人FAM172A基因慢病毒载体的构建及其在巨噬细胞中的表达

Construction of the Lentivirus Vector of Human FAM172A Gene and Its Expression in Human Macrophages

目的构建人FAM172A基因慢病毒载体,并包装慢病毒FAM172A感染人巨噬细胞予以鉴定。方法以PDC315-FAM172A为模板扩增FAM172A基因全长序列后,与慢病毒载体pLenti6.3/V5-GFP经酶切、连接转化大肠杆菌,挑取阳性克隆,行菌液PCR、酶切及测序鉴定。将鉴定正确的FAM172A基因慢病毒载体和病毒包装质粒共转染FT293细胞包装慢病毒FAM172A,测定病毒效价。Western blot和QPCR验证慢病毒FAM172A感染巨噬细胞效果。结果菌液PCR、酶切及测序结果显示FAM172A基因成功插入到慢病毒载体中。病毒包装后荧光显微镜观察到FT293细胞中有大量绿色荧光,病毒效价为1.2×108TU/ml。Western blot和QPCR结果显示,慢病毒FAM172A侵染靶细胞后可明显增加目的基因FAM172A的表达。结论成功构建慢病毒FAM172A,为后续FAM172A基因功能研究奠定了基础。

Objective To construct the lentivirus vector of human FAM172A gene,package corresponding virus and identify its expression in human macrophages.Methods After PDC315-FAM172A being taken as a template to amplify overall length of FAM172A gene,the products for amplification and lentiviral vector pLenti6.3/V5-GFP were digested,linked together and transformed into E.coli cells.The candidate clones were picked and identified by bacterial PCR,enzyme digestion and sequence.The correctly confirmed lentivirus vector of FAM172A gene and packing mix were co-transfected into FT293 cells to package lentivirus FAM172A and the corresponding infection titer was measured.Western blot and QPCR verified the effects of transferring lentivirus FAM172A into macrophages.Results The FAM172A gene was successfully inserted into the lentivirus vetor confirmed by bacterial PCR,enzyme digestion and sequence identification.A lot of green fluorescence proteins in FT293 cells were found under fluorescent microscope.Viral titer was 1.2 × 108TU/ml.Western blot and QPCR confirmed that the expression of target gene FAM172A obviously increased after lentivirus FAM172A infecting macrophages.Conclusion The lentivirus virus for FAM172A was successfully constructed,which established the foundation for further function research of FAM172A gene.