猪生产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

利用含有强启动子PAOX1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基因的重复组质粒pPICZαA－pST。通过电击将经SacI酶后线化的pPICZαA－pST质粒转化到巴斯德毕赤酵母X－33菌中，并筛选Mut^ 表型的重型的生组菌。表达产物的SDS－PAGE和Western blot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Western blot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956mg/L。将发酵液上清进行N－糖基化分析，显示rPST无N－糖基化加工修饰。

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

The porcine somatoropin gene was inserted into the Pichia pastoris expression vector of pPICZ alpha A which contains AOX I promoter and alpha-factor signal sequence. The recombinant plasmid of pPICZ alpha A-pST was linearnized by Sac I 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 weight 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 pPICZ alpha-pST showed the expression level was improved greatly and rPST has the same antigenicity as natural one. The expressed rPST accumulated up to about 956 mg/L. The N-glycosylation analysis showed rPST had no N-glycosylation.