rhTPO的克隆及其在原核细胞中的表达  

Cloning and prokaryotic expression of rhTPO in prokaryotic cells

作　　者：曹华[1] 葛忠良[1] 张群伟[1] 孙丽亚[1] 张雪峰[1] 丁清明 

机构地区：[1]北京放射医学研究所

出　　处：《中华放射医学与防护杂志》1997年第5期316-318,共3页Chinese Journal of Radiological Medicine and Protection

摘　　要：为了更加简便有效地得到重组人血小板生长因子（ｒｈＴＰＯ），以治疗放射及化学药物等所致骨髓损伤造成的血小板减少与缺乏，缓解临床上的出血症状，降低死亡率。方法本实验从６月龄人胚胎肝脏细胞中分离ｍＲＮＡ，设计合成特异性引物，经逆转录，套式ＰＣＲ等步骤克隆出了人ＴＰＯ全基因。将人ＴＰＯ基因亚克隆至原核细胞表达载体ｐＢＶ２２０，形成了重组表达质粒ｐＢＶ２２０／ＴＰＯ，以该重组质粒转化了原核细胞大肠杆菌ＤＨ５α，应用４２℃热诱导表达目的蛋白。结果经ＳＤＳ－聚丙烯酰胺凝胶电泳检测，得到了ＴＰＯ在原核细胞中的表达，其分子量与目的一致。结论ｈＴＰＯ可以在原核细胞得到良好的表达，为临床血小板减少症的生物治疗奠定了基础。Objectives\ Thrombopoietin (TPO) is a normal physiological hematopoietic factor which is able to stimulate the hematopoietic progenitor cells to proliferate and differentiate into megakaryocytes and to induce the production and release of platelets. In this study rhTPO was cloned to be potentially used for treatment of radiation induced thrombocytopenia, we studied the prokaryotic expression of rhTPO.Methods\ According to the cDNA sequence encoding hTPO published elsewhere, we designed two sets of PCR primers at first, and cloned hTPO gene from fetal liver mRNA by means of RT PCR, then subcloned it into prokaryotic expression vector pBV220 to form recombinant expression plasmids pBV220/TPO, and to transfer it into prokaryotic cells escherichia coli, DH5α. Expression was induced by heat of 42℃. Results\ We obtained the aimed prokaryotic expression of recombinant(r) hTPO by assessment of SDS Page. Conclusion\ hTPO can be expressed completely in prokaryotic cells. The prokaryotic expression of rhTPO might be a basis for the bio treatment of radiation induced thrombocytopenia.

关 键 词：血小板生长因子 克隆 原核表达 

分 类 号：Q78[生物学—分子生物学] R973[医药卫生—药品]