Expression of a Novel Gene, Biot2-L and Generation of Its Antibodies

Biot2-L is a novel mouse cancer-testis (CT) antigen gene that was first identified with cross-reactive serological analysis of recombinant cDNA expression libraries technique in our laboratory. To express and purify the CT antigen Bio2-L for further investigation as tumor vaccine, the full-length of Biot2-L gene was cloned and the recombinant expression vector pET28a(+)-Biot2-L was constructed. The full-length gene was confirmed by restriction enzyme digestion and sequence analysis. After optimization, E. coli BL21 (DE3) bearing the recombinant plasmid was induced with 0.8 mM IPTG for 4 h at 37 °C, and the recombinant Biot2-L was produced at the level of about 28% of total bacterial proteins. Target protein was purified by Ni²⁺ affinity chromatography and characterized by western blot with anti-His antibody. The purified recombinant protein was further confirmed by ESI-Q-TOF-MS. After protein purification, Biot2-L was used to immunize adult rabbits and its antibodies were generated. The titer was 6.4×105. This study provided a valuable foundation for further researches on the novel gene, Biot2-L.