rhTPO的分子克隆表达及活性测定

EXPRESSION AND BIOLOGICAL ACTIVITY IDENTIFICATION OF rhTPO

目的；分子克隆并表达具有生物学活性的人血小板生成素（rhTPO）。方法：采用PCR、序列测定、原核表达及亲和层析法。结果：以人全长的TPOCDNA为模板[1]，用PCR法获得了编码195个氨基酸残基的hTPO195cDNA并克隆至原核表达质粒pMal－p2中，以麦芽糖融合蛋白（MBP）方式进行了表达。经Amylose树脂系和层析纯化获得了较高纯度的rhTPO195融合蛋白。生物学活性测定结果显示，其促进体外培养的巨核细胞集落形成与日本麒麟（Kirin）公司产品类似。结论；获得了具有天然生物学活性rhTPO，并在原核细胞中获得表达。

Objective: Expression of recombinant human thrombopoietin (rhTPO)in prokaryotic system.Preparation and biological actiyity identification of rhTPO. Methods: PCR,molecular cloning, prokaryotic expressing system and affinity chromatography were used.Results:The cDNA of hTPO was inserted into upstream of an expression vector pMal-p2 and expressed in E. coli as a Maltose-binding protein fusion (MBP-TPO),which can be induced by IPTG. the high expression of rhTPO has been achieved. The purification of the fusion protein was very convenient and quick with MBP-Frotein purification system (Affinity column).Comparing our rhTPO with the product of the Kirin Co,we have found that it has similar biologicaly activities in stimulating the formation of CFU-Meg alone or in combining with other cytokines. Conclusion: A biologically active rhTPO has been obtained,which may provide a material to explore its function and clinical usage.