人脑髓鞘碱性蛋白cDNA重组质粒pRK41转化及全长编码区体外扩增

Transformation of Recombinant Plasmid pRK 41 and Amplification of Full-Length Coding Region of Human Brain Myelin Basic Protein cDNA

作者将含人脑髓鞘碱性蛋白(MBP)编码区和3’端非翻译区(1.2kb)的重组质粒pRK41—1.2转化宿主菌E.coli JM109,用非同位素标记鼠MBP cDNA克隆片段作探针,经菌落原位杂交筛选出阳性克隆并以此为模板,采用聚合酶链反应(PCR)技术扩增出含21.5kd MBP全长编码序列(600bp),又经限制性内切酶BamHⅠ和KpnⅠ消化,电泳结果进一步证实该PCR产物的特异性,为人MBP全长编码序列。

Transformation of the re-combinant plasmid pRK41 containing 2.15kd human brain myelin basic proteinMBP)-coding sequence and 3' untrans-lated region(1.2kb) into the E.coli JM109was made by using Hanahan's method.Positive colonies were sereened withdigoxigenin oligo labelled rat brain MBPcDNA fragment (1.2kb). To remove 3'untranslated region and obtain the full-length coding sequence of MBP cDNA. apair of specific DNA primers was desig-ned and synthesized. A 600 bp fragmentwas amplified from the recombinant plas-mid, extracted from the positive colonyby using polymerase chain reaction(PCR).The PCR fragment was isolated, and thendigested with BamH I, Kpn I and BamHI+Kpn I .The results of restriction ana-lysis indicate that the PCR amplifiedfragment is desirable and can be useddirectly to constract expression vectors.