rhTPO在大肠杆菌中的表达、纯化及生物活性分析

目的：探讨人促血小板生成素(human thromlbopoietin，hTPO)在大肠杆菌中表达、分离纯化及生物学活性初步鉴定。方法：利用RT-PCR法从人胎肝细胞中扩增目的基因片段，将其定向插入pQE30表达质粒T5启动子下游的多克隆区，转化大肠杆菌M15，得到pQE30-TPO的工程菌，诱导目的蛋白表达、纯化。将表达产物给血小板减少模型小鼠尾静脉注射，观察注射后不同时间血小板量的改变。结果：经异丙基硫代-β-D-半乳糖苷(isopropylthio-β-D-galactoside IPTG)诱导培养，该工程菌可以产生单一特异性的高表达蛋白条带。将纯化后的表达蛋白注射小鼠，结果显示对实验性小鼠血小板减少症具有一定的治疗作用。结论：在大肠杆菌中成功地高效表达了重组hTPO，该产物具有促血小板生成的活性。

Expression, purification and activity identification of hTPO in Escherichia coli

Objective:To express and purify human thrombopoietin in Escherichia coli.Methods:The TPO cDNA coding was amplified from the human fetal liver cell by RT-PCR,the target cDNA was inserted into the polycloning region of pQE30expression vector,then transferred into the E.coli M15.The expression products was injected into the mice with thrombocytopenia and the change of the platelet' level in peripheral blood was detected.Results:SDS-PAGE analysis showed that the rhTPO protein was ex-pressed especially in E.coli M15harboring pQE30-TPO recombinant plasmid induced by IPTG,and the target protein was purified with histidine-tail through Ni-NTA affinity gel.The rhTPO was effective in treating carboplatin-induced thrombocytopenia in mice.Conclusion:The recombinant human thrombopoietin was successfully expressed in Escherichia coli.