慢病毒载体介导CD1d和GFP融合基因转染PANC-1细胞的实验研究
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摘要
目的构建人免疫基因CD1d与绿色荧光蛋白(green fluorescent protein,GFP)融合基因慢病毒载体并转染人胰腺癌细胞(PANC-1).方法实时定量聚合酶链反应(RT-PCR)方法扩增获得CD1d的全外显子片段,使之克隆到带GFP荧光报告基因慢病毒表达载体质粒中,慢病毒包装质粒和穿梭质粒转染293T细胞,包装成功后收集上清,浓缩,鉴定.通过慢病毒转染预实验确定MOI,进行人CD1d重组慢病毒载体转染胰腺癌PANC-1细胞株.结果电泳鉴定结果与目的基因表达条带完全吻合,克隆测序结果与NCBI收录的CD1d基因序列完全一致.重组慢病毒质粒可高效转染PANC-1细胞,荧光显微镜下可观察到大量绿色荧光.结论成功构建了CD1d与GFP融合基因慢病毒表达载体并转染人胰腺癌细胞.
Objective To construct lentiviral vector of human immunogene CD1d and green fluorescent protein(GFP) fusion gene and transfect PANC-1 cell.Methods The fragments containing all the exons of CD1d were amplified by RT-PCR and were cloned into the lentiviral expression vectors labeled with GFP.The lentivirus was packaged and used to transfect 293T cells together with shuttle plasmid.The supernatant of virus-producing cells was collected,concentrated and identified,then was used to infect 293T cells.MOI was determined by preliminary experimental,and pancreatic cancer cell lines PANC-1 mediated by human restructured gene CD1d lentiviral vector was transfected.Results Electrophoresis showed that the sequence of the RT-PCR product was consistent with the data of NCBI by DNA sequencing analysis.The lentivirus coud effectively transfect PANC-1 cells.Strong green fluorescence was observed by fluorescent microscopy.Conclusion The lentivirus vector containing CD1d-GFP recombinant gene have been successfully constructed,and the fusion gene could effectively transfect PANC-1 cells.
Objective To construct lentiviral vector of human immunogene CD1d and green fluorescent protein(GFP) fusion gene and transfect PANC-1 cell.Methods The fragments containing all the exons of CD1d were amplified by RT-PCR and were cloned into the lentiviral expression vectors labeled with GFP.The lentivirus was packaged and used to transfect 293T cells together with shuttle plasmid.The supernatant of virus-producing cells was collected,concentrated and identified,then was used to infect 293T cells.MOI was determined by preliminary experimental,and pancreatic cancer cell lines PANC-1 mediated by human restructured gene CD1d lentiviral vector was transfected.Results Electrophoresis showed that the sequence of the RT-PCR product was consistent with the data of NCBI by DNA sequencing analysis.The lentivirus coud effectively transfect PANC-1 cells.Strong green fluorescence was observed by fluorescent microscopy.Conclusion The lentivirus vector containing CD1d-GFP recombinant gene have been successfully constructed,and the fusion gene could effectively transfect PANC-1 cells.
引文
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[2]WUN K S,ROSS F,PATEL O,et al.Human and mousetype I natural killer tcell antigen receptors exhibit differentfine specificities for CD1d-antigen complex[J].J BiolChem,2012,287(46):39 139-39 148.
[3]RAGHURAMAN G,GENG Y,WANG C R.IFN-b-Me-diated Up-Regulation of CD1d in Bacteria-Inf-ectedAPCs[J].J Immunol,2006,177(11):7 841-7 848.
[4]QASIM W,VINK C A,THRASHER A J.Hybrid lentiviralvectors[J].Mol Ther,2010,18(7):1 263-1 267.
[5]SHINOHARA E T,KAMINSKI J M.Active integration:new strategies for transgenesis[J].Transgenic Res,2007,16:333-339.
[6]KOO B,KWON M,ROH J,et al.336 quantitative analysisof tetracycline-inducible expression of the green fluorescentprotein gene in transgenic chickens,2012,25(1):315-316.