生物技术通报Issue(9)：244-250,7.DOI:10.13560/j.cnki.biotech.bull.1985.2015.09.035
重组人白细胞介素-1α在毕赤酵母中的表达、纯化及生物学活性检测
Expression,Purification and Bioactivity of Recombinant Human Interleukin-1αExpressed in Pichia pastoris
摘要
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-1αamplified 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-1αwas 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-1αby 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-1αwas detected;the purity of the rhIL-1αreached about 95%and the yield was about 40%;rhIL-1αinhibited the proliferation of Bel-7402 cells. In conclusion, recombinant engineering vector of rhIL-1αwas successfully constructed, and it was highly expressed in P. pastoris, which lays groundwork for further study of its function and bioactivity.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Key words
recombinant human interleukin-1α/Pichia pastoris/expression/purification/Bel-7402 cell引用本文复制引用
李斌,许晓亚,杨刚刚,崔晴晴,杨亚娟,徐存拴..重组人白细胞介素-1α在毕赤酵母中的表达、纯化及生物学活性检测[J].生物技术通报,2015,(9):244-250,7.基金项目
国家“973”前期研究专项（2012CB722304），河南省自然科学基金项目（142300413212，132300413208），河南省重大科技攻关项目 （）
