论文部分内容阅读
目的：建立人血小板互补脱氧核糖核酸（ｃＤＮＡ）文库，筛选出血小板内皮舒张因子合酶（ＮＯＳ）ｃＤ－ＮＡ克隆。方法：用新鲜血小板悬液纯化血小板，提取总核糖核酸（ＲＮＡ），以ＲＮＡ为模板合成ｃＤＮＡ，将ｃＤＮＡ连接λｇｔ１１臂，用噬菌体包装蛋白包装，然后转染宿主菌Ｙ１０９０建库。用地高辛标记探针进行杂交，挑出阳性克隆，扩增后纯化脱氧核糖核酸（ＤＮＡ）。将重组ＤＮＡ用ＥｃｏＲＩ酶解，电泳，并将插入片段切下，纯化得到内皮舒张因子合酶ｃＤＮＡ。结果：从血小板中获得ＲＮＡ约１．５ｍｇ，ＲＮＡ变性电泳可见较清晰的１８Ｓ和２８Ｓ条带且无ＲＮＡ降解。第一条ｃＤＮＡ链产量为１．８２μｇ，第二条链为３．７９μｇ，放射性自显影显示双链ｃＤＮＡ分子大小范围在０．５～５．０ｋｂ，大部分位于１．０～４．０ｋｂ之间。人血小板内皮舒张因子合酶ｃＤＮＡ克隆约３．０ｋｂ左右。结论：人血小板内皮舒张因子合酶ｃＤＮＡ克隆约３．０ｋｂ，与其它组织的相似。
OBJECTIVE: To establish a cDNA library of human platelet-complementing cDNA and screen clonal cD-NA of platelet-derived endothelial relaxing factor synthase (NOS). Methods: Platelets were purified with fresh platelet suspension. Total RNA was extracted and RNA was used as a template to synthesize cDNA. The cDNA was ligated to λgt11 arm, packaged with phage packaging protein, and then transfected into host strain Y1090. Digoxigenin-labeled probes were used for hybridization, positive clones were picked out and DNA was purified after amplification. The recombinant DNA was digested with EcoRI, electrophoresed, and the inserted fragment was cut and purified to obtain cDNA of endothelial relaxing factor synthase. Results: Approximately 1.5 mg of RNA was obtained from platelets, and RNA denaturing electrophoresis showed clearer 18S and 28S bands without RNA degradation. The first cDNA strand yield was 1.82 μg and the second strand was 3.79 μg. Radioactive autoradiography revealed that the size of the double-stranded cDNA molecule ranged from 0.5 to 5.0 kb, mostly at 1.0-4.0 kb between. Human platelet endothelial relaxing factor synthase cDNA clone is about 3.0kb. Conclusion: The cDNA clone of human platelet endothelial relaxing factor synthase is about 3.0kb, which is similar to other tissues.