新型毕赤酵母分泌表达载体的构建与功能验证

巴斯德毕赤酵母(Pichia pastoris)是目前应用最广泛的外源蛋白表达系统之一。用于毕赤酵母表达系统的表达载体种类繁多,却因需要甲醇诱导表达或者基因转化过程中引入抗生素基因而限制了其在食品行业中的应用。研究设计以毕赤酵母组成型启动子-GAP启动子替换毕赤酵母表达载体pPIC9上的甲醇诱导型启动子AOX,成功构建了毕赤酵母组成型表达载体pGAPHα。并以里氏木霉木聚糖酶基因xyn2为报告基因进行功能验证,结果表明,该载体可实现xyn2基因在毕赤酵母中的高效表达。在此基础上以经密码子优化后的克鲁维酵母菊粉酶基因信号肽INU替换载体上原有的α-Factor信号肽,结果表明,INU信号肽能够有效引导XYNII的分泌。

Construction and probation of neotype secreting expression vectors for Pichia pastoris

Pichia pastoris is one of the foreign protein expression system which has been widely used. The expression vectors for Pichia pastoris are varied, but they can not be used in food vocation, because of the need of methanol in expression and the leading-in of antibiotic gene by transformation. This research designed to replace the methanol induced AOX promoter by Pichia pastoris's constitutional promoter, GAP promoter, and successfully constructed the secreting expression vector pGAPH`, for Pichia pastoris. The gene encoding endo-β-1, 4-xylanase from Trichoderma Reesei, and xyn2 gene were used as a reporter gene, and then the function probation indicated that the xyn2 gene could be overexpressed in Pichia pastoris. Based on this neotype secreting expression vector pGAPHαⅡ, we replaced the α-Factor by inulase gene's signal peptide sequence which had been optimized in codon, the results showed that the showed gene's signal peptide could also utility guide the secreting expression of xyn2 gene.