β-Trcp载体构建及其应用

Construction of β-Trcp expression vector and its application

目的:构建pc DNA3.1-β-Trcp真核表达质粒及鉴定,检测上调β-Trcp对胃癌细胞AGS的β-catenin表达量和亚细胞定位的影响。方法:应用PCR方法钓取β-Trcp片段与pc DNA3.1载体连接,鉴定重组质粒pc DNA3.1-β-Trcp。在胃癌细胞AGS中分别转染pc DNA3.1和pc DNA3.1-β-Trcp,利用Real-time PCR和Western blot方法检测β-Trcp表达情况,免疫荧光检测胃癌细胞ACS中,β-catenin表达量和亚细胞定位的变化。结果:pc DNA3.1-β-Trcp重组质粒构建成功,并在胃癌细胞AGS中成功表达。转染pc DNA3.1-β-Trcp质粒的胃癌细胞AGS中β-catenin整体表达量下调,在细胞核内的表达量下调尤为明显。结论:pc DNA3.1-β-Trcp质粒为进一步研究β-Trcp在胃癌中的作用提供实验基础。β-Trcp抑制胃癌细胞AGS的β-catenin表达,核内抑制尤为明显。

Objective： To construct an eukaryotic expression vector pcDNA3.1-Trcp and study its effect on the expression and localization of β-catenin. Methods： The full length of β-Trcp cDNA was amplified from colorectal cancer cell by RT-PCR and cloned into empty vector pcDNA3.1, which was identified by restriction enzyme digestion and DNA sequencing. The expression of β-Trcp was examined by real-time and Western blot in AGS cells transfectd with pCDNA3.1 or pCDNA3.1-β-Trcp, and its effect on the expression and subcellular localization of β-catenin was detected by immunofluorescence staining. Results： The recombinant plasmid pcDNA3.1-β-Trcp was successfully constructed and expressed in gastric cancer cell. β-Trcp overexpression decreased the expression of β-catenin, especially within the nucleus. Conclusion： The vector pcDNA3.1-β-Trcp provides a basic tool for further study the function of β-Trcp in gastric cancer. β-Trcp inhibits the expression of β-catenin, especially within the nucleus.