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.

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.