Nested polymerase chain reaction amplification and sequencing analysis of the light-chain and heavy-chain variable regions in the influenza A H1N1 virus hemagglutinin monoclonal antibody gene.

Abstract

The nested polymerase chain reaction (PCR) method was used for the amplification of the influenza A H1N1 virus hemagglutinin monoclonal antibody light-chain and heavy-chain genes. Sequence analysis of the obtained genes was then used to identify common cloning methods of the mouse immunoglobulin-kappa (Igκ) light-chain and heavy-chain variable gene regions. Twenty-two pairs of amplification primers for the mouse Igκ light-chain and heavy-chain variable gene regions were designed, and 6 mouse anti-human H1N1 influenza virus hemagglutinin monoclonal antibody light-chain and heavy-chain variable gene regions were cloned and sequenced. Comparative analysis was conducted between our results and the mouse Ig sequences published in the National Center of Biotechnology Information (NCBI). The nested PCR method effectively avoided cloning the pseudogenes of the monoclonal antibody, and the amino acid sequence obtained was consistent with the characteristics of the mouse Ig variable region. A general method of cloning the mouse Ig light-chain and heavy-chain variable gene regions was established, which provides a basis for further cloning of mouse monoclonal antibody variable gene regions. This study also provides data for further studies of H1N1 influenza virus hemagglutinin antibody binding sites.