Screening and characterization of monoclonal antibodies with neutralizing activity against porcine transmissible...

Porcine Transmissible Gastroenteritis Virus (TGEV) is widespread and frequently involved in co-infections, leading to high mortality rates in piglets and significantly constraining the swine industry. Based on the genetic sequence of TGEV strain HB-1, we designed and constructed a eukaryotic expression plasmid, pCAGGS-TGEV-CTD, encoding the CTD gene of TGEV. The recombinant CTD protein was successfully expressed and purified using the HEK-293 F suspension cell expression system. Balb/c mice were immunized with the purified CTD protein.

Upon achieving a serum antibody titer of 10⁵, antigen-specific memory B cells were isolated using single-cell flow cytometry sorting. A total of 83 matched antibody light- and heavy-chain gene pairs were obtained via single-cell RT-PCR. These gene pairs were cloned into pCAGGS eukaryotic expression vectors containing murine constant light (CL) and heavy (CH) chain regions, resulting in the construction of 42 recombinant plasmids for antibody expression. Following transfection into HEK-293T cells, antibody supernatants were harvested at 48 h post-transfection. ELISA screening identified 18 antibodies reactive to the TGEV CTD protein, five high-titer antibodies were confirmed to cross-react with TGEV virions through indirect immunofluorescence assay (IFA). Further neutralization assays demonstrated that all five monoclonal antibodies exhibited in vitro neutralizing activity, effectively inhibiting TGEV infection and replication in swine testicular (ST) cells.

This study successfully generated TGEV-specific antibodies, providing critical support for fundamental research on TGEV research and the development of clinical diagnostic reagents.