【摘要】 【目的】以Split-GFP双荧光互补技术检测鸡肌肉生长抑制素基因（MSTN）RNA干涉（RNAi）效果，并与其他常用检测方法比较，以验证Split-GFP双分子荧光互补技术在RNAi效果评估中的有效性和可行性。【方法】将合成的3个shRNA慢病毒载体（shRNA-a、shRNA-b和shRNA-c）分别转染稳定表达GFP11-MSTN融合蛋白的HEK293T^GFP11-MSTN细胞，经实时荧光定量PCR筛选出最佳shRNA慢病毒载体并包装为慢病毒，然后以慢病毒感染HEK293T^GFP11-MSTN细胞，采用潮霉素B筛选m Cherry阳性（mCherry⁺）细胞，再以实时荧光定量PCR和Western blotting检测RNAi效果；得到的m Cherry⁺细胞再转染pcDNA3.1（+）-GFP1-10质粒，通过荧光显微镜观察和流式细胞术评估RNAi效果。【结果】3个shRNA慢病毒载体对MSTN基因表达均有极显著的抑制作用（P<0.01，下同），其中又以Anti-MSTN shRNA-a慢病毒载体的干涉效果最佳。Anti-MSTN shRNA-a慢病毒感染HEK 293T^GFP11-MSTN细胞的最适MOI=3，该条件下MSTN基因相对表达量及GFP11-MSTN融合蛋白表达量均受到抑制；得到的mCherry⁺细胞再转染pcDNA3.1（+）-GFP1-10质粒，荧光显微镜观察和流式细胞术检测结果表明，GFP⁺细胞数量明显减少，GFP⁺细胞百分率由31.1%降至11.5%。Split-GFP检测结果与实时荧光定量PCR及Western blotting检测结果相符，说明Anti-MSTN shRNA-a慢病毒能有效抑制GFP11-MSTN融合蛋白表达，发挥了RNAi作用。【结论】以Anti-MSTN shRNA-a慢病毒对MSTN基因的干涉效果最佳，其感染HEK 293T^GFP11-MSTN细胞后MSTN基因表达极显著下调，且再转染pcDNA3.1（+）-GFP1-10质粒后细胞中的GFP⁺细胞百分率明显下降，与实时荧光定量PCR和Western blotting检测结果相符，证实Slipt-GFP双分子荧光互补技术是一种可靠的可视化RNAi检测方法。更多还原

【Abstract】 【Objective】The purpose of this study was to detect the efficacy of RNA interference（RNAi）of chicken myostatin gene（MSTN）by Split-GFP bimolecular fluorescence complementary technique and to compare it with commonly used detection methods in order to validate the effectiveness and availability of the Split-GFP bimolecular fluorescence complementary technique in the assessment of RNAi efficacy.【Method】The three synthesized shRNA lentivira cloning vectors（shRNA-a,shRNA-b and shRNA-c）were respectively transfected to HEK 293T^GFP11-MSTNcells which stably expressed the GFP11-MSTN fusion protein.The optimal shRNA lentiviral cloning vector was screened by realtime fluorescence quantitative PCR and packaged as lentivirus.Subsequently,the HEK 293T^GFP11-MSTNcells were infected with the lentivirus,and mCherry positive（m Cherry⁺）cells were screened using hygromycin B,and the RNAi efficiency was detected by real-time fluorescence quantitative PCR and Western Blotting.The obtained mCherry⁺cells were transfected with a pcDNA3.1（+）-GFP1-10 plasmid,and the RNAi efficiency was assessed by fluorescence microscopy observation and flow cytometry.【Result】All of three shRNA lentiviral vectors had extremely significant inhibitory effects on MSTN gene expression（P<0.01,the same below）,among which Anti-MSTN shRNA-a lentiviral vector had the best interfering efficacy.Anti-MSTN shRNA-a lentivirus infected HEK 293T^GFP11-MSTNcells at the optimal MOI=3,and the relative expression of the MSTN gene and the expression of the GFP11-MSTN fusion protein were inhibited under this condition The obtained mCherry⁺cells were re-transfected with pcDNA3.1（+）-GFP1-10 plasmid,and both fluorescence microscopy observation and flow cytometry assay results showed that the number of GFP⁺cells was decreased greatly,and the percentage of GFP⁺cells was reduced from 31.1%to 11.5%.The results of Split-GFP assay were consistent with those of realtime fluorescence quantitative PCR and Western Blotting detections,indicating that Anti-MSTN shRNA-a lentivirus could inhibit GFP11-MSTN fusion protein expression,thus playing a RNAi role.【Conclusion】Anti-MSTN shRNA-a lentivirus has the highest interfering efficacy on MSTN gene,which extremely significantly down-regulates the expression of MSTN gene after infection of HEK 293T^GFP11-MSTNcells,and the percentage of GFP⁺cells in the cells is greatly decreased after re-transfection of pcDNA3.1（+）-GFP1-10 plasmid,which is consistent with the results of real-time fluorescence quantitative PCR and Western blotting detection results,confirming that Slipt-GFP bimolecular fluorescence complementary technique is a dependable and visualized method for RNAi detection.更多还原