A truncated p40 subunit of porcine interleukin-12 (pIL-12) gene without the N-terminal signal peptide sequence was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1. The resulting recombinant plasmid pGEX-IL12-40 was transformed into host cells BL21(DE3)pLysS, and the expression of the p40 subunit was induced using isopropyl β-D-thiogalactoside (IPTG). An anti-p40 polyclonal antibody was generated by immunizing a rabbit with the purified protein. Immunoreactivities of the p40 protein and the antibody were confirmed by immunoblotting. At the same time, a recombinant plasmid expressing the entire pIL-12 consisting of p35 and p40 genes was constructed by splicing by overlap extension (SOE)-PCR and transiently transfected into BHK-21 cells. Expression of p40 subunit on the surface of the transfected cells was identified using the anti-p40 antibody. The p40 protein and the specific antibody are biologically active and can be used as detecting reagents.
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