人毛乳头细胞HSPC016基因在毕赤酵母中的表达和鉴定  

Expression and identification of HSPC016 gene of human dermal papilla cell by Pichia pastoris expression system

作　　者：邹锋[1] 郝飞[1] 宋志强[1] 易勇[2] 杨希川[1] 杨斌[1] 叶庆佾[1] 

机构地区：[1]第三军医大学西南医院皮肤科 重庆市皮肤性病研究所,重庆400038 [2]第三军医大学医学检验系临床微生物及免疫学教研室,重庆400038

出　　处：《第三军医大学学报》2005年第14期1481-1483,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目(30200249)~~

摘　　要：目的构建人毛乳头细胞HSPC016基因的真核表达载体,诱导其表达并对目的蛋白基本生物学特性进行鉴定。方法用PCR从pET-28a(+)/HSPC016中扩增出195bp目的基因,克隆至酵母表达载体pPIC9K质粒,经酶切鉴定后转化毕赤酵母菌GS115,G418-YPD平板筛选高拷贝转化子,甲醇诱导表达后进行Westernblot检测和目的蛋白N-端氨基酸测序鉴定。结果用PCR成功扩增出目的基因;pPIC9K/HSPC016转化GS115用G418-YPD平板筛选高拷贝转化子,甲醇诱导后,SDS-PAGE电泳鉴定:目的蛋白相对分子量约为7·2×103,与理论预测值相吻合;UVP扫描表明目的蛋白表达率约18%;Westernblot检测具有良好的免疫反应性;N-端氨基酸测序结果与预期序列完全一致。结论HSPC016基因能通过酵母表达载体pPIC9K及宿主菌GS115进行高效、正确地表达,为研究HSPC016基因在真核细胞水平表达的生物学意义奠定了基础。Objective To construct and express the gene of HSPC016, and to study the basic biological properties /characteristic of the HSPC016 gene expression products. Methods The HSPC016 gene was amplified from the plasmid of pET-28a(+)/HSPC016 by PCR. The gene was subcloned into the plasmid pMD-18T and digested by two restriction enzymes of NotⅠ and EcoR Ⅰ. After purified, the HSPC016 gene was inserted into the prokaryotic expression vector pPIC9K. The pPIC9K/HSPC016 was transformed into P. pastoris GS115 strain by electroporation and recombinant protein was expressed after induced by methanol. The N-terminal amino acid residual sequencing of the protein was made after SDS-PAGE analysis. Results The HSPC016 gene was successfully cloned into pPIC9K. The results of SDS-PAGE showed that the molecular weight of the expressed product was 7.2×103, and the expression rate was about 20%. The result of the N-terminal amino acid residual sequencing is identical to that of the molecular design. Conclusion HSPC016 gene was successfully constructed and well expressed in Pichia pastoris expression system.

关 键 词：毛乳头细胞 HSPC016基因 毕赤酵母 真核表达 

分 类 号：R322.991[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]