作　　者： ; ; ; ; ;

机构地区： 中国医学科学院北京协和医学院血液学研究所

出　　处： 《细胞与分子免疫学杂志》 2009年第11期991-993,997,共4页

摘　　要： 目的:为了得到可溶性表达的人促血液血管细胞生成素(HAPO)蛋白,构建含HAPO基因片段的pET22b(+)表达载体,获得纯化的HAPO蛋白并检测其生物学活性。方法:利用RT-PCR技术从人胎肝中获得HAPO cDNA片段,并克隆至表达载体pET22b(+)中,利用基因工程菌BL21(DE3)进行表达,产物利用Ni2+-螯合亲和层析及SP Sepharose FF柱层析分离纯化。采用黏附实验检测HAPO对人脐静脉内皮细胞(HUVEC)黏附性的影响。结果:从人胎肝中克隆出长为897 bp HAPO目的片段,成功构建重组质粒pET22b(+)-HA-PO,其表达的融合蛋白以可溶状态存在,表达量占菌体总蛋白的10%,经分离纯化融合蛋白的纯度可达80%。活性测定结果表明HAPO以剂量依赖性方式增加HUVEC的总黏附性。结论:可溶性表达了HAPO蛋白,并用体外实验证实HAPO对造血干/祖细胞归巢有一定促进作用。 AIM： To prepare a soluble hemangiopoietin （HAPO） protein and to construct pET22b（ ＋ ） expression vector, to obtain pure recombinant HAPO protein and to measure its bioactivity. METHODS： HAPO cDNA was amplified using RT-PCR method from a commercial human fetal liver cDNA library. The resulting product was cloned into pET22b（＋） vector and transformed into E. coli BL21 （DE3）. The recombinant protein was isolated and purified by Ni2＋-NTA chelating resin and the chromatographies of SP Sepharose FF. The adhesion of human umbilical vein endothelial cells （HUVEC） were measured by adhesion assay. RESULTS： HAPO gene with a reading frame of 897 bp was successfully cloned from human fetal liver cDNA library, the expressed pET22b（ ＋ ）-HAPO fused protein existed in a soluble form, with the yield above 10% total bacterial protein and its purity achieved above 80%. The activity assay showed that the treatment of HAPO enhanced total adherence of HUVEC in a concentration-dependent manner. CONCLUSION： HAPO protein can be expressed in a soluble form. HAPO may facilitate the homing of hematopoietic stem/progenitor cells in vitro.

关 键 词： 促血液血管细胞生长素 可溶性表达 分离纯化 人脐静脉内皮细胞

领　　域： [生物学]