人血小板内皮舒张因子合酶互补脱氧核糖核酸克隆  

Nitric Oxide Synthase Complementary Deoxyribonucleic Acid Clone in Human Platelets

作　　者：王华梁 龚兰生[1] 孔宪涛 蔡伟箐 于金德[1] 戚文航[1] 吴春芳[1] 王海英 赵函芳[2] 陈诗书[2] 程枫[2] 王亚新[2] 卢健[2] 

机构地区：[1]上海第二医科大学附属瑞金医院心内科 [2]上海第二医科大学生化教研室

出　　处：《中国循环杂志》1996年第10期622-625,共4页Chinese Circulation Journal

摘　　要：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 construct cDNA library and screen nitric oxide synthase (NOS) cDNA clone in human platelets. Methods:Human platelets were purified from fresh blood,total RNA was extracted and was used as a template to synthesize cDNA.The cDNA was ligated to λgt11 arms,packed with the packing proteins,transinfected to Y1090.The library was scanned by dig labeled probe.The DNA was purified from individual recombinant plaques and was digested with EcoRI.After electrophoresis on agarose gel,the‘insert fragment’was cut and purified,the NOS cDNA clone was obtained. Results:RNA purified from human platelets was 1.5 mg.18 S and 28 S bands were clear in RNA electrophoresis.The first strand cDNA was 1.82 μg and the second strand 3.79 μg.Autoradiograph showed that the synthetic cDNA molecule was 0.5-5.0 kb(mostly 1.0-4.0 kb).NOS cDNA clone in human platelets was about 3.0 kb. Conclusion:NOS cDNA clone in human platelets is about 3.0 kb.It is similar to that in the other tissues.

关 键 词：血小板 内皮舒张因子 合酶 DNA 克隆 

分 类 号：Q592.1[生物学—生物化学]