摘要
目的:构建人白介素18(hIL-18)真核表达载体,在毕赤酵母中高效分泌表达hIL-18。方法:通过PCR法扩增得到hIL-18基因,构建其真核表达载体pPICZaC/hIL-18,电转化毕氏酵母X-33,PCR、SDS-PAGE、Western blot等方法筛选获得高效表达rhIL-18的工程菌株,疏水层析和阴离子交换层析纯化表达产物,并初步测定其生物学活性。结果:rhIL-18经甲醇诱导后其表达产量约为202mg/L。Western blot检测了rhIL-18的特异性结合活性;rhIl-18纯化后纯度达95%左右,并证明rhIL-18能够协同IL-2诱导PBMC分泌IFN-γ。结论:构建了重组hIL-18的基因工程菌,并在毕赤酵母中实现了高效分泌表达,为进一步研究其活性和功能奠定了基础。
AIM： To construct eukaryotic expression vector and express human interleukin 18 （hIL-18） in Pichia pastoris. METHODS： The gene encoding of hIL-18 was amplification by PCR. The recombinant pPICZaC/hIL-18 was transformed into the Pichia pastoris X-33 strain via electroporation. The high level expression was selected and assayed by the methods of PCR, SDS-PAGE and Western blot. The rhIL-18 was purified by the methods of hydrophobic chromatography and anion exchange chromatography. The bioactivity of it was initially assayed. RESULTS： The rhIL-18 was secreted into the supernatant and the concentration reached to 202 mg/L. The rhIL-18 was further identified by Western blot with specific antibody binding activity. The purity of the rhIl-18 reached about 95%. And rhIL-18 can synergistically induce PBMC to produce IFN-γ with IL-2. CONCLUSION： A rhIL-18 is successfully constructed and expressed in Pichia pastoris. And this contributes to further study of its function and activity.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2008年第11期1040-1043,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
天津市科技创新专项资金项目(06FZZDSF1500)
关键词
人白细胞介素18
毕赤酵母
表达
纯化
human interleukin 18（hIL-18）
Pichia pastoris
expression
purification
