钝顶螺旋藻sod基因真核表达载体的构建与表达  

Construction of Eukaryotic Expression Vector of sod Gene from Spirulina platensis and Its Expression in Pichia pastoris

作　　者：姜晓杰[1] 王文杰[1] 高金亮[1] 李炜[1] 祁美荣[1] 

机构地区：[1]鄂尔多斯市疾病预防控制中心,内蒙古鄂尔多斯017000

出　　处：《生物技术》2014年第5期17-21,共5页Biotechnology

基　　金：内蒙古自然科学基金项目("钝顶螺旋藻重组超氧化物歧化酶生产工艺的建立";No.2013MS0521);内蒙古自治区应用技术与研究开发资金计划("重组钝顶螺旋藻超氧化物歧化酶大规模生产优化";2013年度)资助~~

摘　　要：目的:在毕赤酵母中表达钝顶螺旋藻超氧化物歧化酶SOD。方法:以质粒p ET30-sod为模板,采用PCR扩增sod基因,并将其与表达载体p PIC9K相连,构建重组表达质粒p PIC9K-sod。将重组质粒p PIC9K-sod用限制性内切酶PmeⅠ线性化,并电转化入毕赤酵母GS115。利用单菌落PCR筛选整合有重组质粒的阳性转化子,用甲醇进行诱导表达,并在5L发酵罐内进行发酵表达目的蛋白。用改进的邻苯三酚自氧化法测定重组SOD的活性。结果:成功构建了钝顶螺旋藻的sod的真核表达载体,并在毕赤酵母中表达了分子量为22k Da的重组蛋白SOD。发酵罐高密度发酵所获目的蛋白的平均浓度为0.36±0.4 mg/m L,比活性为409±17U/mg。结论:在毕赤酵母中表达了分子量为22k Da的钝顶螺旋藻SOD。Objective： To express the superoxide dismutase（ sod） gene of Spirulina platensis in Pichia pastoris. Method： The sod gene of S. platensis was amplified from p ET30- sod by PCR and cloned into expression vector p PIC9 K. The constructed recombinant plasmid p PIC9K- sod was digested by PmeⅠ and further transformed into P. pastoris GS115 by electroporation. Positive Pichia strains with genomes integrated by the linearized recombinant plasmid were screened by PCR. Recombinant SOD was expressed under induction of methanol in an orbital shaker and further in a 5L- fermentor. Antioxidant activity of the expressed SOD was determined by pyrogallol autoxidation. Result： Recombinant plasmid p PIC9K- sod was correctly constructed and a recombinant protein with a relative molecular mass of about 220 000 was successfully expressed in P. pastoris. Average concentration of the recombinant SOD produced in the fermentor was 0. 36± 0. 4 mg·m L- 1,with a specific activity of 409 ± 17U·mg- 1in pyrogallol autoxidation assay. Conclusion： Recombinant SOD with a relative molecular mass of about 220 000 of S. platensis was successfully expressed in P. pastoris.

关 键 词：钝顶螺旋藻 超氧化物歧化酶(SOD) 毕赤酵母 发酵 比活性 

分 类 号：Q554.6[生物学—生物化学]