猪IL-18在毕赤酵母中的表达及活性分析

Expression and Analysis of Recombinant pIL-18 in Pichia pastoris

摘要:

采用PCR方法从pGEM-IL-18重组质粒中扩增出IL-18基因并构建真核融合表达载体pPIC9K-IL-18，电激法转化入毕赤酵母GS115，采用G418抗性梯度法筛选得到多拷贝重组菌株，甲醇诱导表达，应用SDS-PAGE分析重组蛋白的表达情况，并将表达蛋白用凝胶层析柱纯化后，用MTT法检测其生物学活性。实验结果表明重组的GS115酵母菌株可表达分泌pIL-18，其表达在72h时达高峰，分泌量可达160mg/L，纯化的重组pIL-18蛋白具有显著的促进淋巴细胞增殖的活性，说明本试验已在毕赤酵母中在国内首次成功表达了具有生物学活性的pIL-18。

Abstract:

The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR，then the pPIC9K-IL-18 of fusion expression vector was constructed by inserting IL-18 fragment，and was transformed to GS115 by electroporation，multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol，SDS-PAGE was used to analyze expression product，fusion protein was purified by Sephadex G-100 column，bioactivity of IL-18 was measured by MTT assays. Experiment results show fusion protein of pIL-18 secreted by GS115，expression reaches the secretion peak of 160mg/L at 72h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in Pichia pastoris.