Primer Design and Amplification of IgM Antibody Repertoire of Philippine Carabao (Bubalus kerabau Fitzinger, 1860) for Construction of Phage Display Library

Antibody display library is a robust platform for the production of recombinant or monoclonal antibodies that are of great value in diagnostics and therapeutics. A naïve library constructed based on immunoglobulin M (IgM) can reflect the natural immunity of the animal and can offer broad antigen targets. As a natural host of various zoonotic pathogens, the Philippine carabao (Bubalus kerabau Fitzinger, 1860) offers great potential as a surrogate model for molecular biology and biomedical applications. However, studies on its antibody repertoire are very scarce, if not available. Hence, this study was undertaken to design and evaluate degenerate primers targeting the antigen-binding paratope of B. kerabau Fitzinger, 1860 IgM – specifically, lambda variable light chain (VLL), kappa variable light chain (VLK), and variable heavy chain (VH). These primers were mainly based on carabao’s close relative in the Bovidae family, the cattle (Bos taurus). A total of 32 degenerate primers, 14 designed and 18 adapted from literature, were used to amplify the target antibody fragments using a high-fidelity DNA polymerase with a modified touchdown–polymerase chain reaction method. Results demonstrate the amplification of VLL, VLK, and VH genes derived from B-cells of carabao splenic tissues using the degenerate primers. The amplicons will be used in the construction of an antibody phage library to elucidate the IgM repertoire of the Philippine carabao. In addition, DNA sequencing surprisingly revealed longer VLL and VLK genes than VH genes in contrast to what is found in other bovines, suggesting a novel organization of immunoglobulin genes of B. kerabau Fitzinger, 1860 that merits further investigation.