Attenuated Chimpanzee Adenovirus Oxford (ChAdOx1) was modified to express each antigen of the Esx-5a secretion system separately. The fusion of four antigens, PE8, PPE15, EsxJ, and EsxI, was prepared using GeneArt Technology (ThermoFisher Scientific, UK). Ag85A was subsequently added at the C-terminus by PCR. The generation of ChAdOx1 and MVA constructs was performed as previously described (14 (link), 28 (link)). Briefly, the antigens were cloned into a Gateway entry vector, under a modified human cytomegalovirus major immediate early promoter (CMV). This was followed by recombination into the ChAdOx1 destination DNA BAC vector, with E1 and E3 regions deleted and E4 modified. The ChAdOx1 genome was excised with PmeI, and the virus was rescued and propagated in human embryonic 293 (HEK293 cells). For MVA, antigens were subcloned into a poxvirus shuttle vector under the control of the vaccinia virus p7.5 promoter, which was later recombined at the TK locus of MVA in chicken embryo fibroblasts (CEFs). Rescue and purification of the viruses were performed by the Viral Vector Core Facility, University of Oxford.
Geneart technology
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The GeneArt Technology is a suite of tools and services designed for DNA assembly and modification. It provides a comprehensive solution for the construction of genetic constructs, synthetic genes, and DNA fragments. The core function of the GeneArt Technology is to enable efficient and reliable DNA engineering for various applications in fields such as molecular biology, synthetic biology, and genomics.
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Lab products found in correlation
4 protocols using geneart technology
1
Multivalent Chimpanzee Adenovirus Vaccine
2
Cloning and Production of Viral Vectors
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ChAdOx1 and MVA expressing each antigen were cloned using GeneArt Technology (ThermoFisher Scientific, UK). Four mammalian codon-optimised antigens (fasD1, fasD2, glfT2, iniB) and flanked by a Kozak consensus sequence, a tPA leader sequence, a GS linker at the 5′-end and a PK tag, Histidine tag and STOP codon at the 3′ end, were cloned into a GeneArt entry vector and then recombined into ChAdOx1 or MVA destination plasmids as previously described.63 (link),64 (link) ChAdOx1 and MVA expressing Ag85A and PPE15 were produced as previously described.24 (link),25 Construct sequences are available upon request.
3
Mouse Fosl2 and Peli1 3' UTR Assay
4
Recombinant Glycosyltransferase Enzyme Assay
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Full-length UGT91R1 cDNA synthesized using GeneArt Technology (Life Technologies, Carlsbad, CA, USA) was subcloned into the pET15b expression vector (Merck Millipore, Darmstadt, Germany) and transformed into E. coli BL21 (DE3). Recombinant protein expression was induced by incubating 200 mL of culture with 0.1 mM IPTG at 22 °C for 24 h. Proteins were harvested using ultrasonic disruption and were purified using 1 mL of HisTrap HP (GE Healthcare, Chicago, IL, USA). The purified enzyme was incubated with 100 mM sugar acceptor (HexGlc) and 2 mM sugar donor (UDP-arabinose (chemically or enzymatically synthesized), UDP-xylose (Complex Carbohydrate Research Center, GA, USA), or UDP-galactose, UDP-glucose, UDP- glucuronic acid (Sigma Aldrich, Tokyo, Japan)) in 50 mM potassium phosphate buffer (pH 7.5) at 30 °C for 15 min. The reaction was stopped by adding 50 μL of ice-cold methanol and quickly cooled with liquid nitrogen. The enzymatic reaction mixtures were analyzed using HPLC-MS with a Shimadzu LCMS-2020 system, coupled to a Capcell Pak UG120 C18 reversed-phase column (2.0 mm i.d. × 150 mm, 5 μm) under the following conditions: 5% solvent B (0–1 min), followed by a linear gradient flow up to 20% in 34 min (solvent A: H2O containing 0.05% (v/v) formic acid, B: acetonitrile).
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