Generation and Characterization of Polyclonal Antibodies Against Mouse T-cell Immunoglobulin and Immunoreceptor Tyrosine-based Inhibitory Domain by DNA-based Immunization

Abstract

Mouse T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory domain (TIGIT) is a newly identified surface protein expressed in regulatory, memory, natural killer (NK), and activated T cells. Several studies indicate that mouse TIGIT is a vital immunomodulator that can control the activities of both NK and T cells and plays an important role in transplantation tolerance. In this study, we designed a vector, TIGIT-pcDNA3.1 (+), that encodes the complete coding sequence of mouse TIGIT. The vector was intramuscularly injected into rats, and then the specific antisera were harvested and purified using a protein A/G PLUS-agarose affinity column. Western blot and immunohistochemistry analyses revealed that the antibodies generated by DNA immunization can bind with the mouse TIGIT. Using these antibodies in immunoblots, TIGIT was detected in lysates of mouse organs, T cells from mouse lymph nodes, and recombinant mouse fusion protein of TIGIT and Fc fragment. Immunohistochemistry analysis of normal mouse kidney showed that immunoreactivity was located on endothelial cells of glomerular capillary loops and peritubular capillaries. Our results demonstrated that the DNA immunization of rats through intramuscular injection was a simple and easily available method of producing polyclonal antibodies that can be used to detect and analyze mouse TIGIT expression in mouse systems.