拟多形假丝酵母的双营养缺陷型工程化改造及鉴定

Construction and identification of the double auxotrophic Candida parapolymorpha

目的以拟多形假丝酵母菌（ATCC26012）为材料，构建遗传稳定性良好的尿嘧啶和亮氨酸双营养缺陷型工程化菌株。方法利用物化及基因工程等方法对拟多形假丝酵母菌株的染色体进行改造，将ura3基因和leu2基因进行定向诱变，以获得尿嘧啶和亮氨酸双营养缺陷型突变菌株，并对其进行生物学鉴定，包括菌株的遗传稳定性、基因序列及生理生化特征分析等。结果筛选得到尿嘧啶和亮氨酸双营养缺陷型突变菌株，鉴定结果显示该菌株遗传稳定性良好，标志基因均发生定向改变，生理生化结果显示该菌株能利用多种碳源和氮源，且生长状况良好。结论拟多形假丝酵母的双营养缺陷型工程化改造为进一步研究该菌株的遗传学奠定了基础，为多价蛋白组成类病毒颗粒的外源表达提供了工程化宿主。进而，为复杂疫苗抗原的重组表达和功能研究提供了技术手段。

Objective To construct a genetically-stable double auxotrophic, in which the uracil and leueine were mutated,using the Candida parapolymorpha ATCC26012 as materials. Methods Based on the physical and genetic engineering methods, the chromosome of the C. parapolyrnorpha strain was modified, where the ura3 and /eu2 genes were directly mutated, to obtain the uracil and leucine double auxotrophic strain. Then the constructed strain was identified by the analysis of its biological properties, such as genetic stability, the change of the genes, and the physiologic and biochemical characteristics. Results The uracil and leucine double auxotrophic strain is obtained by screening. The biological identification results show that the obtained strain is genetically stable and the targeted genes are directly altered. In addition, the physiologic and biochemical analyses indicate that the auxotrophie can utilize various kinds of carbon and nitrogen nutri- ent sources, and its growth is good. Conclusion The successful construction of double auxotrophic mutant strains facilitated the genetic studies on C. parapolymorpha to meet various investigational purposes. Moreo- ver,the constructed auxotrophie strains can be applied as advantageous host ceils to express multiple pro- teins/antigens simultaneously ,which is of great significance in the development of vaccines.