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WO2008146019A2 - Génération de baculovirus - Google Patents

Génération de baculovirus Download PDF

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Publication number
WO2008146019A2
WO2008146019A2 PCT/GB2008/001867 GB2008001867W WO2008146019A2 WO 2008146019 A2 WO2008146019 A2 WO 2008146019A2 GB 2008001867 W GB2008001867 W GB 2008001867W WO 2008146019 A2 WO2008146019 A2 WO 2008146019A2
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cells
virus
baculovirus
bacmid
insect cells
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WO2008146019A3 (fr
WO2008146019A8 (fr
Inventor
Kari Juhani Airenne
Seppo Ylä-Herttuala
Hanna-Riikka KÄRKKÄINEN
Olli Laitinen
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Ark Therapeutics Ltd
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Ark Therapeutics Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/103—Plasmid DNA for invertebrates
    • C12N2800/105—Plasmid DNA for invertebrates for insects

Definitions

  • This invention relates to baculovirus generation.
  • Background of the Invention Various on-going and finished genome projects are sources of novel open reading frames, which are likely to provide protein drug candidates or targets for medical use. The vast amount of data requires efficient high-throughput methods to turn the genetic code into functional information. Thus, the need for a simple and flexible protein production technology with a high diversity and capacity exists.
  • Baculoviruses are potential tools for this purpose since during the last 20 years, thousands of proteins from different species have been produced in insect cells using baculovirus expression system.
  • the versatility of baculoviruses has become even more apparent from their use as successful gene transfer vehicles in multiple vertebrate cell lines and in vivo. In addition, recent developments in the baculovirus production methods have rendered them more suitable for high-throughput screening purposes (Airenne, 2003).
  • amplification and titer determination of recombinant baculoviruses viruses are critical steps in obtaining accurate and consistent results.
  • the titer determination is done by detecting morphological changes in infected cells by using plaque formation and or point dilution assays.
  • plaque formation and or point dilution assays are laborious and plaque assay, in particular, is highly dependent on technical expertise. Indeed, several improved protocols for virus titering have been described. Some of these methods are based on immunological assays, which recognize the viral proteins on the surface of infected cells.
  • Bac-PAK baculovirus rapid titer kit (BD Biosciences; Clontech) is based on the immunological assay against gp64 glycoprotein present on the virus surface.
  • the kit reduces the time required for titer determination to 2 days, however with high cost and manual labor.
  • Mulvania and colleagues detected anti-gp64 labeled cells by flow cytometry 18 hours post infection.
  • Flow cytometry has also been used to detect labeled viral DNA as well as to detect DNA and capsid proteins simultaneously.
  • Titer has also been determined by analyzing cell size after infection and amplifying fragments of selected viral genes by quantitative PCR. As a disadvantage, false positive results may occur when fragments of baculoviral genome are detected.
  • Some methods are based on the expression of the GFP marker protein driven by a viral promoter in infected insect cells.
  • the fluorescent marker is transferred to the baculovirus genome together with the transgene from the donor vector, an arrangement which increases the size of the donor vector, possibly leading to problematic cloning and lack of space for long human genes.
  • McCaII et al, 2005 disclose transfection in suspension, but in 24-well plates.
  • Bahia et al, 2005 disclose a method to culture insect cells in deep 24-well plates for small-scale optimization for baculovirus-mediated protein expression experiments. It is stated that "The capacity of insect cells to grow in 48- and 96-well format was also investigated, but we were unable to obtain reliable growth of cells in these systems.”
  • Garzia et al, 2003 show no virus generation in HTS format, but an automated method to express and purify a protein in a 96-well plate format.
  • the present invention is based on the discovery that baculoviruses can be generated in high-throughput plates.
  • the present invention is a method of baculovirus generation from insect cells, using a high-throughput plate, wherein the insect cells are transfected or infected by a virus construct, in suspension, and wherein the plate comprises wells which have a substantially square cross-section. Description of Preferred Embodiment
  • a "virus construct” is any baculovirus genome vector that can generate a baculovirus.
