人胚鼻咽上皮细胞cDNA文库的构建及鼻咽癌相关基因的筛选

Construction of Directional cDNA Library from Human Embryo Nasopharyngeal Epithelia and Screeming a Candidate Tumor Suppressor Gene Related with Nasopharyngeal Carcinoma

为了进一步分离人鼻咽组织特异性表达基因和鼻咽癌特异相关基因 ,采用SMART (switchingmechanismat 5′endofRNAtranscript)技术 ,构建了人胚鼻咽上皮cDNA文库 .从原代培养的人胚鼻咽上皮分离总RNA并纯化mRNA ,利用经修饰的oligo (dT)引物 (含sfiⅠB酶切位点 )合成cDNA第一链 ,同时根据真核生物mRNA5′端帽子结构特点 ,利用SMART核苷酸 (含sfiⅠA酶切位点 )作为cDNA第一链在mRNA 5′端延伸出去的模板 ,进而以此序列为引物利用LD PCR (long distance PCR)合成双链cDNA ,双链cDNA经sfiⅠ (ⅠA和ⅠB)酶切和过柱分级分离后 ,克隆入经sfiⅠ酶切的λTrip1EX2载体后经体外包装而成cDNA文库 .结果表明 ,原始人胚鼻咽上皮cDNA文库获得 1 0× 10 6个重组子 ,重组率达 96 % .文库扩增后 ,滴度达 7 8× 10 9pfu ml,插入cDNA平均长度为 1 2kb ,用PCR从该文库扩增出本实验室新克隆的鼻咽癌相关基因NAG4的全长cDNA .构建的人胚鼻咽上皮cDNA文库具有良好的质量 ,该cDNA文库为进一步筛选。

In order to isolate and screem tissue-specific genes of human nasopharynx and new tumor suppressor genes of nasopharyngeal carcinoma (NPC), a directional cDNA library from human embryo nasopharyngeal epithelia was constructed by SMART (switching mechanism at 5' end of RNA transcript) technique. The total RNA and mRNA were separated from primary cultural human embryo nasopharyngeal epithelia and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo (dT) primer (contained sfi I B site) while the SMART oligonucleotide (contained sfi I A site) was utilized as a template so that the first-strand cDNA could be extended over the 5' end of mRNA. The double-strand cDNA was amplified by LD-PCR (long-distance PCR) with the above two primers and then digested by sfi I (I A & I B) restriction enzyme. After cDNA size fractionation through CHROMASPIN column, the double-strand cDNA was ligated into the sfi I digested lambdaTripIEx2 vector and then the recombinant DNA was packaged in vitro. The unamplified human embryo nasopharynx cDNA library consists of 1.0x10(6) independent clones in which the percentage of recombinant clones is about 96%. The titer of the amplified cDNA library is 7.8x10(9) pfu/ml and the average exogenous inserts of the recombinants is 1.2 kb. The full-length cDNA of NAG4, a candidate tumor suppressor gene related with nasopharyngeal carcinoma, was amplified in the cDNA library by PCR. These results show that the human embryo nasopharynx cDNA library has an excellent quality and lays solid foundation for screening and cloning new tumor suppressor genes of nasopharyngeal carcinoma and tissue-specific genes of human nasopharynx.