作　　者： ; ; ; ; ; ; ; ;

机构地区： 军事医学科学院生物工程研究所

出　　处： 《生物技术通讯》 2006年第3期325-327,共3页

摘　　要： 目的:提高外源蛋白可溶性肿瘤坏死因子相关促凋亡配体(sTRAIL)在巴斯德毕赤酵母中的分泌表达。方法:根据GenBank公共数据库中公布的模式生物酿酒酵母的分子伴侣(Ssa1p、YDJ1、Kar2p和PDI)基因序列设计引物,利用PCR方法从酿酒酵母基因组中得到各基因片段,并将单独Ssa1p或Kar2p、组合YDJ1+PDI、Kar2p+PDI或YDJ1+PDI+PDI分别构建到pPIB2Z表达载体中,并整合到外源蛋白sTRAIL工程菌(毕赤酵母GS115)中进行筛选和诱导表达。结果:SDS-PAGE分析表明,sTRAIL的表达量明显提高,特别是整合了分子伴侣组合YDJ1+PDI的工程菌。Western印迹分析整合的分子伴侣基因后,分子伴侣蛋白在工程菌中的表达量得到了提高。结论:提高细胞内分子伴侣的表达,可以增加外源蛋白的分泌表达,为进一步研究巴斯德毕赤酵母奠定了基础。 Objective： To increase the secretory expression of heterologous protein sTRAIL（soluble tumor necrosis factor related apoptosis inducing ligand） in Pichia pastoris. Methods： The DNA sequences encoding the different Saccharomyces cerevisiae chaperone proteins were synthesized from the genome of S.cerevisiae by polymerase chain reaction. The genes of chaperone proteins Ssalp, Kar2p, and pairs recombinations YDJI＋PDI, Kar2p＋PDI, YDJI＋PDI＋PDI were separately constructed into expression vector pPIB2Z and then were integrated in sTRAIL expression strain （P.pastoris GSll5）. Results： SDS-PAGE analysis shows that the expression level of sTRAIL was improved through integrated genes of chaperones, especially chaperones pair of YDJI＋PDI. Western-blot observes that integrated chaperone genes result in over-expression of chaperone protein in vivo. Condusion： To increase the level of the chaperones in vivo in P.pastoris may improve the secretory expression of heterologous protein. So this established a solid basis for research of P.pastoris.

关 键 词： 分子伴侣 毕赤酵母 可溶性肿瘤坏死因子相关促凋亡配体

领　　域： [生物学] [生物学]