类人胶原蛋白表达载体的构建及在毕赤酵母中的表达  

Construction of Human-like Collagen Expression Vector and Its Expression in Pichia Pastoris

作　　者：高力虎[1] 杨树林[1] 储卫华[2] 李新柱[1] 钮小松[1] 杨艳[1] 

机构地区：[1]南京理工大学化工学院,江苏南京210094 [2]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《南京理工大学学报》2008年第2期252-256,共5页Journal of Nanjing University of Science and Technology

基　　金：国家“863”计划(2004AA2Z3C60)

摘　　要：基于人胶原蛋白胶原域序列特征,设计合成了一段类人胶原蛋白基因单体gel。采用同向串联方式,构建了含6个单体的类人胶原蛋白表达载体pPIC9KG6,并经DNA测序鉴定。将pPIC9KG6电转化毕赤酵母(Pichia pastoris)经G418筛选得到多拷贝转化子,通过甲醇诱导表达类人胶原蛋白,SDS-PAGE检测表明:初步表达的类人胶原蛋白为胞外产物,其表观分子量为112 ku。A human-like collagen monomeric gene （gel） is designed and synthesized according to the unique character of collagenous domain, which is made up of total repeat of ＂Gly-X-Y＂ unit. The gel repeats in the same orientation forming six times ploygene, which is inserted into the EcoRI/NotI sites of vector pPIC9K with a HIS4 selectable marker through designed adaptors. After verifying the validity of construction by DNA sequencing, the recombinant vector pPIC9KG6 is electroporated into the Pichia pastoris GS115, and the transformants are screened on YPD medium with gradient concentration of G418 to determine the copy number of the human-like collagen ploygene. The human-like collagen is expressed and secreted with methanol fed-batch inducement by adding per 24 hours for maintaining at 1% concentration. The SDS-PAGE result shows that the expressed human-like collagen is extracellular protein and its apparent molecular weight is 112 ku.

关 键 词：类人胶原蛋白 基因串联 毕赤酵母 分泌表达 

分 类 号：Q789[生物学—分子生物学]