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按Bishop方法给新西兰兔注射盐酸苯肼(2.5％)0.3ml/kg体重/d,连续6d致贫血,收集血液用生理盐水洗去白细胞后,得到的网织红细胞用冻融法使细胞裂解,用乙醇、氯信选择性变性除去血红蛋白,再于1OO℃加热变性2min,去除大部分杂蛋白,经Sephadex G-100凝胶过滤,再连续经HPLC反相柱,HPLC monoQ阳离子交换柱后,提到一活性蛋白质,分子量7～8kD,在SDS-PAGE电泳图中为单一蛋白条带。将此条带印迹在immobiton-P膜上,进行蛋白质的氨基酸序列分析,得到8个氨基酸的序列为VRIRQTIK(477A氨基酸测序仪,美国),经计算机检索分析,未发现同源序列。
New Zealand rabbits were injected intraperitoneally with phenylhydrazine hydrochloride (2.5%) 0.3ml / kg body weight / d according to Bishop’s method for successive 6 days to cause anemia. Blood was collected and the white blood cells were washed with saline. The resulting reticulocytes were lysed by freeze- Hemoglobin was selectively denatured with ethanol and chlorhexidine, then denatured at 100 ° C for 2 min to remove most of the hybrid proteins, then gel-filtered through Sephadex G-100 and then continuously by HPLC reversed-phase column and HPLC monoQ cation exchange column To an active protein, molecular weight 7 ~ 8kD, SDS-PAGE electrophoresis in a single protein band. The band was blotted on immobiton-P membrane and the amino acid sequence of the protein was analyzed. The 8 amino acid sequence was VRIRQTIK (477A amino acid sequencer, USA). No homologous sequences were found by computer search.