鸡源志贺氏菌IpaC基因新型表达载体构建及其在毕赤酵母中的表达  

Construction of new expression vector of chicken-derived Shigella IpaC gene and its expression in Pichia pastoris

作　　者：马金玉[1] 韩志霞[1] 蒋大伟[1] 王亚宾[1] 许兰菊[1] 苗银萍[1] 孔江南[1] MA Jinyu HAN Zhixia JIANG Dawei WANG Yabin XU Lanju MIAO Yinping KONG Jiangnan(College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, Chin)

机构地区：[1]河南农业大学牧医工程学院,郑州450002

出　　处：《黑龙江畜牧兽医》2016年第10期36-41,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31272568)

摘　　要：为使鸡源志贺氏菌IpaC蛋白保持天然的生物学活性,在毕赤酵母表达系统中实现分泌表达,进一步研究IpaC蛋白的生物学功能,本研究在不改变IpaC蛋白氨基酸序列的情况下,根据酵母密码子偏爱性优化合成IpaC#基因,利用真核表达载体p GAPZA和8种真核信号肽,分别构建真核表达载体pGAPZA-Inu/Inv/Kil/Lys/Mat/Amy/Glu/Ser-IpaC#,然后将线性化的重组质粒电转化至毕赤酵母X-33中,对阳性转化子进行表达,并用SDS-PAGE及Western-blot检测其表达产物。结果表明:在8种重组菌表达产物的菌体沉淀中检测到大小约为60 ku的目的蛋白。说明鸡源志贺氏菌IpaC基因成功实现了在毕赤酵母的胞内表达。To keep the natural biological activities of Chicken -derived Shigella IpaC protein to achieve secretory expression in Pichia pastoris expression system and further study biological function of IpaC protein, the IpaC# gene was optimized and synthesized according to the yeast codon preference without changing the amino acid sequence of IpaC protein in this research. The eukaryotic expression vectors pGAPZA - Inu/ Inv/Kil/Lys/Mat/Amy/Glu/Ser - IpaC# were constructed using eukaryotic expression vector pGAPZA and eight kinds of eukaryotic signal pep- tides, respectively. And then the linearized recombinant plasmids were electrotransformed into Pichia pastoris X -33. The positive transforma- nts were expressed and the expression products were detected by SDS - PAGE and Westem- blot. The results showed that a target protein of a- bout 60 ku was detected in bacter/al precipitation from the expression products of eight kinds of recombinant bacteria. The result indicates that the chicken - derived Shigella IpaC gene successfully achieves intracellular expression in Pichia pastoris.

关 键 词：鸡源志贺氏菌 IpaC基因 毕赤酵母 pGAPZA 真核信号肽 

分 类 号：S852.61[农业科学—基础兽医学] Q786[农业科学—兽医学]