BTF红色荧光蛋白表达载体的构建及其在大鼠血管平滑肌细胞中的表达和定位研究  

Constructionof BTF Red Fluorescent Protein Expression Plasmid and Its Expression in Rat VSMCs

作　　者：马云[1] 王昌博[1] 杨满君 秦凌雪[1] 李斌元[1] 何淑雅[1] 

机构地区：[1]南华大学生物化学与分子生物学研究所,湖南省衡阳市421001

出　　处：《医学分子生物学杂志》2014年第3期161-164,共4页Journal of Medical Molecular Biology

基　　金：国家自然科学基金青年项目（No.81200881）,湖南省自然科学基金（No.12JJ6073）

摘　　要：目的构建Bcl-2相关转录因子1（BTF）与红色荧光蛋白（RFP）融合表达载体并在大鼠血管平滑肌VSMC细胞中表达，为进一步研究BTF的相互作用蛋白及作用机制奠定实验基础。方法以人体外周血白细胞cDNA为模板，PCR扩增Btf基因，插入红色荧光蛋白表达载体pDsRed-N1中．构建重组表达载体pDsRed-N1-Btf，转染大鼠血管平滑肌VSMC细胞，通过激光共聚胶显微镜观察其在转染细胞中的表达及定位。结果DNA测序、酶切鉴定的结果显示，红色荧光蛋白表达载体pDsRed-N1-Btf构建成功，且序列正确。转染后经激光共聚胶显微镜观察到VSMC细胞中有红色荧光。且BTF主要定位于细胞质中。结论成功构建了pDsRed-N1-Btf表达载体，并主要定位于VSMC的胞质中，为后期研究BTF的相互作用蛋白及其与相互作用蛋白之间的作用机制奠定了良好的基础。Objective To construct the expression vector of Bcl-2-associated transcription factor 1 （BTF） fused to red fluorescent protein （RFP） and examine its expression in rat vascular smooth muscle cells （VSMCs） in order to provide an experimental foundation for further studying the interaction of BTF and other proteins in cells and the action mechanism. Methods Bff gene was amplified by PCR with the cDNA of peripheral white blood cells as the template, and inserted into the red fluorescent protein expression vector pDsRed-N1 to obtain the recombinant plasmid pDsRed- N1-Btf, which was then transfected into the VSMCs. The laser confocal microscopy was employed to investigate the expression and subcellular distribution of pDsRed-N1-Btf. Results Restriction enzyme analysis and sequencing showed that the red fluorescent protein expression vector pDsRed-N1- Btf was constructed successfully. The red fluorescent protein could be observed in VSMCs by laser confocal microscopy after the transfection, and BTF was mainly located in the cytoplasm. Conclusion The recombinant plasmid pDsRed-N1-Btf was successfully constructed, and it was predominantly located in the cytoplasm of VSMCs. This study lays a foundation for further studying the interaction of BTF and other proteins in cells and the action metabolism.

关 键 词：Bcl-2相关转录因子1 红色荧光蛋白 融合蛋白 

分 类 号：R34[医药卫生—基础医学]