人TPON端结构域在毕赤酵母中的分泌表达及产物鉴定  

Secretion expression and identification of thrombopoietin in Pichia pastoris

作　　者：田生礼[1,2] 刘丽[1,2] 孟岩[1,2] 芦兴武[1,2] 谢宝树[1,2] 刘立忠[1,2] 冷爱军[1,2] 王颖[1,2] 

机构地区：[1]空军总医院空军临床分子生物学研究中心 [2]东北师范大学遗传与细胞研究所

出　　处：《中华微生物学和免疫学杂志》1998年第5期397-400,共4页Chinese Journal of Microbiology and Immunology

基　　金：全军"九五"医药卫生科研基金

摘　　要：目的在酵母系统中分泌表达人ＴＰＯＮ－端结构域，对其生物学活性进行初步研究，为与其它ＴＰＯ分子进行ＴＰＯ糖基化生物学意义的研究打基础。方法将编码人ＴＰＯ成熟肽Ｎ端１９５个氨基酸的ｃＤＮＡ与酵母整合型载体ｐＰＩＣＺαＡ重组，构建的重组质粒用ＤｒａⅠ消化成线性ＤＮＡ，转染酵母细胞ＧＳ１１５，使ＴＰＯ基因整合到酵母染色体上；用ＴＰＯ抗体筛选获得遗传性分泌稳定表达的重组酵母细胞株；用ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ鉴定ＴＰＯ产物及分子量；用对小鼠骨髓细胞形成巨核细胞集落形成单位（Ｍｅｇａｋａｒｙｏｃｙｔｅｃｏｌｏｎｙｆｏｒｍｉｎｇｕｎｉｔ，ＣＦＵ－Ｍｅｇ）的方法测定活性。结果获得了ＴＰＯＮ－端结构域在酵母系统中的分泌表达，表达产物可被ＴＰＯ抗血清识别，分子量约为２２０００；其产物对小鼠骨髓细胞形成ＣＦＵ－Ｍｅｇ具有明显的刺激作用。Objective To preliminarily study secretion expression of human TPO N terminal domain in Pichia pastoris and its activity was tested for further studying the biological significance of the glycosylation with other TPO molecules. Methods The cDNA for the N terminal 195 amino acids were recombinated with the integrated plasmid of the yeast pPICZαA.The recombinated plasmid was digested with Dra I and formed a linear DNA, with which the yeast cell GS115 was transfected and the genes of TPO were integrated into the chromosomes of the yeast.The strain of the yeast cells which can obtain genetically the steady expression was screened with TPO antibody. The product of TPO and its molecular weight were determined with ELISA and Western blot, and its activities were assayed with the method of Megakaryocyte clony forming unit (CFU-Meg).Results Western blot analysis showed that the expressed products showed positive bands with 22kD molecular weight. Activity assay indicated that the culture supernatant can significantly stimulate murine CFU Meg colony formation in vitro. Conclusion The N terminal of human TPO with biological activities can be expressed successfully in Pichia pastoris.

关 键 词：血小板生成素 毕赤酵母 N端结构域 分泌 表达 

分 类 号：Q592.1[生物学—生物化学] Q78