Split-GFP双分子荧光互补技术在鸡MSTN基因RNAi检测中的应用  

Application of Split-GFP bimolecular fluorescence complemen⁃tary technique in detecting RNAi of chicken MSTN gene

作　　者：杨磊[1] 朱正 王博永 梁谦学 吴文德[1] 李恭贺[1] 郑喜邦[1] YANG Lei;ZHU Zheng;WANG Bo-yong;LIANG Qian-xue;WU Wen-de;LI Gong-he;ZHENG Xi-bang(College of Animal Science and Technology,Guangxi University,Nanning,Guangxi 530004,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004

出　　处：《南方农业学报》2023年第8期2444-2453,共10页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32060738,31660653);广西自然科学基金重点项目(2018GXNSFDA281026);广西重点研发计划项目(桂科AB16380098)。

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HEK 293TGFP11-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HEK 293TGFP11-MSTN细胞,采用潮霉素B筛选mCherry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mCherry+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 293TGFP11-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 293TGFP11-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。【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 lentiviral cloning vectors(shRNA-a,shRNA-b and shRNA-c)were respectively transfected to HEK 293TGFP11-MSTN cells 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 293TGFP11-MSTN cells were infected with the lentivirus,and mCherry positive(mCherry+)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 293TGFP11-MSTN cells 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 c

关 键 词：肌肉生长抑制素(MSTN) RNA干涉(RNAi) shRNA 慢病毒 Split-GFP双分子荧光互补技术 

分 类 号：S814.8[农业科学—畜牧学]