Yang Li-li, Wei Feng, Liu Hong, Li Hui, Yu Jin-pu, Ren Xiu-bao
Department of Immunology, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Nov;24(11):1040-3.
To construct eukaryotic expression vector and express human interleukin 18 (hIL-18) in Pichia pastoris.
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.
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-gamma with IL-2.
A rhIL-18 is successfully constructed and expressed in Pichia pastoris. And this contributes to further study of its function and activity.
构建真核表达载体并在毕赤酵母中表达人白细胞介素18（hIL-18）。
通过PCR扩增hIL-18编码基因。将重组pPICZaC/hIL-18经电穿孔法转化至毕赤酵母X-33菌株。采用PCR、SDS-PAGE和Western印迹法筛选并检测其高水平表达。通过疏水层析和阴离子交换层析法纯化重组人白细胞介素18（rhIL-18）。初步检测其生物活性。
rhIL-18分泌至上清液中，浓度达202 mg/L。通过具有特异性抗体结合活性的Western印迹法进一步鉴定rhIL-18。rhIL-18纯度达约95%。并且rhIL-18可与IL-2协同诱导外周血单个核细胞（PBMC）产生γ干扰素。
成功在毕赤酵母中构建并表达了rhIL-18。这有助于对其功能和活性进行进一步研究。
