CAJ | 학술논문

【目的】构建人脑源性神经营养因子（BDNF）基因真核表达载体，并在小鼠耳蜗毛细胞与螺旋神经节细胞（SGCs）表达。【方法】应用RT‐PCR从人外周血中扩增BDNF基因开放阅读框（ORF），采用 TA克隆技术，将目的片段插入到pUCm‐T载体中进行鉴定，重组的质粒命名为pUCm‐T‐BDNF。随后，将BDNF进一步克隆到真核表达载体pT racer‐CM V／Bsd中。用脂质体将经过测序、验证的重组pT racer‐CM V／Bsd‐BD‐NF质粒转染小鼠耳蜗SGCs细胞，荧光显微镜下观察绿色荧光蛋白的表达，转染SGCs细胞，经杀稻瘟菌素（Blasticidin）筛选4周，用Western blot检测BDNF的表达。【结果】成功构建了pTracer‐CMV／Bsd‐BDNF真核表达质粒，转染SGCs细胞，发现转染成功的细胞均发绿色荧光；Western blot检测结果表明，所转染的BD‐NF在SGCs细胞中成功表达。【结论】成功构建了能够同时表达绿色荧光蛋白和BDNF的真核表达质粒pTracer‐CMV／Bsd‐BDNF ，为进一步研究BDNF在内耳基因治疗中的作用奠定了基础。
【목적】구건인뇌원성신경영양인자（BDNF）기인진핵표체재체，병재소서이와모세포여라선신경절세포（SGCs）표체。【방법】응용RT‐PCR종인외주혈중확증BDNF기인개방열독광（ORF），채용 TA극륭기술，장목적편단삽입도pUCm‐T재체중진행감정，중조적질립명명위pUCm‐T‐BDNF。수후，장BDNF진일보극륭도진핵표체재체pT racer‐CM V／Bsd중。용지질체장경과측서、험증적중조pT racer‐CM V／Bsd‐BD‐NF질립전염소서이와SGCs세포，형광현미경하관찰록색형광단백적표체，전염SGCs세포，경살도온균소（Blasticidin）사선4주，용Western blot검측BDNF적표체。【결과】성공구건료pTracer‐CMV／Bsd‐BDNF진핵표체질립，전염SGCs세포，발현전염성공적세포균발록색형광；Western blot검측결과표명，소전염적BD‐NF재SGCs세포중성공표체。【결론】성공구건료능구동시표체록색형광단백화BDNF적진핵표체질립pTracer‐CMV／Bsd‐BDNF ，위진일보연구BDNF재내이기인치료중적작용전정료기출。
[Objective] To construct an eukaryotic expression vector of human brain‐derived neurotrophic factor (BDNF) and explore its expression in spiral ganglion cells (SGCs) of cochlear in mice .[Methods] The open reading frame (ORF) of BDNF was amplified from human peripheral blood by reverse transcription‐poly‐merase chain reaction (RT‐PCR) .TA cloning strategy was employed to insert target fragments into pUCm‐T vector .The recombinant plasmid was identified and noted as pUCm‐T‐BDNF .Then BDNF was subcloned into pTracer‐CMV/Bsd ,an eukaryotic expression vector .The plasmid of pTracer‐CMV/Bsd‐BDNF was sequenced and introduced into SCGs of cochlear by LipofectamineTM 2000 .The expression of green fluorescence protein (GFP) was observed by fluorescence microscope .The expression of BDNF was detected by Western blot after screening by blasticidin for 4 weeks .[Results]The eukaryotic expression plasmid pTracer‐CMV/Bsd‐BDNF was successfully constructed .GFP was found in transfected cells by fluorescence microscope .And the expres‐sion of BDNF was detected in transfected cells by Western blot .[Conclusion] The recombinant eukaryotic ex‐pression vector of pTracer‐CMV/Bsd‐BDNF has been constructed successfully .It can co‐express GFP and BD‐NF so as to serve as a tool for further gene therapy study .