摘要
目的构建人源组蛋白H2A.X和核仁磷蛋白NPM1哺乳动物双杂交表达载体,并实现其在乳腺癌MCF-7细胞中的表达。方法PCR扩增H2A.X及NPM1基因表达序列,TA克隆后连接至p BIND和PACT载体,双酶切和测序验证后转染MCF-7细胞,RT-PCR检测表达效果。结果PCR结果产生与H2A.X和NPM1编码序列大小一致DNA片段;目的片段的TA,p BIND,PACT克隆及双酶切验证后均产生预期大小DNA片段;测序结果表明所构建的H2A.X-pBIND和NPM1-PACT产生的DNA序列同H2A.X和NPM1编码序列完全匹配;转染H2A.X-pBIND和NPM1-PACT表达载体的细胞相应mRNA表达水平明显增加。结论成功构建H2A.X和NPM1哺乳动物双杂交表达载体并实现其在乳腺癌MCF-7细胞中的表达,为研究乳腺癌中二者的相互作用及功能提供实验基础。
Objective To construct human histone H2 A.X(H2 A.X)and nucleophosmin(NPM1)mammalian two-hybrid vectors and to realize their expression in MCF-7 cells.Methods H2 A.X and NPM1 coding sequences were amplified by PCR and cloned into p BIND and PACT vectors via TA cloning and validated by restriction enzyme mapping and sequencing.MCF-7 cells were then transfected with H2 A.X-pBIND and NPM1-PACT.Overexpression of H2 A.X and NPM1 was detected using RT-PCR method.Results PCR amplified the expected H2 A.X and NPM1 coding sequence fragments.Restriction mapping of TA,p-BIND and PACT clones produced the predicted DNA bands.Sequencing showed the two vector constructs to completely align with H2 A.X and NPM1 sequences.mRNA levels of H2 A.X and NPM1 were increased in cells transfected with H2 A.X-pBIND and NPM1-PACT,respectively.Conclusions Human H2 A.X and NPM1 mammalian two-hybrid vectors were successfully constructed and expressed in MCF-7 cells.
作者
高秀丽
段文博
王婧
王涛
任珊
GAO Xiuli;DUAN Wenbo;WANG Jing;WANG Tao;REN Shan(Research Institute of Medicine and Pharmacy,Qiqihar Medical University,Qiqihar 161006,China;Department of Medical Technology,Qiqihar Medical University,Qiqihar 161006)
出处
《中国比较医学杂志》
CAS
北大核心
2021年第2期24-29,共6页
Chinese Journal of Comparative Medicine
基金
国家自然科学基金(81802834,81972491)
黑龙江省教育厅青年创新人才项目(UNPYSCT-2020085)。
