人促血液血管生成素的原核表达、分离纯化及活性分析

Expression,purification and characterization of humanhemangiopoietin protein

目的利用基因工程技术获得重组的人促血液血管生成素(HAPO),以探讨其对骨髓细胞的作用。方法提取人胎肝总RNA,利用RT-PCR、克隆HAPO cDNA插入表达载体pET32c,使之在大肠杆菌BL21(DE3)中表达;用DEAESepharose Fast Flow阴离子交换柱、Ni-Chelating Sepharose Fast Flow亲和柱以及SP Sepharose Fast Flow阳离子交换柱分离纯化,肠激酶酶切,液体培养小鼠骨髓细胞。结果经IPTG诱导HAPO实现了在大肠杆菌中的可溶性表达,经一系列层析获得融合的rh-HAPO,肠激酶酶切除去N-端融合部分后,获得高纯度的rh-HAPO。液体培养小鼠骨髓实验发现rh-HAPO可促进CD34+细胞和flk-1细胞的增殖。结论重组蛋白rh-HAPO可促进造血干/祖细胞的增殖。

Objective To investigate the effect of Hemangiopoietin （HAPO） on bone marrow cells by gene engineering Methods The eDNA of HAPO was cloned from total RNA of human fetal liver cells by RT-PCR. The eDNA of HAPO was inserted into the pET32c （ ＋ ） expression vector downstream to the his-tag site. After transformation into E. coli （BL21）, the recombinant human HAPO（rh-HAPO） protein was expressed in E. coli cells. The rh-HAPO was isolated by DEAE sepharose Fast Flow column, his-tag affinty column and SP sepharose Fast Flow column and digested by enterokinase. Using liquid culture, the biological activities of rh-HAPO was tested. Results After induced by IPTG, the rh-HAPO was expressed efficiently as soluble protein in E. coli cells. Isolation of HAPO protein required a series of purification procedures. After enterokinase digestion, the high purity of rh-HAPO was obtained. In liquid culture, rh-HAPO increased the number of the number of CD34 ＋ and Flk-1 ＋ cells of murine bone marrow cells. Conclusion rh-HAPO promotes the proliferation of hematopoietic stem / progenitor cells.