【摘要】 目的构建带绿色荧光蛋白报告载体(pEGFP-C1)的死骨片1(SQSTM1/P62)真核表达载体,在获得pEGFPSQSTM1融合表达蛋白后,对其功能进行验证。方法以人乳腺文库为模板,通过聚合酶链反应(PCR)扩增人SQSTM1基因编码序列,插入到pEGFP-C1载体中,把测序正确的重组质粒转染人胚肾293T细胞,蛋白质印迹法(Western blotting)检测该融合蛋白表达情况;分别转染pEGFP-C1空载体、pEGFP-SQSTM1至乳腺癌细胞ZR75-1,荧光显微镜观察SQSTM1蛋白在细胞中的定位情况;荧光漂白后恢复实验(FRAP)验证乳腺癌细胞ZR75-1中SQSTM1小体荧光漂白后恢复情况。结果重组质粒pEGFP-SQSTM1的基因序列与目的序列完全一致,Western印迹检测蛋白表达成功;荧光显微镜观察SQSTM1的表达,结果显示,表达产物主要以点状小体形式定位于乳腺癌ZR75-1细胞的胞质中;FRAP实验验证乳腺癌细胞ZR75-1中SQSTM1小体激光漂白后绿色荧光可恢复。结论成功构建pEGFPSQSTM1真核表达载体,证实了SQSTM1蛋白在人乳腺癌细胞ZR75-1中表达,表达蛋白以点状小体形式定位于细胞质中,为进一步研究SQSTM1小体的功能提供基础。更多还原

【Abstract】 Objective To construct the eukaryotic expression vector of sequestosome 1(SQSTM1/P62)with green fluorescent protein reporter(pEGFP-C1)and to verify the function of pEGFP-SQSTM1 protein. Methods Using the human mammary library as the template,the SQSTM1 gene was amplified by polymerase chain reaction(PCR)and inserted into pEGFP-C1 vector. The recombinant plasmid was transfected into human embryonic kidney 293 T cells after confirming the sequence. The expression of the pEGFP-SQSTM1 protein was detected by Western blotting. pEGFP-C1 empty vector and pEGFP-SQSTM1 were transfected into breast cancer cell ZR75-1 respectively. The localization of SQSTM1 protein in breast cancer cells was observed by fluorescence microscopy. The recovery of SQSTM1 body was confirmed after fluorescence bleaching of SQSTM1 body in breast cancer cells by the technique of fluorescence recovery after photobleaching(FRAP). Results The gene sequence of recombinant plasmid pEGFP-SQSTM1 was identical with the target sequence.The expression of SQSTM1 protein was successfully detected by Western blotting. Fluorescence microscopy demonstrated that the localization of SQSTM1 protein was expressed in the cytoplasm in the form of scattered bodies in breast cancer ZR75-1 cell line. FRAP assay indicated that the fluorescence of SQSTM1 body could be recovered in breast cancer cells after bleaching. Conclusion The eukaryotic expression vector of pEGFP-SQSTM1 is successfully constructed. The expression of SQSTM1 protein in human breast cancer cell ZR75-1 is confirmed and the expressed product is localized in the form of scattered bodies(sequestosome)in the cytoplasm,which can contribute to sequestosome functional studies.更多还原