  • a suitable virus construct is a BVboostFG virus.
  • Another example is F-Bacmid.
  • a "high-throughput plate” is a plate suitable for high- throughput screening (HTS).
  • a HTS plate contains multiples of 96-wells. In the present invention, the HTS-plate is preferably a 96-well plate.
  • the insect cells are transfected or infected, in suspension. This may improve the baculovirus generation, and help make it possible in 96-well plates. Preferably, they are kept in suspension throughout the transfection process. This may be achieved by shaking the plate.
  • the plate contains wells which have a substantially square cross-section. This may aid the baculovirus generation.
  • the F-bacmid contins a fluorescent protein expression cassette.
  • the expression cassette is an enhanced green fluorescent protein expression cassette.
  • the insect cells are spodoptera frugiperda (sf cells) or Trichoplusia ni (Tn) cells. More preferably, the cells are sf9 or BTI-TN-5B1- 4 (High Five).
  • F- bacmid a novel baculoviral genome shuttle vector, F- bacmid, by introducing an enhanced green fluorescent protein (EGFP) gene under the control of polyhedrin promoter into the baculovirus genome.
  • EGFP cassette was inserted by MuA transposase random reaction and found to be located into the ODV-E66 gene of the baculovirus genome.
  • This novel baculovirus vector enables the easy detection of recombinant virus generation in insect cells by fluorescence.
  • a simple and rapid 18 hour titer determination protocol compatible with F-bacmid or any fluorescent protein expressing baculovirus involves flow cytometry and miniaturization of the baculovirus generation to 96-well format.
  • the F- bacmid viruses produce efficiently desired recombinant proteins in insect cells.
  • the baculoviruses generated by the F-bacmid system also function efficiently as gene delivery vehicles in vertebrate cells.
  • the F-bacmid vector is based on Ark's improved Tn7 mediated BVboost baculovirus generation system, further supporting the suitability of the system for recombinant protein production, high-throughput screening (HTS), functional genomics and gene delivery.
  • HTS high-throughput screening
  • F-bacmid recombinant baculovirus
  • This expression cassette was introduced into the baculovirus genome by using a random MuA transposase reaction.
  • the newly constructed F-bacmid is compatible with transposition-based baculovirus generation systems (6,7,21). Consequently, the infected insect cells can be visualized by fluorescence microscopy or flow cytometry, making the infection monitoring and titer determination substantially faster and more convenient as compared to traditional methods.
  • aspects of this invention that may be critical to success are the use of substantially square wells and/or cell culture on bottles followed by transfer to plate, before transfection, and/or transfection of the cells in suspension. Rapid expression of recombinant proteins remains challenge in the post- genomic era, since most of the current protocols are time consuming and laborious, limiting the number of constructs and expression conditions which can be studied simultaneously.
  • a high-throughput method based on established baculovirus system, would be highly useful for this purpose, since baculovirus-insect cell system has capability for eukaryotic protein processing and efficient protein production. However, so far attempts to convert baculovirus system into 96-well format have been unsuccessful.
  • baculovirus generation was shown to be successful in deep 96- well plates. Using green fluorescent protein we could reliably detect progression of the infection as well as protein production in the 96-well plate format. This miniaturization is a vital milestone in developing a high-throughput screening systems, allowing extensive and rapid optimization of multiple expression conditions before initiation of large-scale experiments. Miniaturisation also enables automation. Most of the previous studies describing virus generation in multiwell format, show formation of viruses in 24-well plates. We also observed that the increase of the incubation time from 3 days to 7 days in 96 well plates increased the amount of EGFP positive cells, supported by McCaII et al. Miniaturisation to a 96- well plate platform enables the creation of recombinant baculoviruses and proteins in true high-throughput format for highly efficient screening purposes.
  • plaque assay and end-point titering methods may give lower titers compared to methods developed more recently. Shen and colleagues obtained, on average, 3.7 times higher titers with flow cytometric titering compared to plaque assay and end-point titering. The traditional titering methods are known to be less sensitive and the difference was considered to be a combination of this and the use of SYBR Green I to stain viral DNA, making it impossible to distinguish between infective and non infective virus particles.
