摘要
在毕赤酵母中分泌表达重组人白细胞介素-1α(rh IL-1α),优化rh IL-1α的发酵工艺及纯化方法,以获得高表达、高纯度具有生物学活性的rh IL-1α。通过PCR扩增获得h IL-1α基因,构建其真核表达载体p PICZαA/h IL-1α,电转化至毕赤酵母X-33,用PCR和SDS-PAGE方法筛选高效表达rh IL-1α的工程菌株并进行Western blot鉴定,DEAE弱阴离子离子交换层析一步纯化表达产物,并用MTT法初步检测其对人肝癌细胞7402的生物学作用。rh IL-1α在摇瓶规模下,经甲醇诱导4 d后表达量约为30 mg/L。Western blot检测rh IL-1α的特异性结合,获得纯度约95%,收率40%左右的rh IL-1α,并证明rh IL-1α能够抑制人肝癌细胞7402的增殖。构建了重组h IL-1α的基因工程菌,并在毕赤酵母中实现了高效表达,为进一步研究其生物学活性和功能奠定了基础。
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.
出处
《生物技术通报》
CAS
CSCD
北大核心
2015年第9期244-250,共7页
Biotechnology Bulletin
基金
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(111100910600)
