摘要
目的构建重组质粒TAZ-pcDNA3.1及TAZ-pEGFP-C2,并应用Western Blot检测TAZ蛋白在细胞内的表达情况,初步探索TAZ分子促进细胞增殖和迁移的作用机制。方法通过PCR扩增获得TAZ基因片段,胶回收后连接T载体,蓝白斑筛选,转化,提质粒,酶切,用T4DNA Ligase连接,亚克隆进入pEGFP-C2和pcDNA3.1获得新的重组质粒,分别转染HEK293细胞,智能型荧光细胞监测仪观察TAZ分子在细胞内的分布情况,Western Blot检测其在细胞内的表达情况。结果重组质粒经双酶切鉴定和测序证明构建成功,荧光照片显示TAZ分子分布在细胞核和细胞浆中,Western Blot检测结果表明转染有重组质粒TAZ-pcDNA3.1的HEK293细胞中有大量TAZ蛋白表达。结论成功构建了两种重组质粒,并初步验证了TAZ蛋白的细胞定位,为进一步研究其促进增殖和迁移的作用机制奠定了基础。
Objective Two recombinant plasmids,TAZ-pcDNA3.1and TAZ-pEGFP-C2,were established.The protein expression of TAZ in HEK293 cells was detected by Western Blot and the roles of TAZ in promoting cell proliferation and migration were further explored.Methods AZ gene was amplified by PCR,fragments were recovered followed by connection with glue T carrier,blue-white screening,transformation and extraction of plasmid DNA.Then the plasmid DNA was digested,connected by T4 DNA Ligase,and then sub-cloned into pEGFP-C2 and pcDNA3.1 to construct new recombinant plasmids.These plasmids were transfected into HEK293 cells to observe the distribution of TAZ using a fluorescence detector.The protein expression was detected by Western Blot.Results By restriction enzyme identification and sequence analysis,the recombinant plasmids were successfully constructed.Fluorescent photos show that the distribution of TAZ molecule was in the nucleus and cytoplasm.Western Blot test results showed that TAZ molecule could induce over-expression of specific proteins.Conclusion Two recombinant plasmids were successfully constructed.The effects of TAZ overexpression were validated,which will lay a foundation for revealing the mechanism of TAZ in promoting cell proliferation and migration.
出处
《河南大学学报（医学版）》
CAS
2015年第2期111-114,共4页
Journal of Henan University:Medical Science
基金
河南大学校内科研基金(2011YBZR007)
