猪生长激素基因在巴斯德毕赤酵母中的高效分泌表达及产物的N-糖基化分析  

High Level Secretion Expression of Porcine Somatoropin Gene in Pichia pastoris and N-Glycosylation Analysis of Products

作　　者：欧阳菁[1] 杨林[1] 龙綮新[1] 王珣章[1] 邢珂[1] 魏平华[2] 

机构地区：[1]中山大学生物防治国家重点实验室,生物医药中心,广州510275 [2]广东省农业科学院,广州510650

出　　处：《生物工程学报》2001年第5期520-525,共6页Chinese Journal of Biotechnology

基　　金：广东省自然科学基金资助 (96 30 0 7)&&

摘　　要：利用含有强启动子PAOX 1 和α MF信号肽序列的巴斯德毕赤酵母载体质粒pPICZαA构建出含PST基因的重组质粒pPICZαA pST。通过电击将经SacⅠ酶切后线性化的pPICZαA pST质粒转化到巴斯德毕赤酵母X 33菌中 ,并筛选Mut+ 表型的重组菌。表达产物的SDS PAGE和Westernblot结果表明 ,分泌于胞外的PST蛋白分子量比天然PST分子量稍大 ,而胞内的PST蛋白分子量与天然PST大小相同。将经SacⅠ酶切后线性化的pPICZαA pST再次转化重组酵母细胞X 33 pPICZαA pST(Mut+ ) ,所得表达产物的SDS PAGE和Westernblot结果显示 ,PST基因的表达水平明显提高 ,且表达产生的蛋白均可发生正确的抗原 抗体结合反应 ,表达量达 95 6mg L。将发酵液上清进行N 糖基化分析 ,显示rPST无NThe porcine somatoropin gene was inserted into the \%Pichia pastoris \%expression vector of pPICZαA which contains AOX Ⅰ promoter and α\|factor signal sequence.The recombinant plasmid of pPICZαA\|pST was linearnized by SacⅠ\ and transformed into X\|33 by electroporation.The multi\|copy insert transformants were selected and cultivated in flasks.SDS\|PAGE and Western blot analysis showed that PST gene products were observed in the supernants with a little larger molecular weihgt size than the natural PST's,however,the molecular weight size of the PST gene products in the soluble cellular proteins were identical to the natural PST's.Retransformation of the linearnized pPICZα\|pST showed the expression level was improved greatly and rPST has the same antigenicity as natural one.The expressed rPST accumulated up to about 956mg/L.The N\|glycosylation analysis showed rPST had no N\|glycosylation.

关 键 词：猪生产激素 巴斯德毕赤酵母 表达载体系统 基因表达 高效分泌表达 N-糖基化分析 

分 类 号：Q786[生物学—分子生物学] TQ467.2[化学工程—制药化工]