摘要
利用甲醇毕赤氏酵母系统对改造的TRAIL基因进行优化表达研究及产物活性分析，确定改造后的基因表达量是否提高。应用电转化技术将4种重组表达载体转化毕赤氏酵母菌GS115，经Zeocin抗生素筛选、PCR检测获得阳性酵母工程菌，采用摇瓶发酵及SDS-PAGE进行高效表达菌株的筛选，经CM-柱层析、Western blot及MTT法对重组蛋白进行初步纯化、抗原活性检测和生物学活性分析，确定了最佳摇瓶发酵条件：培养基最适pH值5．5～6．5，最佳甲醇诱导浓度为1％，最适甲醇诱导周期为96h。筛选到高效表达菌株pPICZα-A—sTRAIL／GS115、pPICZα-A—sTRAILK／GS115、pPICZα-A—sTRAILA／GS115、pPICZα-A—sTRAILAK／GS115，其表达量分别达到67，113．4，98，145mg／L，改造后的TRAIL具有抗原活性和生物学活性。对TRAIL基因的改造可以提高其在甲醇毕赤氏酵母系统中的表达。
Four kinds of recombinant P. pastoris were used to express modified sTRAIL gene and to analysis the activity of the recombinant sTRAIL to confirm whether the modified gene has a higher expression yield than the wild gene. The recombinant expression vectors were transformed to strain P. pastoris GS115 by electrical transformation. After screening with Zeocin and PCR, positive recombinant strains were obtained. The optimized fermentation condition and the highly expressed strains were confirmed by SDS-PAGE analysis. Chromatography, western blot and MTT method were used for further study of this protein. It was confirmed that the optimal pH value was from 5.5-6.5, the optimal methanol inducing concentration was 1.0% and the optimal inducing cycles were 96 hours. Four kinds of highly expressed recombinant strains were also screened as follows, pPICZα-A-sTRAIL/GS115, pPICZα-A-sTRAILK/GS115, pPICZα-A-sTRAILA/GS115 and pPICZα-A-sTRAILAK/GS115, and their expression yields were 67mg/L, 113.4mg/L, 98mg/L, 145mg/L , respectively. It was also confirmed that products of modified sTRAIL gene had antigen activity and biological activity as wild gene. It was feasible to increase expression yield of exogenous protein by modifying genes.
出处
《药物生物技术》
CAS
CSCD
2007年第1期5-9,共5页
Pharmaceutical Biotechnology
基金
海南省教育厅科研基金项目(No.Hjkj200507)
