| 摘要: |
| 【目的】构建能够在神经系统中高效表达的小鼠cofilin-1野生型(Cfl1-wt)、活化型(Cfl1-S3A)、失活型(Cfl1-S3D)3种真核表达载体，并研究其在鸡胚脊髓神经元中的表达情况，为进一步探讨cofilin-1在大脑发育过程中对神经元迁移的影响奠定基础。【方法】从成年小鼠大脑组织中提取RNA，经反转录获得cDNA样本，RT-PCR扩增小鼠cofilin-1基因的CDS区，经引物设计引入点突变，扩增了野生型、活化型及失活型的cofilin-1基因片段，克隆入pCAG-MCS-myc真核表达载体中。经双酶切和测序鉴定后，转染CHO细胞，通过半定量RT-PCR和免疫荧光染色检测其在细胞中的表达情况。利用鸡胚活体原位电转的方法，将重组质粒转染胚胎发育第3天(E3)的鸡胚脊髓，在胚胎发育第6天（E6）取材，切片后经免疫荧光染色观察并统计分析重组质粒在脊髓中的表达情况。【结果】克隆并模拟了小鼠cofilin-1野生型、活化型和失活型的cDNA片段，成功构建了pCAG-cfl1-wt、pCAG-cfl1-S3A和pCAG-cfl1-S3D 3种真核表达载体。体外转染CHO细胞，半定量RT-PCR检测及统计学分析表明，转染重组质粒的CHO细胞中，cofilin-1 mRNA相对表达量与对照组（未转染质粒的正常CHO细胞）存在显著性差异；免疫荧光染色后观察到了cofilin-1融合蛋白的表达。对经活体原位电转转染重组质粒的鸡胚脊髓切片进行免疫荧光染色，检测到cofilin-1融合蛋白的表达，且重组质粒转染效果与pCAG-EGFP转染效果无明显差异。【结论】pCAG-cfl1-wt、pCAG-cfl1-S3A和pCAG-cfl1-S3D 3种真核表达载体的成功构建及其在体内外的高效表达，为进一步研究cofilin-1及其Ser 3位点的磷酸化在神经元迁移中的作用机制奠定了基础。 |
| 关键词: cofilin-1 Ser 3 磷酸化 去磷酸化 质粒构建 活体原位电转 |
| DOI： |
| 分类号: |
| 基金项目:西北农林科技大学人才专项基金项目（Z111021101） |
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| Construction of mouse cofilin-1 gene eukaryotic vectors and their expression in vitro and in vivo |
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LI Ling-ling,ZHANG Wei,DU Rui,et al
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| Abstract: |
| 【Objective】This study constructed eukaryotic vectors that could highly express mouse cofilin-1 wildtype (Cfl1-wt),continuing activated (Cfl1-S3A) and continuing inactivating (Cfl1-S3D) in neurons and detected their expressions in chick spinal cord to improve the understanding of the role of cofilin-1 in neuronal migration during brain development.【Method】Total cDNA was reverse transfected from the RNA collected from adult mouse brain.Mouse cofilin-1 genetic fragment was amplified and introduced into pCAG-MCS-myc vector.After being confirmed by EcoRⅠand BglⅡ digestion analyses and DNA sequencing,the recombinant pCAG-cfl1-myc plasmids were transfected into CHO cells.The expressions of the recombinant plasmids were detected by RT-PCR and immunofluorescence.Furthermore,in ovo culture technique was used to transfer the recombinant plasmids into spinal cord of chick until the embryos developed to E3 and the expression levels of the plasmids in neuron at E6 were detected.【Result】pCAG-cfl1-wt,pCAG-cfl1-S3A and pCAG-cfl1-S3D eukaryotic vectors were constructed successfully.After transfecting CHO cells in vitro,the expression of cofilin-1 at mRNA level was detected by semi-quantitative RT-PCR and it was significantly different between control and treatment groups.The expression of cofilin-1 through immunofluorescence in transfected CHO cells was also observed.In ovo electroporation to developmental spinal cord in chick embryo proved that the recombinant plasmids could express in neurons and their expressions were almost same as pCAG-EGFP.【Conclusion】The recombinant plasmids pCAG-cfl1-wt,pCAG-cfl1-S3A and pCAG-cfl1-S3D were constructed and expressed successfully,which laid foundation for further studies on the role of cofilin-1 and phosphorylation in Ser 3 during neuronal migration. |
| Key words: cofilin-1 Ser3 phosphorylation dephosphorylation plasmid constructed in ovo electropration |
