inserts cloned downstream of the polyhedrin pr
Analyzing transformants by PCR
You may also analyze positive transformants using PCR. Use the appropriate PCR primers and amplification conditions for your insert. If you are using this technique for the first time, you may want to perform restriction analysis in parallel. Artifacts may be obtained because of mispriming or contaminating template. The protocol below is provided f
IMPORTANT
Insertions of the mini-Tn7 into the mini-attTn7 attachment site on the bacmid disrupt the expression of the LacZα peptide, so colonies containing the recombinant bacmid are white in a background of blue colonies that harbor the unaltered bacmid. Select white colonies for analysis. True white colonies tend to be large; therefore, to avoid selecting
Transforming DH10BacTM E. coli, continued
Note: It is possible to verify successful transposition to the bacmid by using agarose gel electrophoresis to look for the presence of high molecular weight DNA. This method is less reliable than performing PCR analysis as high molecular weight DNA can be difficult to visualize. tools.thermofisher.com
Isolating recombinant bacmid DNA
Introduction Before starting Equilibrating the column Preparing the cell lysate tools.thermofisher.com
Transfecting insect cells
Introduction Plasmid preparation Transfection method Cellfectin® II reagent Insect cell lines tools.thermofisher.com
Positive control
If you have generated a recombinant bacmid from one of the pFastBacTM control plasmids (i.e., pFastBacTM-Gus, pFastBacTMHT-CAT, or pFastBacTM Dual-Gus/CAT), we recommend including this positive control in your transfection and expression experiments to help you evaluate your results. In these bacmids, the gene encoding β-glucuronidase (Gus) and/or
Introduction
Follow the guidelines and instructions provided below to: Determine the titer of your baculoviral stock Plaque purify the virus (optional) tools.thermofisher.com
Plaque purification
You may generate a viral stock from a single viral clone by plaque purifying your baculovirus, if desired. Use a protocol of your choice or the procedure below. tools.thermofisher.com
Baculovirus Expression System for Recombinant Proteins Expression Protocol Preview
Recombinant protein production in Baculovirus Insect expression system
Baculovirus insect cell expression system
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Pamukkale Üniversitesi Mühendislik Bilimleri Dergisi Pamukkale
Production of recombinant protein is widely studied in recent years. Spodoptera frugiperda insect cell lines preferred as a host for baculovirus (BV) because of |
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Guide to Baculovirus Expression Vector Systems (BEVS) and Insect
Generating a recombinant baculovirus by homologous recombination. Clone the gene to be expressed into the transfer vector. DAY 0: Co-transfect insect cells with |
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Use of Baculovirus as Expression Vector in Biotechnology
1 Oca 1998 virus in insect cells by a recombinant baculovirus and analysis of its immunogenic property. Virus Res.. 12:169-180 |
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Active baculovirus recombinant p70s6k and p85s6k produced as a
23 Eki 1992 Among these systems infection of Sf9 cells by baculovirus recombinants ... 1 recombinant virus (lane 3 ) or clone 2 recombinant virus (lane 6). |
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Engineering of the baculovirus expression system for optimized
18 Eki 2018 Initially recombinant baculoviruses were obtained by homol- ogous recombination between the viral genome and a donor plasmid into the insect ... |
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Generation of a Baculovirus Recombinant Prostate-Specific
24 Kas 1999 Prostate-specific membrane antigen (PSMA) is a 100-. kDa transmembrane glycoprotein identified by the monoclonal antibody 7E11-C5.3 from the ... |
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Screening Recombinant Baculovirus Plaques In Situ with Antibody
A procedure that allows one to directly detect baculo- virus recombinants that express foreign proteins of in- terest in situ using antibody probes is |
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Characterization of baculovirus recombinant wildâ•type p53
Purification of recombinant nuclear p53. The recombinant baculovirus used was vEVSSpS3 (O'R- eilly and Miller 1988). All the purification steps were per |
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Baculovirus Recombinant Expressing a Secreted Form of a
nien nuclear polyhedrosis virus by homologous recombination. The full- length cDNA baculovirus recombinant directed the expression of a 40-. kDa glycoprotein |
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Immunogenicity of baculovirus expressed recombinant proteins of