  • infected cells are the only indication of infective viruses, Mulvania et a/, labelled cells with anti-gp64 and phycoerythrin-conjugated secondary antibody followed by detection by flow cytometry to determinate the titers in less than 24 hours. Yet, the cost of antibodies renders this method rather expensive and tedious.
  • the amount of infective viruses can also be detected by analyzing the size of infected cells in suspension. Janakiraman and colleagues measured cell- diameter 24 hours post infection and applied statistical modeling techniques to obtain virus titer, which were three-fold higher as compared to titers obtained with the plaque assay method. This was explained with the lower sensitivity of the plaque assay as compared to the cell-dimension measurement.
  • VSV-G and VSV-GED have shown to significantly enhance gene delivery
  • avidin-display has enabled biodistribution imaging and capsid protein vp39 modifications have enabled protein delivery.
  • transgene expression levels have been enhanced by introducing the Woodchuck Hepatitis Virus Post-transcriptional Regulatory element (WPRE) in to expression cassettes (Mah ⁇ nen, A unpublished). Since the accurate viral titer is essential for both protein production and gene therapy, we assessed the accuracy of the flow cytometric method by comparing it to end-point dilution method.
  • WPRE Woodchuck Hepatitis Virus Post-transcriptional Regulatory element
  • the presented 96-well plate format offers an efficient virus generation platform enabling high-throughput virus and protein production.
  • the novel F- bacmid based protocol allows direct measuring of infective titer by using a simple and fast flow cytometric method. Steps of bacmid isolation, baculovirus generation and virus titering are adapted into true high throughput format so that the system can be automated.
  • the following study illustrates the invention. Study Construction of modified baculovirus shuttle vector (bacmid)
  • baculovirus genome was obtained by extracting the plasmid DNA from DHIOBac cells (Invitrogen, Carlsbad, CA, USA) and subsequently transforming DH10B cells (Invitrogen). Colonies resistant for kanamycin and sensitive to tetracycline (no helper plasmid) were selected.
  • the cassette expressing EGFP was prepared by using the following procedure: Plasmid pfastbad EvolEGFP was digested with Swal and SnaBI. The cassette containing the EGFP gene under the polyhedrin promoter was isolated and blunted with T4 DNA polymerase. This was ligated with EcoRV digested pEntranceposon(cam r ) plasmid (Finnzymes, Espoo, Finland). The accomplished plasmid was digested with BgIII and the fragment containing the pPohl-EGFP cassette flanked by Mu transposon inverted terminal repeats was used in transposition reaction (Figure 1) according to manufacturer's instructions (Finnzymes). The EGFP expression cassette containing bacmids were screened for antibiotic resistance by using chloramphenicol.
  • Inverted PCR was used to determine the integration site of the EGFP expression cassette in the bacmid DNA from the validated clone.
  • the DNA was isolated from bacteria and amplified by PCR using 5'- ATACTCGTCGACAAGCTTCTCG-3' and ⁇ '-GTATCAACAGGGACACCAGGAT-S' primers, which amplified the DNA outwards from the EGFP expression cassette.
  • the amplified fragments were purified using Wizard ® SV Gel and PCR clean up system (Promega, Madison, Wl, USA) and cloned into pGEM ® -T Easy vectors (Promega) according to manufacturer's instructions. Correct clones were screened for by blue-white screening and restriction analysis with EcoRI.
  • the TA- cloned inserts were sequenced with primers ⁇ '-AATACGACTCACTATAGGG-S' and ⁇ '-CATACGATTTAGGTGACACTATAG-a 1 .
  • the obtained nucleotide sequences were fed into the NCBI-BLAST ® (National Centre of Biotechnology, USA- Basic
  • F-bacmid containing DH10B cells were transformed with helper plasmid and the resulting bacterial clone was further transformed by pBVboost ⁇ amp and selected for as described in Airenne 2003.
  • the strain was named as DH10Bac ⁇ Tn7EGFP.
  • Generation of recombinant baculoviruses Recombinant baculoviruses expressing transgene under universal promoter were prepared as described in Laitinen, 2005 by using E. coli strain DH10Bac ⁇ Tn7EGFP.
  • Titers were determined by infecting insect cells in suspension with a series of virus dilutions and determining the percentage of infected cells by flow cytometry.
  • the virus samples were serially diluted in duplicates and 0.5 ml of each dilution was used to infect 0.5 ml of Sf9 cells (approximately 2 x 10 6 cells/ml). Incubation was allowed to continue for 18 hours at 27°C with shaking at 270 rpm, before the samples were centrifuged for 5 minutes at 500 x g.
