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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。
Objective: To construct eukaryotic expression vector of human interleukin 18 (hIL-18) and express hIL-18 efficiently in Pichia pastoris. Methods: The hIL-18 gene was amplified by PCR and its eukaryotic expression vector pPICZaC / hIL-18 was constructed. The recombinant plasmid was transformed into Pichia pastoris X-33 by PCR, SDS-PAGE and Western blot. 18 engineering strains, hydrophobic chromatography and anion exchange chromatography purification of the expression product, and preliminary determination of its biological activity. Results: The expression of rhIL-18 after induction with methanol was about 202 mg / L. The specific binding activity of rhIL-18 was detected by Western blot. The purity of rhIL-18 after purification was about 95%. It was also demonstrated that rhIL-18 could induce IL-2-induced PBMCs to secrete IFN-γ. CONCLUSION: Recombinant hIL-18 genetically engineered bacteria were constructed and highly secreted and expressed in Pichia pastoris, which laid the foundation for further study of its activity and function.