Amplification of a minimally biased antibody repertoire for in vitro display using a universal primer-based amplification method.
J Immunol Methods
; 496: 113089, 2021 09.
Article
em En
| MEDLINE
| ID: mdl-34181966
ABSTRACT
Immune hosts are valuable sources for antibody discovery. To construct in vitro display antibody libraries from immune repertoires, singleplex or multiplex PCR amplification were employed using primers targeting multiple immunoglobulin genes. However, during this process, the B cell receptor repertoire is distorted due to interactions between multiple target genes and primers. To minimize this alternation, we devised a new method for harvesting immunoglobulin genes and tested its performance in rabbit variable heavy chain (VH) and variable kappa light chain (VK) genes. Double-stranded cDNA was synthesized using primers containing V/J gene-specific regions and universal sequence parts for in vitro display. VH and VK gene libraries were obtained through subsequent PCR amplification using primers with universal sequences. Next-generation sequencing analysis confirmed that universal PCR libraries had more diverse VH and VK clonotypes, and a less biased clonal distribution, than conventional singleplex or multiplex gene-specific PCR libraries.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Região Variável de Imunoglobulina
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Receptores de Antígenos de Linfócitos B
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Biblioteca Gênica
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Cadeias Pesadas de Imunoglobulinas
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Cadeias kappa de Imunoglobulina
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Reação em Cadeia da Polimerase Multiplex
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Anticorpos
Limite:
Animals
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
2021
Tipo de documento:
Article