  • the cells were resuspended in 2% FBS in PBS and analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NY, USA) to reveal the percentage of the cells which were infected by EGFP expressing baculoviruses.
  • the percentage of cells emitting green fluorescence for each sample was analyzed by counting 10000 cells per measurement in triplicates. To ensure optimal infection time and to ascertain its effect on viral titer, titer was also determined 24 hours post infection. In addition, the virus titers were also determined by using end-point dilution method. Protein production efficiency of F-bacmid derived viruses in insect cells
  • Sf9 cells 25 ml of Sf9 cells (1 x 10 6 cell/ml) were infected with avidin and VEGF-D ⁇ N ⁇ C viruses [F-bacmid derived and standard BVboost viruses] at a multiplicity of infection (MOI) 5. Infection was accomplished with secondary virus preparation and allowed to continue for 4 days at 27 Q C, shaking at 110 rpm. The infected cells were lysed and total protein amount was quantified with Coomassie Plus- The Better BradfordTM Assay Reagent (Pierce, Rockford, IL, USA).
  • Lysates and supematants were analyzed by Western blotting using polyclonal rabbit anti-avidin and mouse anti-hVEGF-D (R&D systems, Minneapolis, MN, USA) antibodies.
  • the concentration of VEGF-D ⁇ N ⁇ C in the supernatant was also analyzed by ELISA (Quantikine® Human VEGF-D kit, R&D Systems) according to manufacturer's instructions.
  • Protein production was also performed in larger scale with F-bacmid derived baculovirus expressing VEGF-D ⁇ N ⁇ C .
  • Sf9 cells (1 x 10 6 cells/ml, 500 ml) were infected with the virus at MOI 5. The cells were incubated at 27 Q C, shaking at 110 rpm for 4 days prior to harvesting the medium for protein purification. Purification was accomplished with metal affinity chromatography (IMAC). The protein was attached to 3 ml of cobalt containing resin (BD Talon Metal Affinity Resin, BD Biosciences Clontech, Palo Alto, CA, USA) and subsequently transferred to purification columns (EconoPac, Bio-Rad, Hercules, CA, USA).
  • cobalt containing resin BD Talon Metal Affinity Resin
  • the protein containing column was washed 3 times with washing buffer (50 mM Sodium phosphate, 300 mM NaCI , pH 7.0) prior to collection of 12 eluates (elution buffer; 50 mM HEPES, 20 mM NaCI, 450 mM imidazole) comprising 1 ml each.
  • Eluate fractions 2-4 were dialyzed (Slide-A-LyzerDialysis Casette, 3.5K MWCO, Pierce) twice for 6 hours and concentrated against buffer containing 50 mM HEPES, and 20 mM NaCI, 30% PEG at pH 7.4.
  • the concentration of the purified protein was determined using DC protein assay kit (Bio-Rad) according to manufacturer's instructions. Testing F-bacmid derived baculoviruses in mammalian cells Human hepatoma (HepG2), rat glioma (BT4C), and human ovarian cancer
  • SKOV-3 cells were transduced with avidin and VEGF-D ⁇ N ⁇ C viruses (F-bacmid derived and standard BVboost viruses).
  • HepG2, BT4C and SKOV-3 cells were plated into a 6 cm plate containing 5 x 10 5 , 6 x 10 5 , and 1.5 x 10 5 cells, respectively, and transduced after 24 hours. It has been shown that mammalian cell cultures are not disturbed by small amounts of insect cell medium when transducing mammalian cells with unconcentrated baculoviruses. Thus, mammalian cells were transduced with unpurified secondary virus preparations in insect cell medium.
  • Baculovirus generation in 96-well plate format Baculovirus generation was carried out in deep 96-well plate format by transfecting Sf9 cells in suspension with F-bacmid expressing VEGF-D ⁇ N ⁇ C .
  • the transfection solution consisted of 40 ⁇ l of Insect X-press medium, 0.6 ⁇ l of cationic lipid reagent Cellfectin ® (Invitrogen) and 5 ⁇ l of F-bacmid DNA.
  • the bacmid DNA was isolated with Montage BAC 96 Miniprep Kit according to the manufacturer's instructions (Millipore, Billericia, MA, USA) and the DNA concentrations were measured by NanoDrop (NanoDrop Technologies, Wilmington, DE, USA).
  • the generated baculoviruses were amplified and proteins were produced by infecting in 750 ⁇ l (approximately 1 x 10 6 cells/ml) Sf9 cells in 96-well plate, with 250 ⁇ l of primary virus supernatant (collected by centrifuging samples at 500 x g for 5 minutes). The cells were incubated at 27°C, shaking at 270 rpm for 7 days. Virus amplification was analyzed by fluorescence microscopy. The virus containing medium was collected by centrifugation as described previously, and stored at 4 5 C. Protein production was analyzed by Western blotting using polyclonal rabbit anti- avidin and mouse anti-hVEGF-D (R&D systems) antibodies.
