毕赤酵母重组菌株直接基因缺失方法的研究

Application of Direct Gene Disruption Method in Recombinant Pichia pastoris

摘要:

毕赤酵母重组菌在表达外源蛋白时往往存在比较严重的蛋白降解现象，一种可供选择的解决办法就是将其中起作用的某种蛋白酶基因缺失, 但目前国内外研究主要集中于非重组毕赤酵母的基因缺失，直接对重组菌进行基因缺失的研究较少。以现有的毕赤酵母基因重组菌(GS115Hir)为宿主菌，分别采用有标记的插入替换方法和无标记的Pop-In/Pop-Out方法直接缺失其PRC1蛋白酶基因和KEX1蛋白酶基因。缺失菌株经测序分析，结果显示发生了理论的基因缺失。在此基础上本文比较了这两种方法的各自优缺点，探讨了它们的不同的用途，为直接在毕赤酵母重组菌中进行基因缺失提供了有益的参考。

Abstract:

Proteolytic degradation has been a severe problem when Pichia pastoris is employed to express recombinant proteins.One alternative method to circumvent this problem is to construct protease gene disruptant. However, the main study of gene disruption is focused on nonrecombinant Pichia pastoris rather than recombinant strain. In our study, we established two different methods to directly disrupt PRC1 and KEX1 gene in recombinant Pichia pastoris. On the basis of this, we further discussed and compared the application and advantages of both methods.