Protocol for production and expression of chimeric bovine-human monoclonal antibodies

Abstract

We describe herein a protocol for production of chimeric bovine-human monoclonal antibodies (mAbs) from vaccinated cows. The genes of HIV-1-specific single B cells are amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned into human expression vectors, and expressed in human cell lines. This protocol provides an efficient step-by-step methodology to produce HIV-1 chimeric mAbs and could be widely adapted for other antigens. For complete details on the use and execution of this protocol, please refer to Heydarchi et al. (2022).¹.

Keywords: Antibody; Health Sciences; Immunology; Microbiology; Molecular Biology; Single Cell.

Graphical abstract

Figure 1

FACS sorting gating strategy of HIV-1 specific bovine B cells Cow PBMCs are sorted for live IgG+ cells that bind to biotinylated AD8 SOSIP-AviTag conjugated to PE and APC fluorophores.

Figure 2

Strategy to construct chimeric bovin-human mAb Bovine heavy and light mAb variable genes are amplified in nested RT-PCR (including PCR1 and PCR2). Then, the genes are cloned into the expression vectors containing human heavy and light antibody constant genes.

Figure 3

Gel electrophoresis of PCR products PCR products of bovine heavy and light variable genes (PCR2) on 2% agarose gel with the size standard markers of defined base pair length (bp) indicated on the right.

Figure 4

Linearization of expression vectors pFUSE2ss-CLIg-hL2 (light) and pFUSEssCHIg-hG1 (heavy) expression vectors containing human antibody constant genes are digested and linearized with EcoRI-HF/AvrII and EcoRI-HF/NheI-HF, respectively. The linearized vectors are dephosphorylated and run on 1% gel to cut out a band of 3,837 bp and 4,481 bp for pFUSE2ss-CLIg-hL2 and pFUSEssCHIg-hG1, respectively.

Figure 5

Heavy and light mAb chains on 12% SDS-PAGE reducing gel with apparent molecular weight marker protein positions annotated in Dalton units on the right