  • the ODV-E66 protein is not included in the structure of budded baculovirus (the form used in protein production and gene therapy applications). Thus, the integration site should not disturb the function of the viral vector. To support this hypothesis, F-bacmid derived viruses were produced, and it was shown that EGFP expression cassette integrated in ODV-E66 gene does not affect the titer (Table 1) or function of the virus. Titer determination by flow cytometry To determine titer, Sf9 insect cells were infected with serial dilutions of
  • EGFP expressing baculoviruses and the percentage of infected insect cells was determined by flow cytometry at 18 and 24 hours post infection. However, the 24 h incubation led to overestimation of virus titer due to secondary infections (Table 3).
  • Calculations of virus titers were carried out by modifying the normalization method described by Mulvania et al. (Tables 2 and 3). First, the EGFP percentages of duplicate samples were averaged and normalized by dividing the averaged percentage by the maximum EGFP percentage (EGFP percentage of 1 :500 dilution of concentrated virus or undiluted secondary virus preparation). The value represents a corrected percentage of the EGFP expression. The corrected percentage was then multiplied by the dilution factor and by the number of cells per ml, to obtain the titer of the virus stock.
  • Table 1 Virus constructs and their titers obtained with end-point dilution and flow cytometer methods. Titers are shown as averages of multiple analyses.
  • the F-bacmid derived viruses are secondary virus preparations and the two0 last ones are concentrated viruses.
  • Table 2 Titer determination of a concentrated virus after 18 hours incubation.5
  • Seri ⁇ 1 Serle 2 (average/average of 1:500) x 100 (DF) cell number
  • titers were calculated from dilutions which contained 1 to 10% of EGFP positive cells following normalization (bolded in Tables 2 and 3). Using dilutions that produced higher0 EGFP percentages led to an underestimation of the virus titer and vice versa. If more than one dilution had a corrected percentage of between 1 to 10%, these corrected percentages were averaged for titer determination. To confirm the results obtained from the flow cytometric assay, viral titers were also determined with end- point dilution method based on the detection of morphological changes and EGFP5 fluorescence of cells.
  • BVboostFG viruses containing avidin and VEGF-D ⁇ N ⁇ C expression cassettes under universal promoter. Protein production was analyzed by Western blotting and ELISA. Western blot analysis showed high levels of VEGF-D ⁇ N ⁇ C expression when Sf9 cells were infected with F-bacmid derived or normal BVboostFG viruses. These0 results were confirmed by ELISA. As in the case of VEGF-D ⁇ N ⁇ C , avidin production did not significantly differ between the F-bacmid derived and standard BVboostFG viruses, with results within the normal variation.
  • Protein production was also performed in larger volume (0.5 I) with F-bacmid derived baculovirus expressing VEGF-D ⁇ N ⁇ C .
  • concentration of purified VEGF- D ⁇ N ⁇ C was comparable to the production and purification of VEGF-D ⁇ N ⁇ C by using the traditional pBVboost system.
  • F-bacmid viruses resulted in efficient gene expression in human hepatoma (HepG2), rat glioma (BT4C), and human ovarian cancer (SKOV-3) cells also.
  • Virus generation in 96-well plate format Virus generation was carried out in 96-well plate format by transfecting 0.5 x
  • the polyhedrin promoted expression of EGFP enabled detection of infection by fluorescence microscopy, showing that 88-100% of the 96 wells contained viruses.
  • the EGFP percentage of F-bacmid transfected Sf9 cells increased daily between days 3 to 7. On day 7, the percentage of positive cells per well varied between 70 to 85%. Protein production was successfully detected with Western blotting.

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Abstract

La présente invention concerne une méthode de génération de baculovirus à partir de cellules d'insectes, à l'aide d'une plaque à haut rendement, dans laquelle les cellules d'insectes sont transfectées ou infectées par un virus en suspension. Ladite plaque comprend des puits présentant une section transversale sensiblement carrée.
PCT/GB2008/001867 2007-06-01 2008-06-02 Génération de baculovirus Ceased WO2008146019A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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CN115791575A (zh) * 2022-08-26 2023-03-14 武汉华美生物工程有限公司 一种基于流式细胞术测定重组杆状病毒滴度的方法

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CA2321261A1 (fr) * 1998-03-04 1999-09-10 Onyx Pharmaceuticals, Inc. Systeme et procede d'expression de baculovirus pour l'expression a haut rendement de materiel genetique
US7319001B2 (en) * 2002-03-09 2008-01-15 Neurogenex Co., Ltd. High throughput system for producing recombinant viruses using site-specific recombination

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115791575A (zh) * 2022-08-26 2023-03-14 武汉华美生物工程有限公司 一种基于流式细胞术测定重组杆状病毒滴度的方法

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