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.
A series of DNA primers specific for human brain myelin basic protein (MBP) gene was designed and synthesized. MBP cDNA fragment about 600bp in length was amplified from human brain cDNA library by using polymerase chain reaction (PCR) with the specific primers P1 and P2. The recovered PCR product was flushed by klenow fragment and inserted into pGEM-3Zf (+) vector pretreated with SmaⅠand calf intestinal alkaline phophatase. The recombinant plasmid was used to trans-form competent cell JM 109. The positive colonies were directly screened on indicator plates. The recombinant plasmid DNA and insert fragment isolated from four positive colonies were analyzed by digestion with EcoR Ⅰ,Kpn Ⅰ and Taq Ⅰ.The different coding sequences including MBP exon Ⅰ—Ⅶ, Ⅰ—Ⅲ,Ⅲ—Ⅶ and Ⅰ—Ⅴ were amplified from these clones with their corresponding nested sets of primers respectively. These results show that these cDNA clones contain full-length coding sequence for 21.5kD human MBP.
陈俊杰,王若菡,程汉华,陈朴,李昌隆,杨鲁川.人脑髓鞘碱性蛋白cDNA体外扩增、克隆和鉴定[J].生物化学与生物物理进展,1994,21(3):244-247
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