关键词: 重组人白细胞介素-1α, 毕赤酵母, 表达, 纯化, 人肝癌细胞

Abstract: This work is to secret and express human interleukin-1α(rhIL-1α)in Pichia pastoris and optimize the fermentation and purification process of rhIL-1α for obtaining the rhIL-1α with high-purity, high-expression and owing biological activity. The gene hIL-1α amplified by PCR was constructed into the eukaryotic expression vector pPICZαA/hIL-1α, and then it was transformed into the P. pastoris X-33 strain via electroporation．The engineering strain with high-expression of rhIL-1α was screened and assayed by the methods of PCR and SDS-PAGE, further indentified by Western blot. The expressed product was purified by the DEAE Sepharose Fast Flow ion exchange chromatography, and the bioactivity of it to human cancer Bel-7402 cell was initially assayed. Results showed that inducing rhIL-1α by methanol for 4 d at shaking flask level, the expression reached 30mg/L, the test by Western blot revealed that specific binding activity of rhIL-1α was detected；the purity of the rhIL-1α reached about 95％ and the yield was about 40%；rhIL-1α inhibited the proliferation of Bel-7402 cells. In conclusion, recombinant engineering vector of rhIL-1α was successfully constructed, and it was highly expressed in P. pastoris, which lays groundwork for further study of its function and bioactivity.

Key words: recombinant human interleukin-1α, Pichia pastoris, expression, purification, Bel-7402 cell