Prokaryotic expression, polyclonal antibody production, and application of yak TGF-β2

Chen, YamingPan, YangyangYu, SijiuWang, JingleiFan, Jiangfeng

Abstract This study aimed to generate yak-specific polyclonal antibodies against transforming growth factor beta 2 (TGF-β2). Specific primers targeting the TGF-β2 coding sequence (CDS) were designed, and the gene was amplified via RT-PCR. The amplified product was cloned into the pET-32a(+) vector to construct the recombinant plasmid pET-32a(+)-TGF-β2. This plasmid was transformed into Escherichia coli BL21(DE3) for protein expression. Isopropyl β-D-1-thiogalactopyranoside (IPTG) induced TGF-β2 production, and the recombinant protein was purified. New Zealand rabbits were immunized with the purified protein to generate polyclonal antibodies. Polyclonal antibody titers were determined using ELISA, while specificity was assessed through Western blot and immunohistochemistry. The recombinant plasmid was successfully constructed, and IPTG induction yielded a 63 kDa protein. Optimal expression occurred at 25 °C with 0.5 mmol·L−1 IPTG and a 10-hour induction period. ELISA confirmed an antibody titer of 1:106. Western blot and immunohistochemistry demonstrated TGF-β2 expression in female yak ovaries, oviducts, and uteri across reproductive stages, with significantly elevated ovarian levels during pregnancy. This study successfully produced and validated a highly specific anti-yak TGF-β2 polyclonal antibody, providing a vital tool for investigating its role in yak reproductive physiology.