摘要
目的:探索人IL-18成熟蛋白(mhIL-18)在毕赤酵母中的高效表达.方法:采用SOE ing及不对称PCR方法扩增出mhIL-18基因并构建融合型表达载体pPIC9-IL-18-Inte in,转化入毕赤酵母GS115,甲醇诱导表达,运用SDS-PAGE和W estern B lot分析重组蛋白的表达,并经亲和层析后用MTT法检测表达的mhIL-18生物活性.结果:成功构建载体并转化入毕赤酵母,经甲醇诱导,重组的GS115可分泌mhIL-18,其表达在96 h时达高峰,分泌量可达100 mg/L.亲和层析后的mhIL-18纯度可达95%,并具有显著的IL-18的生物学活性.结论:在毕赤酵母中成功表达具有显著生物学活性的mhIL-18.
AIM： To achieve high level expression and purification of recombinant mature human IL-18 （mhlL-18） protein in pichia pastoris. METHODS： An Intein Tag was added to the vector. The gene of mhlL-18-Intein was amplified by SOEing and asymmetric PCR. The recombinant expression plasmid pPIC9-IL-18-1ntein was constructed by cloning mhIL-18-1ntein into pPIC9 vector and was transformed to GS115. The expression of recombinant fusion protein was induced by methanol under an optimum inducing condition. After the protein excreted out of the cells was harvested, SDS-PAGE and Western Blot were used to analyze the expression of recombinant fusion protein. The activity of mhIL- 18 purified by affinity chromatography was measured by MTT assays. RESULTS： The cloned sequence of mhlL-18 was identical with the sequence in GenBank. After methanol induction, the fusion protein of mhIL-18 reached the secretion peak of 100 mg/L at 96 h. The purity of the recombinant expressed mhIL-18 was 95% by means of affinity chromatography and the purified mhIL-18 had the biological activity of IL-18. CONCLUSION： We have succeeded in expressing and purifying the recombinant human IL-18 with obvious biological activity in pichia pastoris.
出处
《第四军医大学学报》
北大核心
2005年第22期2057-2061,共5页
Journal of the Fourth Military Medical University
基金
渝教科2001[12]重庆医科大学重点基金(Cx200101)
