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Order Blubervirales: Surface Protein
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Recently, Kingston et al. (2019) proposed an interesting alternative to the RTS,S vaccine by retaining the specific malaria antigen but changing the site of the epitope insertion within the HBs molecule and expression platform. Thus, the authors inserted the four or nine NANP repeats into the SHBs immunodominant region between aa residues 127 and 128 and achieved large-scale production of the chimeric nonmosaic HBs-malaria VLPs in human embryonic kidney cells but not in yeast. An N-terminal myc-tag sequence was included into the chimeric molecules to allow detection of the VLPs independent of the SHBs backbone. As a result, the VLPs with NANP9 repeats induced antibodies with the ability to activate complement, which could contribute to the inactivation of invading sporozoites. It is highly important that the expression of the chimeric VLPs in mammalian cell lines allowed additional manipulations of the HBs backbone to further enhance immunogenicity, as it was established earlier by the Hans J. Netter’ team (Cheong et al. 2012; Hyakumura et al. 2015).
Molecular Farming Antibodies in Plants: From Antibody Engineering to Antibody Production
Published in Maurizio Zanetti, J. Donald Capra, The Antibodies, 2002
Rainer Fischer, Ricarda Finnern, Olga Artsaenko, Stefan Schillberg
fragments are monovalent [20], which may be essential when selecting antibodies of higher affinity. Both scFv and Fab phage-display libraries have been shown to perform well. Purification and detection of scFv is normally achieved by adding short tag sequences (e.g., myc-tag, His-tag) to either the N-terminus or the C-terminus [18, 46].
Reciprocal SH2-SH3 Domain Contacts between ITK Molecules Limit T Cell Receptor Signaling in Th2-type CD4+ T Cells
Published in Immunological Investigations, 2022
Ji-Long Chen, Jennifer Y. Barr, Jonathan J. Zuk, Jacob V. Gorman, John D. Colgan
Published NMR data (Brazin et al. 2000; Severin et al. 2009) suggest amino acids in the CD loop of the ITK SH2 domain and the ITK SH3 domain mediate contacts that promote binding between ITK molecules (Figure 1a). A co-immunoprecipitation (co-IP) assay was developed to determine the importance of specific amino acids in the ITK SH2 and SH3 domains for intermolecular interaction (Figure 1b). Expression plasmids were constructed that encode wild-type (WT) or mutant forms of mouse ITK fused at the C-terminus to either the Myc or FLAG epitope tag. Plasmids encoding a Myc and a FLAG-tagged version of ITK were mixed and transiently co-transfected into human embryonic kidney 293 T (293 T) cells so that the 2 epitope-tagged forms of ITK were co-expressed. Immunoblot analysis of whole-cell lysates prepared from transfected cells was performed to confirm co-expression of ITK-Myc and ITK-FLAG proteins. Cell lysates were also used to carry out immunoprecipitations (IPs) with anti-Myc tag antibodies. Immunoblot analysis of recovered material was performed to show ITK-Myc was pulled down and to detect ITK-FLAG protein that was co-IP’d due to intermolecular interaction with ITK-Myc. That co-IP of ITK-FLAG depended on interaction with ITK-Myc was confirmed by control IPs, which showed that ITK-FLAG was not pulled by anti-Myc tag antibodies when ITK-Myc was absent (see Figure 2b for example).
cGAS-STING effectively restricts murine norovirus infection but antagonizes the antiviral action of N-terminus of RIG-I in mouse macrophages
Published in Gut Microbes, 2021
Peifa Yu, Zhijiang Miao, Yang Li, Ruchi Bansal, Maikel P. Peppelenbosch, Qiuwei Pan
DMXAA, H151 and 2ʹ3ʹ-cGAMP were purchased from InvivoGen. JAK inhibitor 1 (SC-204021) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). N-Acetyl-L-cysteine was purchased from Sigma-Aldrich. Rabbit polyclonal antisera to MNV NS1/2 was kindly provided by Prof. Vernon K. Ward (School of Biomedical Sciences, University of Otago, New Zealand).47 Rabbit polyclonal antisera to MNV NS7 was kindly provided by Prof. Ian Goodfellow (Department of Pathology, University of Cambridge, UK).48 Antibodies against STAT1 (#9172), pSTAT1 (Ser727, #9177), RIG-I (D14G6, #3743), MDA5 (D74E4, #5321), cGAS (D3O8O, #31659), STING (D2P2F, #13647), Myc-tag (71D10, #2278) and Myc-Tag (9B11, #2276) were purchased from Cell Signaling Technology. Rabbit anti-GBP2 (11854-1-AP) and anti-GBP5 (13220-1-AP) antibodies were purchased from Proteintech. Mouse anti-Flag (F1804, Sigma-Aldrich) and anti-β-actin (#sc-47778, Santa Cruz Biotechnology) antibodies were used. Anti-rabbit and anti-mouse IRDye-conjugated secondary antibodies (Li-Cor Bioscience, Lincoln, USA) were used.
A bispecific nanobody approach to leverage the potent and widely applicable tumor cytolytic capacity of Vγ9Vδ2-T cells
Published in OncoImmunology, 2018
Renée C. G. de Bruin, John P. Veluchamy, Sinéad M. Lougheed, Famke L. Schneiders, Silvia Lopez-Lastra, Roeland Lameris, Anita G. Stam, Zsolt Sebestyen, Jürgen Kuball, Carla F. M. Molthoff, Erik Hooijberg, Rob C. Roovers, James P. Di Santo, Paul M. P. van Bergen en Henegouwen, Henk M. W. Verheul, Tanja D. de Gruijl, Hans J. van der Vliet
FITC-labeled anti-TCR Vδ2 (catalog #555738), FITC-labeled anti-IFN-γ (catalog #554700), FITC-labeled anti-CD69 (catalog #347823), PE-labeled anti-CD107a (catalog #555801), PE-labeled anti-CD25 (catalog #55542), PE-labeled pan γδ-TCR (catalog #333141), APC-labeled anti-CD25 (catalog #340907), and 7-AAD (catalog #559925) were obtained from BD Biosciences. PerCP-labeled anti-TCR Vδ2 (catalog #331410), PE-labeled anti-TCR Vγ9 (catalog #331308) and APC-labeled anti-TCR Vγ9 (catalog #331310) were from Biolegend. RPE-labeled goat-anti-mouse F(ab’)2 fragment (catalog #R0480) was obtained from Dako. Anti-Myc tag mAb clone 4A6 (catalog #05-724) was obtained from Merck Millipore and anti-Myc tag mAb clone 9E10 was produced in-house. Alexa488-labeled cetuximab was a kind gift of Rens Braster and Yvette van Kooyk (VUmc, Amsterdam, NL). All stainings for flow cytometry were performed in PBS supplemented with 0.1% BSA and 0.02% sodium-azide. Intracellular IFN-γ production was determined by adding GolgiPlug to the cell culture for the final 4 hrs of the experiment. Cells were fixed and permeabilized with the Fixation/Permeabilization Solution Kit from BD Biosciences(catalog #555028) and stained with anti-IFN-γ mAb.