CAJ | 학술논문

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。
목적：구건Bcl-2상관전록인자1（BTF）여홍색형광단백（RFP）융합표체재체병재대서혈관평활기VSMC세포중표체，위진일보연구BTF적상호작용단백급작용궤제전정실험기출。방법이인체외주혈백세포cDNA위모판， PCR확증Btf기인，삽입홍색형광단백표체재체pDsRed-N1중，구건중조표체재체pDsRed-N1-Btf，전염대서혈관평활기VSMC세포，통과격광공취효현미경관찰기재전염세포중적표체급정위。결과 DNA측서、매절감정적결과현시，홍색형광단백표체재체pDsRed-N1-Btf구건성공，차서렬정학。전염후경격광공취효현미경관찰도VSMC세포중유홍색형광，차BTF주요정위우세포질중。결론성공구건료pDsRed-N1-Btf표체재체，병주요정위우VSMC적포질중，위후기연구BTF적상호작용단백급기여상호작용단백지간적작용궤제전정료량호적기출。
Objective To construct the expression vector of Bcl-2-associated transcription fac-tor 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 Btf 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 en-zyme 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 cyto-plasm.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 stud-ying the interaction of BTF and other proteins in cells and the action metabolism .