Computational protocol: Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

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Protocol publication

[…] The variable regions of heavy chain (VH) and light chain (VL) of mAb 26A1 were sequenced according the technology established by Fusion Antibodies Ltd. Briefly, frozen cells pellets (~5 × 104 cells) were used for extracting total RNA with STAT-60 RNA extraction reagent. cDNA was produced by reverse transcription with an oligo(d)T primer. PCR reactions were set up to amplify VH and VL regions with a mix of IgG- and Igk/λ specific primers, respectively. The PCR products of two amplification reactions were cloned using a Eco RI restriction site in a sequencing vector (pCR2.1; Invitrogen) and used for transforming TOP10 E. coli cells, following the manufacturer's instructions. A consensus sequence was determined from at least ten clones. The resulting DNA sequences were aligned and translated into protein sequences enabling the generation of consensus DNA and protein sequence for VH 26A1 and VL 26A1. The VH 26A1 and VL 26A1 protein sequences were compared and aligned with sequences present in the GenomeQuest, GeneSeq, and EBI databases. The CDRs characterizing VH 26A1 and VL 26A1 protein sequences were predicted by the IMGT database [].To generate a recombinant IgG1, a consensus 26A1 VH sequence was amplified by PCR with the following primers: 5'-TTTTTTAGATCTCACCATGAACATACTGTGGAGCATGCTC-3'; 5'-TTTTTTAGATCTTGAGGAGACGGTGACCAGGGTTCC-3'. The primers contained a BglII restriction site (underlined) for ligation into the hIgG expression vector already containing human IgG1 heavy chain constant domains. The in-house designed bicistronic mammalian retroviral expression vector pRetMEx (Fusion Antibodies Ltd. Belfast, Ireland) was a dual promoter vector allowing expression of both antibody chains. The VH domain was cloned into the BamH1 site of multiple cloning site (MCS) 1 of the vector.A complete 26A1 recombinant vector was confirmed by DNA sequencing. CHO DG44 cells were adapted to serum-free suspension culture and seeded at 1 × 106 cells/ml in 125 ml spinner flasks. 300 μg of plasmid DNA was mixed with 900 μg of linear 25 kDa PEI and used to transfect the cell culture. The medium was harvested after 6 days.Recombinant mAb was purified with a Protein G column on an Akta Prime chromatography unit following the manufacturer's standard programme (recombinant IgG). […]

Pipeline specifications

Software tools GenomeQuest, GENESEQ
Databases IMGT
Application Information extraction
Organisms Human betaherpesvirus 5, Homo sapiens
Diseases Communicable Diseases, Cytomegalovirus Infections