可直接克隆PCR产物的毕赤酵母分泌型表达载体

A Secretive Pichia pastoris Expression Vector for Direct PCR Product Cloning

从毕赤酵母表达载体pPICZαA出发,构建了可直接克隆PCR产物的毕赤酵母分泌型表达载体(毕赤酵母表达型T载体),并通过表达重组纤维二糖水解酶II对该载体进行了检验。设计合适的引物扩增一DNA片段,使该片段的上游含XhoI和Eam1105I酶切位点,下游含Eam1105Ⅰ和XbaI酶切位点。通过XhoI和XbaI位点将扩增产物与质粒pPICZαA连接形成重组质粒。用Eam1105I酶切重组质粒,回收大片段即得到毕赤酵母表达型T载体pPICZαT。使用该表达型T载体进行了里氏木霉纤维二糖水解酶Ⅱ基因(cbh2)的克隆和在巴氏毕赤酵母中的表达。结果表明,使用表达型T载体可以直接克隆PCR产物,而且可以使外源基因在毕赤酵母中成功表达。另一方面,使用该载体时不需要使用限制性内切酶,从而可以避免在所表达蛋白的N-末端引入多余氨基酸。

A secretive expression vector of Pichia pastoris system which can be used for the direct cloning of PCR products was constructed, and was verified through the expression of recombinant cellobiohydrolase Ⅱ in Pichia pastoris. A randomly selected fragment was amplified with properly designed primers by PCR. The XhoⅠ and Eam1105Ⅰ restriction sites were included in the 5＇ end of the fragment, and the Eam1105 Ⅰand XbaⅠ restriction sites were included in its 3＇ end. The PCR amplified product was inserted into the P. pastoris expression plasmid pPICZaA through XhoⅠ and XbaⅠrestriction sites and the resultant plasmid was digested with Eam1105Ⅰ , and lastly the big fragment was recovered, generating the P. pastoris expressive T-vector pPICZαT. Then the cellobiohydrolase Ⅱ of T. reesei was successfully expressed in P. pastoris with this expressive T-vector. Such results indicated that the constructed expression T-vector was convenient for PCR product cloning, and was effective for heterologous protein expression in P. pastoris. On the other hand, the application of the expression T -vector avoided the introduction of additional amino acids at the N-terminus of the expressed protein, which generally occurred when normal expression vectors were used in secretive expression system.