人脑髓鞘碱性蛋白cDNA体外扩增、克隆和鉴定  

Amplification. Cloning and Identification ofFull-Length Coding Sequence for HumanMyelin Basic Protein.

作　　者：陈俊杰[1] 王若菡[1] 程汉华[1] 陈朴 李昌隆[1] 杨鲁川 

机构地区：[1]华西医科大学生物化学教研室

出　　处：《生物化学与生物物理进展》1994年第3期244-247,共4页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金;纽约中华医学基金

摘　　要：采用聚合酶链反应（ＰＣＲ）从人脑ｃＤＮＡ文库中扩增出６００ｂｐ的髓鞘碱性蛋白（ＭＢＰ）ｃＤＮＡ片段，与载体ｐＧＥＭ－３Ｚｆ（＋）平端连接．重组质粒ＤＮＡ转化宿主菌ＪＭ１０９，在含Ｘ－ｇａｌ和ＩＰＴＧ的平板上直接筛选阳性克隆．限制性内切酶分析和成套引物扩增鉴定证明，该克隆含有７个外显子的２１．５ｋＤ人脑ＭＢＰ全长编码序列．series of DNA primers specific for humanbrain myelin basic protein (MBP) gene wasdesigned and synthesized. MBP cDNA frag-ment about 600bp in length was amplified fromhuman brain cDNA library by using poly-merase chain reaction (PCR) with the specificprimers P_1 and P_2. The recovered PCR productwas flushed by klenow fragment and insertedinto pGEM-3Zf (+ ) vector pretreated withSmaⅠand calf intestinal alkaline phophatase.The recombinant plasmid was used to trans-form competent cell JM 109. The positivecolonies were directly screened on indicatorplates.The recombinant plasmid DNA and in-sert fragment isolated from four positivecolonies were analyzed by digestion with EcoRⅠ, Kpn Ⅰand Taq Ⅰ.The different codingsequences including MBP exon Ⅰ─Ⅶ.Ⅰ─Ⅲ,Ⅲ─Ⅶ and Ⅰ─Ⅴ were amplified fromthese clones with their corresponding nestedsets of primers respectively. These resultsshow that these cDNA clones contain full-length coding sequence for 21.5kD humanMBP.

关 键 词：聚合酶链反应 髓鞘碱性 蛋白 CDNA 

分 类 号：Q510.3[生物学—生物化学]