摘要
用ＲＴ－ＰＣＲ方法快速克隆了Ｗｉｓｔａｒ大鼠脑神经元特异性烯醇化酶（ＮＳＥ）的ｃＤＮＡ，将此包括编码全长ＮＳＥ４３３个氨基醚的ＤＮＡ片段重组入ｐＵＣ质粒，并用ＰＣＲ方法测定了全部顺序，经重复实验，发现Ｗｉｓｔａｒ大鼠与Ｆｏｒｓｓ－Ｐｅｔｔｅｒ报导的ＳＤ大鼠ＮＳＥ基因顺序，有两外单碱基的差别，其中一个涉及氨基酸的改变。同时还对ＲＮＡ的提取及长片段ＤＮＡ的ＲＴ－ＰＣＲ扩增进行了方法学的探讨。
Neuron-Specific Enolase(NSE) is a specific marker protein for the development of nervous system. In mammalian tissues, NSE is mainly present in mature central and peripheral nervous system. So, NSE is an ideal candidate for studying cell type specificity as well as developmental regulation of gene expression. In this paper, we amplified the complete 1305bp coding sequence of NSE by use of RT-PCR and inserted it into PUC vector system for cloning. And then, we determined its nucleotide sequence by the dideoxy chain termination method combined with PCR technique. The results show that there are two single-nucleotide differences from those of Forss-Petter, and one of them involved in an amino acid variation.The methodology of RT-PCR DNA amplification for large gene fragment was also explored.
