人血小板生成素cDNA的克隆及在COS-7细胞中的表达  

The cloning and expression of human thrombopoietin cDNA in COS7 cells

作　　者：童涌[1,2] 戴建新[1,2] 陈蕊雯[1,2] 周永春[1,2] 孙树汉[1,2] 陆德如[1,2] 

机构地区：[1]第二军医大学基础医学部医学生物技术和分子遗传学研究所 [2]第二军医大学基础医学部分子遗传学教研室

出　　处：《第二军医大学学报》1998年第3期212-214,共3页Academic Journal of Second Military Medical University

基　　金：上海市自然科学基金

摘　　要：目的：克隆人血小板生成素（ｈＴＰＯ）ｃＤＮＡ，并研究其在ＣＯＳ－７细胞中的表达。方法：通过ＲＴ－ＰＣＲ法获得ｈＴＰＯｃＤＮＡ，测序后克隆到真核表达载体ｐＥＤ上，以ＤＥＡＥ－ｄｅｘｔｒａｎ法转染ＣＯＳ－７细胞，巨核细胞集落形成培养法测表达产物活性。结果和结论：获得的ｈＴＰＯｃＤＮＡ和文献报道的序列一致。转染细胞ＲＮＡ斑点杂交结果显示ＴＰＯｃＤＮＡ获得转录，转染后４８和７２ｈ的细胞上清均可测到ＴＰＯ活性。ｐＥＤ是一个表达较高的真核表达载体。Objective: To clone the hTPO cDNA and study its expression level in COS7 cells. Methods:The hTPO cDNA was obtained from 6monthfetal liver RNA by RTPCR. After sequencing, it was inserted into an eukaryotic expressing plasmid pED and transfected into COS7 cells by DEAEdextran. The biological activity of transfected cell supernatant was measured by CFUMeg colony culture. Results and Conclusion: The cloned hTPO cDNA had the same sequence as that reported by Barltey. Transcription of TPO cDNA was detected by RNA dot blot. The biological activity of hTPO was detected 48 and 72 h after transfection. The recombinant expressing plasmid pEDTPO has a high expression level and can be used in stable expression.

关 键 词：人血小板生成素 基因表达 CDNA COS-7细胞 

分 类 号：Q78[生物学—分子生物学]