论文部分内容阅读
目的 筛选胃癌下调基因 ,确定其表达产物的组织分布和细胞定位 .方法 从已成功建立的 5人份正常胃粘膜 m RNA(Tester)抑制消减杂交胃癌 m RNA (Driver)的差异表达文库 ,随机挑取阳性克隆测序为 CA11,RT- PCR证实其为下调基因 .地高辛标记 CA11原位 m RNA杂交 .在其他组织器官中扩增此基因 ,以表明其组织分布特异性 .CA11克隆至载体 pc DNA3.1/Myc- His(- ) A中 ,并瞬时转染 CA11的 COS- 7细胞 .生长至对数生长期时 ,吸取培养液 ,Talon吸附、洗脱后 ,取洗脱液进行SDS蛋白电泳及 Western blot.CA11转染 COS- 7细胞后加抗 Myc标签的一抗 ,最后加 FITC标记的二抗在荧光显微镜下观察 .结果 筛选到胃癌下调基因 CA11,并经 RT- PCR证实 .m RNA原位杂交显示 ,CA11位于胃粘膜上皮细胞 .在其他组织器官 c DNA文库及组织 RNA中均未扩增出该基因 .洗脱液 Western blot未检测到 CA11与 pc DNA3.1/ Myc- His (- ) A的 Myc融合表达蛋白存在 ;CA11表达蛋白亚细胞定位于细胞质 .结论 CA11为一胃癌下调基因 ,它在胃粘膜上皮细胞特异表达 ,表达产物定位于细胞质
Objective To screen down-regulated genes of gastric cancer and determine the tissue distribution and cellular localization of the expressed products.Methods The differential expression libraries of hybridized gastric cancer m RNA were successfully cloned from 5 human normal gastric mucosa m RNAs (Tester) The positive clone was sequenced as CA11 and confirmed by RT-PCR as a down-regulated gene.The digoxigenin-labeled CA11 in situ m RNA was hybridized to amplify this gene in other tissues and organs to show its tissue-specific distribution.CA11 was cloned into vector pc DNA3.1 / Myc-His (-) A, and transiently transfected into COS-7 cells of CA11.When grown to logarithmic growth phase, the culture medium was aspirated, Talon was adsorbed and eluted, and the eluate was used to carry out SDS protein Electrophoresis and Western blot.CA11 transfected COS-7 cells with anti-Myc tag primary antibody, and finally added FITC-labeled secondary antibody observed under a fluorescence microscope.Results Screened to gastric cancer gene CA11 and confirmed by RT-PCR.m RNA in situ hybridization showed that CA11 was located in gastric mucosal epithelial cells, which was not found in other tissues and organs of c-DNA and tissue RNA.Western blotting did not detect CA11 and pcDNA3.1 / Myc-His (-) A Myc financial And the expression of CA11 protein was localized in the cytoplasm.Conclusion CA11 is a gastric cancer down-regulated gene that is specifically expressed in gastric mucosal epithelial cells and localized in the cytoplasm