【摘要】 毕赤酵母是日益受到重视的基因工程受体菌 ,但是目前尚没有一种简便、高效的转化方法 ,也没有一种稳定、可靠的菌落PCR方法。本文通过在通常筛选重组子的MD培养基中增添 1mol/L山梨糖醇使毕赤酵母电穿孔转化效率提高 10倍以上。并比较系统地分析了其他影响电穿孔转化效率的因素 ,如毕赤酵母生长状况即OD60 0 ,不同的整合位点 (BglⅡ、SacⅠ、SalⅠ、StuⅠ ) ,及不同的毕赤酵母受体菌 (GS115和KM71)等。本文描述的转化方法所能达到的转化效率比目前大多数文献报道的效率要高 ,可使每微克质粒DNA产生的整合到受体菌染色体上的转化子述 2 80 0个 ;另外 ,我们经过一种冷热处理毕赤酵母菌落的方法使其细胞壁裂解 ,并改变通常PCR缓冲液的组成成分后 ,毕赤酵母菌落PCR方法几乎和大肠杆菌的操作一样简便可靠。借助本文所描述的电穿孔转化方法和菌落PCR方法 ,成功实现了水蛭素在毕赤酵母中的分泌表达 ,表达量为每毫升培养上清 2 0 μg ,其生物活性达每毫升培养上清 82个国际单位。更多还原

【Abstract】 Pichia pastoris\% has become an increasingly popular host for heterologous protein production. However, there is neither a high\|efficient transformation method nor a fast colony\|PCR assay for the yeast yet. In this paper, we report a transformation procedure by electroporation, which reaches the value of up to 2800 transformants/μg DNA. By using a cold and heat treatment and a modified PCR buffer, we established a simple and reliable colony\|PCR protocol to detect recombinant \%P.pastoris\% clones, which is comparable to the conventional assay for \%E.coli\% colonies. With these two novel techniques, we have successfully achieved the expression of hirudin, an antithrombin agent, in \%Pichia pastoris\%. The secreted hirudin maintains a biological activity of 82 antithrombian units per milliliter supernatant from the media.更多还原