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目的构建NEP1-40(Nogo extra cellular peptide residues 1-40)基因慢病毒表达载体,为后续转染目的细胞奠定基础,并实现在细胞中高效、稳定表达。方法从含有NEP1-40基因的cDNA文库中,利用PCR方法钓取NEP1-40基因编码区片段。将目的基因与酶切线性化的载体pGC-FU进行定向连接,其产物转化细菌感受态细胞。对长出的克隆先进行菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析,比对正确的克隆即为构建成功的目的质粒。将构建成功的目的质粒和两种辅助包装质粒共转染293T细胞,包装成慢病毒,荧光显微镜下观察慢病毒转染293T细胞后荧光表达情况,采用Western blot检测NEP1-40及绿色荧光蛋白融合蛋白表达情况。结果 PCR产物经电泳分析表明成功获取NEP1-40基因cDNA克隆,测序提示慢病毒转染质粒连接构建正确;荧光表达检测显示293T细胞中产生慢病毒颗粒;Western blot显示NEP1-40在细胞内稳定表达。结论成功构建NEP140基因慢病毒表达载体,为后续转染目的细胞后从分子水平探讨NEP1-40基因功能奠定了实验基础。
Objective To construct lentivirus expression vector of NEP1-40 (Nogo extra cellular peptide residues 1-40) gene and lay a foundation for subsequent transfection of target cells and to achieve efficient and stable expression in cells. Methods The cDNA fragment encoding NEP1-40 gene was amplified from the cDNA library containing NEP1-40 gene by PCR. The target gene was ligated with the vector pGC-FU, which was linearized by restriction endonucleases, and its product was transformed into bacterial competent cells. The colonies were cloned first colony PCR identification, and then PCR positive clones were sequenced and compared analysis, the correct clone is the construction of a successful plasmid. 293T cells were co-transfected with the constructed plasmids and the two helper plasmids and then packaged into lentivirus. The fluorescence of lentivirus transfected 293T cells was observed under a fluorescence microscope. Western blot was used to detect the fusion of NEP1-40 and green fluorescent protein Protein expression. Results The electrophoresis analysis of PCR products showed that the cDNA clone of NEP1-40 gene was successfully obtained, and sequencing showed that the lentiviral plasmid was constructed correctly. The expression of lentivirus in 293T cells was confirmed by fluorescent analysis. The expression of NEP1-40 was stable in cells . Conclusion The NEP140 lentiviral vector was successfully constructed, which laid the experimental foundation for the further study on the function of NEP1-40 gene after transfection of target cells.