NEP1-40基因慢病毒载体构建及鉴定

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。

Construction and identification of Nogo extra cellular peptide residues 1-40 gene lentiviral vector

Objective To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40 (NEPl-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells.Methods The DNA fragment of NEP1-40 coding sequence was amplified by PCR with designed primer from the cDNA library including NEP1-40 gene,and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector,pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEPl-40 was confirmed by sequencing.Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40,and packaging plasmids of pHelper 1.0 and pHelper 2.0.Green fluorescent protein(GFP) expression of infected 293T cells was observed to evaluate gene delivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot.Results The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation,and NEP1-40 protein expression was detected in 293T cells by Western blot.Conclusion The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP1-40.