Cloning bovine immunoglobulin VH and VL genes from single B cells

Abstract
A distinct feature of bovine antibodies that has not been observed in any other species, ultralong CDR H3 regions, has the capacity to access epitopes on structurally complex antigens. Bovine immunoglobulins with ultralong CDR H3s have great potential for development as clinical treatments and research tools targeting a broad variety of antigens. However, unlike other species, techniques for studying bovine immunoglobulin genes at the single cell level have not been comprehensively developed. We established a new method for amplification of bovine immunoglobulin VH and VL genes at the single cell level utilizing flow cytometry to sort individual B cells into wells and cDNA production and PCR amplification followed by nested PCR. Optimal primers were designed and ratios of each VH and VL primer, along with other conditions, were optimized for production and amplification of cDNA. The amount of template added to nested PCR reactions and the number of subsequent PCR reactions necessary to procure enough DNA for cloning were also optimized. Results from cells sorted by two types of FACS machines were also compared. Both VH and VL DNA can be obtained from >50% of sorted B cells using this method. This technique enables the production of monoclonal antibodies with heavy and light chain pairs expressed from individual bovine B cells and will be useful for rapid identification of recombinant bovine antibodies.

PST – 001