After 3 days cultured recombinant virus was harvested and the cells were incubated continuously by adding fresh medium. To purify recombinant baculovirus |
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Guide to Baculovirus Expression Vector Systems (BEVS) and Insect
Generating a recombinant baculovirus by homologous recombination. Clone the gene to be expressed into the transfer vector. DAY 0: Co-transfect insect cells with |
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Utilization of Recombinant Baculovirus Expression System to
10 juin 2022 This recombinant baculovirus expression system was then utilized to produce the receptor binding domain (RBD) from the spike protein of SARS-CoV ... |
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La pathologie des insectes au service du génie génétique
QUELQUES PROTÉINES RECOMBINANTES OBTENUES DANS. LE SYSTÈME DE CELLULES D'INSECTES INFECTÉES PAR LE BACULOVIRUS d'un ADN viral recombinant porteur. |
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Optimizing Recombinant Baculovirus Vector Design for Protein
25 nov. 2021 One of the platforms predominantly used for eukaryotic recombinant protein synthesis is the baculovirus expression vector system (BEVS). It ... |
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Bioproduction cellules dinsectes M Cérutti version Web.pptx
10 juil. 2014 Baculovirus et Thérapie. CNRS UPS3044. Production de Protéines Recombinantes en cellules d'insectes. Anticorps recombinants. Martine Cérutti. |
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SARS-CoV-2 Virus-like Particles Produced by a Single
27 avr. 2022 Recombinant Baculovirus Generate Anti-S Antibody and ... structural proteins in insect cells using a recombinant baculovirus [9]. |
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Characterization of baculovirus recombinant wildâ•type p53
Characterization of baculovirus recombinant wild-type p53. Dimerization of p53 is required for high-affinity DNA binding and cysteine oxidation. |
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Active Cross-Protection Induced by a Recombinant Baculovirus
Active Cross-Protection Induced by a Recombinant. Baculovirus Expressing Chimeric Infectious Bursal. Disease Virus Structural Proteins. |
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Impact of Molecular Modification on the Efficiency of Recombinant
13 janv. 2022 Recombinant Baculovirus Vector Invasion to Mammalian ... However low expression of GP64 in recombinant baculoviruses also reduces the ... |
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A Recombinant Baculovirus Efficiently Generates Recombinant
Original Article. A Recombinant Baculovirus Efficiently Generates. Recombinant Adeno-Associated Virus Vectors in Cultured Insect Cells and Larvae. |
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La pathologie des insectes au service du génie - iPubli - Inserm
QUELQUES PROTÉINES RECOMBINANTES OBTENUES DANS LE SYSTÈME DE CELLULES D'INSECTES INFECTÉES PAR LE BACULOVIRUS Gène |
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Bioproduction cellules dinsectes M Cérutti version Webpptx - CNRS
10 juil 2014 · Baculovirus et Thérapie CNRS UPS3044 Production de Protéines Recombinantes en cellules d'insectes Anticorps recombinants |
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Guide to Baculovirus Expression Vector Systems (BEVS) and Insect
Protocols for Generating a Recombinant Baculovirus Protocol 6: Isolation of Bacmid DNA for BAC-TO-BAC® Baculovirus Expression System with the |
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UNIVERSITÉ DU QUÉBEC THÈSE PRÉSENTÉE À L - Espace INRS
de manipulations génétiques, des baculovirus recombinants beaucoup plus efficaces Chez les baculovirus, l'expression des gènes et la réplication de l' ADN |
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Virus - mediaeduscoleducationfr
cellules d'insectes infectées par un baculovirus recombinant 2 2 6 1 La particularité du baculovirus est liée à son cycle de multiplication et à la production de |
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Recombinant Protein Production in Baculovirus/Insect - GenScript
Competent DH 10BacTM E coli Cells Recombinant Donor Plasmid Mini-prep Bacmid Recombinant Bacmid DNA Transfection Adherent sf9 cells Baculovirus |
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Baculovirus as a highly efficient expression vector in insect - Nature
Baculovirus has been widely used for the production of recombinant proteins in insect cells Since the finding that baculovirus can efficiently transduce mamma- |
![PDF] Optimization of the Strategy for Recombinant Baculovirus](https://ars.els-cdn.com/content/image/1-s2.0-S1050464819306400-fx1.jpg "PDF] Optimization of the Strategy for Recombinant Baculovirus")
![PDF] Baculovirus as a Tool for Gene Delivery and Gene Therapy](https://media.springernature.com/w306/springer-static/cover-hires/book/978-1-59745-457-5 "PDF] Baculovirus as a Tool for Gene Delivery and Gene Therapy")
