趋化因子RANTES在毕赤酵母中的表达、纯化与鉴定

Expression, purification and identification of RANTES in Pichia Pastoris

背景:RANTES是典型的趋化性细胞因子,参与很多炎症反应以及器官移植免疫排斥反应。从天然组织中分离RANTES数量有限,难以满足对其生物学功能研究和临床应用的需要;运用基因工程方法生产RANTES具有一定的应用价值。目的:观察人体趋化因子RANTES基因在毕赤酵母体系中的分泌表达,获得重组RANTES蛋白。设计、时间及地点:基因水平的实验观察,于2006-07/2007-10在重庆大学生物工程学院313实验室完成。材料:新鲜人外周血取自志愿者,用以分析人体RANTES基因序列设计引物;质粒pPIC9K、毕赤酵母菌珠Pichia Pastoris GS115均由重庆大学生物工程学院313实验室保存。方法:将人体趋化因子RANTES基因插入含AOX1启动子和α分泌信号肽序列的酵母表达载体pPIC9K中,用SalⅠ将重组质粒线性化,电击转化毕赤酵母GS115感受态细胞,筛选阳性整合子进行甲醇诱导表达。主要观察指标:趋化因子RANTES在毕赤酵母中的表达、纯化与鉴定结果。结果:人体趋化因子RANTES在酵母培养基BMGY中实现了分泌表达,表达产物经SDS-聚丙烯酰氨凝胶电泳及Western blot鉴定为重组RANTES蛋白。结论:在毕赤酵母真核系统中实现了人体趋化因子RANTES的分泌表达,为RANTES蛋白进一步的结构和功能研究打下了基础。

BACKGROUND： RANTES is a typical chematropism cytokines and participates many inflammatory reaction and immunological rejection of organ transplantation. The quantity of RANTES isolated from normal tissues is limited, which is difficult to meet the requirement of studying biological function and clinical application. It is valuable to produce RANTES by gene engineering method. OBJECTIVE： To study the secretion expression of human chemokine RANTES gene in Pichia Pastoris and to obtain recombinant human chemokine RANTES. DESIGN, TIME AND SETTING： The gene level experiment was performed at the 313 Laboratory of College of Bioengineering, Chongqing University from July 2006 to October 2007. MATERIALS： Fresh human peripheral blood was collected from volunteers to analyze RANTES gene sequence, pPIC9K and Pichia Pastoris GS 115 were obtained from the 313 Laboratory of College of Bioengineering, Chongqing University. METHODS： The human RANTES gene was cloned into yeast expression vector pPIC9K containing AOXI promoter and the sequences of secreting α -signal peptides. Recombinant plasmid was linearized by Sal I and transformed into Pichia Pastoris GS115 competent cells. Positive integrated clones were screened out, and RANTES was expressed under the induction of methanol. MAIN OUTCOME MEASURES： Expression, purification and identification of chemokine RANTES in Pichia Pastoris. RESULTS： Human chemokine RANTES was secreted and expressed in BMGY through methanol induction. Sodium dodecyl sulfate polyacrylamide gel electropheresis and Western blot results showed that recombination products were RANTES protein. CONCLUSION： Human chemokine RANTES successfully achieves secretion expression in the Pichia Pastoris system first time, which pave a direct path to further research on structure and function of RANTES protein.