人食管癌cDNA酵母杂交文库的构建与鉴定及其应用

Construction and Identification and Application of cDNA Library From an Esophageal Cancer Cell Line for Yeast-Hybrid System

以往在研究由促癌物 12 o 十四烷酰佛波醇 13 乙酯 (12 o tetradecanoylphorbol 13 acetate,TPA)诱导的人永生化食管上皮细胞恶性变中基因的差异表达情况时 ,曾获得中性粒细胞明胶酶相关lipocalin (neutrophilgelatinase associatedlipocalin ,NGAL)等新的食管癌癌变相关基因 .为深入研究这些癌变相关基因在食管癌中以蛋白质 蛋白质或蛋白质 DNA相互作用为基础的功能网络调控关系 ,建立了一个食管癌cDNA酵母杂交文库 .采用Trizol试剂从一种新的人食管癌细胞 (SHEEC ,由人永生化食管上皮细胞转化而来 )中提取细胞总RNA ,采用OligotexmRNAKit从细胞总RNA中制备PolyA+ mRNA ,采用SuperScriptTM ChoiceSystemForcDNASynthesisKit合成cDNA ,以PolyA+ mRNA为探针 ,化学发光法监测cDNA合成的质量 ,以 pGADT7为载体构建了SHEEC细胞的cDNA酵母杂交文库 .文库的滴度为 1 19× 10 9cfu/ml,重组片段大小主要集中在 0 5～6 0kb ,重组率为 5 0 % .在此基础上 ,应用酵母单杂交技术手段对该文库中的NF κB元件结合因子进行了筛选 ,在SD/ his/ leu/[+15mmol/L 3 AT ]缺陷培养基平板上共获得了约 36 0个单克隆 .按照平板上酵母单克隆直径的大小 ,选择直径大于 2mm者 91个 ,提取质粒进行酶切鉴定和一对一酵母单杂交验?

Many new carcinogenesis-related genes of esophageal cancer, including neutrophil getatinase-associated lipocalin (NGAL), had been cloned when the differentially expressed genes in the process of the esophageal epithelial cell transforming to carcinoma were being looked for. In these genes there might be some control mechanisms of functional networks. In order to further study the network relationships of the genes, a cDNA library from a esophageal cancer cell line (SHEEC), which had been established, for yeast-hybrid system wilt be constructed. Total RNA was extracted from SHEEC by Trizol reagent and PolyA(+) rnRNA was purified from total RNA by Oligotex mRNA Kit. cDNA was synthesized using SuperScript(TM) Choice System For cDNA Synthesis Kit. The quality of synthesized cDNA was detected by chemical tight method using PolyA(+) mRNA probe. The double-strand cDNA was ligated into the EcoR I site of pGADT(7) vector and the cDNA library from the esophageal cancer cell line for yeast-hybrid system was constructed. The titer of the amplified cDNA library was 1.19 x 10(9) cfu/ml, The inserted fragment size of recombinants was from 0.5 kb to 6.0 kb and the percentage of recombinant clones was about 50%. NF-kappaB element binding factors were screened from this cDNA library by yeast one-hybrid technic and more than 360 clones were obtained on the SD/-his/-leu/[ + 15 mmol/L 3-AT]. Plasmids of 91 clones which were larger than 2 mm in diameter were isolated from yeast and transformed to E. coli 30 positive recombinants had been obtained since plasmids were extracted from E. coli and digested by EcoR I and validated by yeast one-hybrid assay, 9 clones of them were sequenced and the sequence was compared with GenBank/BLAST database. Results showed that protein product of some genes obviously conformed to consensus sequence of NF-kappaB element binding domain of p65 or p50 in the key sites of amino acid residues. These results determined that the human esophageal cancer cDNA library for yeast-hybrid system had a higher quality.