应用改良的mRNA差异展示法克隆人鼻咽上皮特异性表达的cDNA片段

Cloning of a Tissue specific cDNA Fragment from Human Nasopharyngeal Epithelium by Means of Improved mRNA Differential Display Method

本文对ｍＲＮＡ差异展示法进行了改良．利用一组随机引物与来源于ｍＲＮＡ翻译起始位点区域的引物组合进行ＲＴ－ＰＣＲ，使差异展示的片段来源于蛋白编码区，并对差异展示的条件进行了优化．应用此法对人鼻咽上皮与软腭口腔粘膜上皮的基因表达进行了比较研究，得到了１０个在鼻咽上皮特异表达的ｃＤＮＡ片段，并对其中的５个片段进行了亚克隆和序列分析．通过与ＧｅｎｅＢａｎｋ数据库中的序列进行同源性比较，确定其中两个片段为未知新序列，Ｎｏｒｔｈｅｒｎ杂交证实其中一个片段ＮＥＳ１为鼻咽上皮特异性表达片段．

The method of mRNA differential display was improved by using a set of arbitrary primers combined with primers from the translation start site.In this case,the cDNA fragments of differential display originated from protein coding region of mRNA.Selecting different RT PCR reaction conditions and different recovery method of fragments optimized differential display.Gene expression profiles of human nasopharyngeal epithelium was compared with that of human oral cavity epithelium of soft palate by this method,and it were found that ten cDNA fragments uniquely expressed in nasopharyngeal epithelium. Five of them were subcloned and sequenced,and two were found to be novel sequences byBLAST sequence similarity searching in GeneBank nucleotide sequence database.Finally,one of them,NES1,was confirmed to be a tissue specific cDNA fragment of human nasopharyngeal epithelium.