利用基因敲除技术缺失毕赤酵母羧肽酶Y基因的初步研究  

Deletion of gene cpy in Pichia pastoris:a preliminary study

作　　者：王文文[1] 郝木强[2] 刘春杰[3] 靳继德[3] 吴祖泽[1,3] 

机构地区：[1]天津大学化工学院,天津300072 [2]北京工业大学生命科学与生物工程学院,北京100124 [3]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《军事医学》2012年第9期673-677,697,共6页Military Medical Sciences

基　　金：国家科技重大专项重大新药创制项目(2012ZX09102301-008)

摘　　要：目的通过基因敲除技术缺失毕赤酵母编码的羧肽酶Y(carboxypeptidase Y,CPY)基因cpy,观察其对毕赤酵母生长和外源蛋白表达的影响。方法通过PCR方法扩增出内部含有潮霉素B抗性基因的cpy同源序列;通过电转法将同源序列转入GS115-EH感受态细胞中,并用潮霉素B筛选出阳性克隆。用平板显色法测定阳性克隆菌的羧肽酶活性,同时用PCR法鉴定阳性克隆菌的cpy基因是否缺失。对cpy基因缺失的GS115-EH△cpy菌,分别用YPD和BMGY-BMMY培养研究其生长特性;用BMMY培养基进行甲醇诱导表达外源蛋白,观察其蛋白表达的特性。结果 GS115-EH△cpy的羧肽酶基因cpy被成功敲除,羧肽酶活性丧失。YPD培养条件下,GS115-EH△cpy生长后期的D600明显低于GS115-EH,而用BMMY诱导培养基,GS115-EH△cpy的生长明显改善,但仍然低于对照菌。蛋白电泳结果显示甲醇诱导GS115-EH△cpy的蛋白表达量也低于对照菌。结论 GS115-EH△cpy的cpy基因被成功敲除,在YPD培养条件下,其生长低于对照菌,在BMMY培养条件下,其生长明显改善,但仍然较对照菌差,而且其外源蛋白的表达也低于对照菌。Objective Carboxypeptidase Y （CPY） is a protease widely existing in yeast, which is encoded by gene cpy and leads to the degradation of exogenous protein. This study is intended to construct a mutant GS115-EH△^cpy by deleting cpy, and evaluate the growth and expression characters of this new mutant strain. Methods The cpy homologous recombi- nation sequence containing hygromycin B resistant gene was amplified by PCR, and then transformed into the recombinant yeast GSll5-EH by LiCl electrotransfection method. Thereafter, the mutant GSll5-EH△^cpy strains were selected in the plate with hygromycin B, and further confirmed with both plate coloration method and PCR method. The growth and protein expression features of the GS115-EH △^cpy were evaluated. Results PCR results showed that the gene cpy of the strains resistant to hygromycin B was deleted and the plate coloration method proved that the protease activities of CPY in GS115- EH A ＂PYdecreased obviously. However, the growth and protein expression of the GS115-EH△^cpy were both inhibited significantly. Conclusion The gene cpy is successfully deleted in the GSll5-EH△^cpy and the growth and exogenous protein expression of the mutant strains are inhibited significantly with cpy deletion.

关 键 词：毕赤酵母 羧肽酶 基因敲除 

分 类 号：Q78[生物学—分子生物学]