可直接克隆PCR产物的毕赤酵母分泌型表达载体

中国生物工程杂志

2007, Vol. 27

Issue (1): 52-58

研究报告

可直接克隆PCR产物的毕赤酵母分泌型表达载体

张超,刘刚,余少文,邢苗

深圳大学

A Secretive Pichia pastoris Expression Vector Capable of Direct PCR Product Cloning

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摘要： 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）。设计合适的引物扩增一DNA片段，使该片断的上游含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-末端引入多余的氨基酸。

关键词： 分泌型表达载体; 巴氏毕赤酵母; T-载体; 里氏木霉; 纤维二糖水解酶

Abstract: It is difficult to obtain N-terminus-authentic recombinant protein in secretive expression system, for cloning of the objective gene through restriction and ligation inevitably introduces additional amino acids between the objective protein and the secretive signal peptide. This work proposed a novel approach to address this problem in the Pichia pastoris secretive expression system through construction and application of an expressive T-vector. A randomly selected fragment was PCR amplified with properly designed primers, such that Xho⒐and Eam1105⒐ restriction sites were included in the 5ˇ end of the amplified product, and Eam1105⒐ and Xba⒐ restriction sites were included in the 3ˇ end. The PCR amplified product was inserted into the P. pastoris expression plasmid pPICZ冄A through Xho⒐ and Xba⒐ restriction sites. The resultant plasmid was digested with Eam1105⒐, and the big fragment was recovered, generating the P. pastoris expressive T-vector pPICZT. The gene of cellobiohydrolase ⒑of T. reesei was expressed in P. pastoris with this expressive T-vector. The results indicated that the constructed expression T-vector was convenient for PCR product cloning, and was effective for heterolgous protein expression in P. pastoris. On the other hand, application of the expression T-vector avoided the introduction of additional amino acids in the N-terminus of the expressed protein, an event that generally occurred when normal expression vectors were used in secretive expression system.

Key words: secretive expression vector Pichia pastoris T-vector Trichoderma reesei cellobiohydrolase

收稿日期: 2006-08-17 出版日期: 2010-05-10

通讯作者: 刘刚 E-mail: zjuliug@szu.edu.cn

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引用本文:

张超刘刚余少文邢苗. 可直接克隆PCR产物的毕赤酵母分泌型表达载体[J]. 中国生物工程杂志, 2007, 27(1): 52-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I1/52

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