| 王小毅,马晓骊,陈 虹,黄秉仁,陈 等.基于VN210/VC210的双分子荧光互补定量分析cofilin-actin相互作用的特异性[J].现代生物医学进展英文版,2022,(4):601-605. |
| 基于VN210/VC210的双分子荧光互补定量分析cofilin-actin相互作用的特异性 |
| Quantitatively Analyzing the Specific Interaction of Cofilin-actin by VN210/VC210-based Bimolecular Fluorescence Complementation Technique |
| Received:June 06, 2021 Revised:June 30, 2021 |
| DOI：10.13241/j.cnki.pmb.2022.04.001 |
| 中文关键词: 双分子荧光互补 定量分析 cofilin-actin相互作用 |
| 英文关键词: Bimolecular Fluorescence Complementary Quantitative analysis Cofilin-actin interaction |
| 基金项目:国家自然科学基金项目(81772986) |
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| 中文摘要: |
| 摘要 目的：利用双分子荧光互补技术定量分析cofilin-actin的特异性相互作用。方法：首先构建表达VN210（编码荧光蛋白Venus 1-210氨基酸）和VC210（编码荧光蛋白Venus 210-238氨基酸）探针的质粒，通过梯度剂量转染检测该探针对的自组装能力；其次构建VN210/VC210与bJun、bFos、Fos△zip、cofilin（WT）、cofilin （S3E）和actin的融合表达载体，在HeLa细胞中分别过表达不同载体组合，以VN210-bJun/bFos-VC210组作为阳性对照，以VN210-bJun/bFos△zip-VC210组作为阴性对照，使用多功能细胞观测显微镜观察并拍摄荧光图像；最后使用多功能微孔板检测仪，对对照组进行波谱扫描，确定最适合检测的激发光波长和发射光波长，再以此选择波长检测实验组中可观察到荧光信号组合的荧光强度，进行统计分析。结果：(1) VN210/VC210在各转染剂量组中均未观察到荧光信号；(2) 阳性对照组可观察到荧光信号，阴性对照组未观察到荧光信号；实验组中，VN210-cofilin(WT)/actin-VC210组荧光信号较强，VN210-cofilin(S3E)/actin-VC210和VN210/actin-VC210组荧光信号较弱，其余组均观察不到荧光信号；(3) 产生荧光信号的实验组中，VN210-cofilin(S3E)/actin-VC210和VN210/actin-VC210组间的荧光信号无显著差异，但分别与VN210-cofilin(WT)/actin-VC210组具有显著差异。结论：VN210/VC210双分子荧光互补技术可定量检测cofilin-actin的特异性相互作用。 |
| 英文摘要: |
| ABSTRACT Objective: To quantitatively analyze the specific interaction of cofilin-actin using bimolecular fluorescence complimentary technique. Methods: Firstly, the plasmids expressing VN210 (coding fluorescent protein Venus 1-210 amino acids) and VC210 (coding fluorescent protein Venus 210-238 amino acids) probes were constructed, and their self-assembly ability was detected by gradient dose of transfection experiment. Then, the fusion expression vectors of VN210/VC210 with bJun, bFos, bFos△zip, cofilin(WT), cofilin(S3E) and actin were constructed. After co-transfections of the different pairs of vector combinations into HeLa cells, their fluorescent signals were measured. Among the combinations, VN210-bJun/bFos-VC210 was used as a positive control and VN210-bJun/bFos△zip-VC210 as a negative control. The fluorescent signals were observed and recorded by the multifunctional cell observation microscope. Finally, the microplate detector was used to scan the spectrum of the control group to determine the optimal excitation and emission wavelengths, and the differences of the fluorescence signals observed in the experimental groups were statistically analyzed. Results: 1 Fluorescent signals were not observed in VN210/VC210 group under various plasmid transfection doses. 2 In the control groups, the fluorescence signals were observed in VN210-bJun/bFos-VC210 group but not in VN210-bJun/bFos△zip-VC210 group. In the experimental groups, the fluorescence signal of VN210-cofilin (WT)/actin-VC210 group was stronger, while those of VN210-cofilin (S3E)/actin-VC210 and VN210/actin-VC210 groups were weaker, and fluorescence signals were not observed in other groups. 3 There was no difference of fluorescence signals between VN210-cofilin (S3E)/actin-VC210 and VN210/actin-VC210 groups, but a significant difference between VN210-cofilin (WT)/actin-VC210 and VN210-cofilin (S3E)/actin-VC210. Conclusion: The specific interaction between cofilin and actin can be quantitatively detected by VN210/VC210 bimolecular fluorescence complementation. |
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