自噬检测质粒pLVX-mRFP-EGFP-LC3的构建和表达鉴定

Construction and Expression Identification of Autophagy Detection Plasmid pLVX-mRFP-EGFP-LC3

细胞自噬的机体重要的一种代谢过程,为了更好地研究不同条件下细胞自噬的水平,构建自噬检测质粒pLVX-mRFP-EGFP-LC3。使用Trizol法提取Hela细胞总RNA,PCR技术特异性扩增LC3基因,将目的片段插入pEGFP-N3载体中。再将mRFP片段插入到EGFP-LC3质粒中,利用PCR技术扩增带有不同酶切位点的mRFP-EGFP-LC3片段,插入到慢病毒载体pLVX-puro中。通过Fugene 6将质粒瞬时转染到293T细胞中,使用荧光显微镜检测荧光蛋白的表达,使用Western blotting检测目的基因的表达。结果显示成功构建自噬检测质粒pLVX-mRFP-EGFP-LC3,荧光显微镜下可见红色和绿色荧光,Western blotting方法检测到LC3的表达。

The autophagy is an important metabolic process.In order to better study autophagy under different conditions,an autophagy detection plasmid pLVX-MRFP-EGFP-LC3 was constructed.The total RNA of Hela cells was extracted by Trizol.LC3 fragment was specifically amplified by PCR technique,and then inserted into the plasmid pEGFP-N3 vector.The mRFP fragment was inserted into the EGFP-LC3 plasmid,and the mRFP-EGFP-LC3 fragment with different restriction sites was amplified by PCR and inserted into the lentiviral vector pLVX-puro.The plasmid was transiently transfected into 293T cells with Fugene 6.The expression of the fluorescent protein was detected by fluorescence microscope.The expression of the target gene was detected by western blotting.The results showed that the autophagy detection plasmid pLVX-mRFP-EGFP-LC3 was successfully constructed.Red and green fluorescence were observed under fluorescence microscope.The expression of LC3 was detected by western blotting.