中国生物工程杂志

Abstract　 Human single chain interleukin-12（hscIL-12）of bioactivity was expressed in methylotrophic yeast Pichia Pastoris expression system , hscIL-12 gene was amplified from plasmid pBI121-IL-12 by PCR. After digested by restricted enzyme, gene of interest was cloned into the yeast vector pPIC9K and obtained recombinant expression vector pPIC9K-hscIL-12. The pPIC9K-hscIL-12 was linearized with SacⅠ and then transformed into the Pichia Pastoris GS115 by PEG1000. Recombinant strains were screened by G418 resistant, and further confirmed by colony PCR. The hscIL-12 was induced to express in yeast by methano1. Western Blot analysis showed that the relative molecular weight of the expressed product was about 70kDa, the expression protein could bind to IL-12 monoclonal antibody specifically. Quantitative analysis showed that the target protein was in a level of 26% of the total protein of the culture supernatant，with a yield of 40mg/L. Bioactivity assay showed that the recombinant hscIL-12 could stimulate the lymphocyte proliferation. The successful expression of the rhscIL-12 in Pichia Pastoris can be potentially used in cancer gene therapy.