TRADD慢病毒载体构建及其对增生性瘢痕成纤维细胞增殖的影响

Construction of lentiviral expression vector containing human TRADD gene and its inhibitory effect on proliferation in hyperplastic scar fibroblasts

目的构建抗食管支架术后再狭窄携人TRADD基因慢病毒表达载体,并检测其对增生性瘢痕成纤维细胞增殖的影响。方法以购买的人TRADD基因为模板,采用PCR法扩增目的基因片段,PCR产物经回收纯化后与线性化的慢病毒表达载体pLVX-EGFP-3FLAG-Puro重组。重组质粒转化DH5α感受态细胞。菌落PCR鉴定转化子,阳性克隆测序无误后,质粒共转染293FT细胞制备慢病毒。Real-time定量PCR法检测病毒滴度。Western blot检测TRADD-GFP-FLAG融合蛋白的表达。MTT法检测TRADD基因慢病毒对增生性瘢痕成纤维细胞增殖的影响。结果菌落PCR扩增产物经凝胶电泳,阳性克隆得到1 200 bp片段,基因测序显示重组质粒中TRADD序列与GenBank中序列一致。包装产生的慢病毒转染293 FT细胞48 h后,荧光显微镜下可见大量绿色荧光,病毒滴度为3.22×108IU/ml,Western blot检测到融合蛋白TRADD-GFP-FLAG在293FT细胞中获得有效表达,MTT法证实该融合蛋白具有生物活性,能有效抑制瘢痕成纤维细胞增殖。结论成功构建并包装了pLVX-TRADD-EGFP-3FLAG-Puro慢病毒表达载体,其可明显抑制增生性瘢痕成纤维细胞的增殖。

Objective To construct and identify a lentiviral expression vector containing the human tumor necrosis factor receptor associated death domain protein（TRADD） gene and to determine the effect of the recombinant lentivirus on the proliferation of fibroblasts derived from hypertrophic scars.Methods The TRADD specific fragment was amplified by PCR and cloned into the EcoRⅠsite of the lentiviral vector pLVX-EGFP-3FLAG-Puro.The recombinant plasmid was obtained and transformed into DH5α competent cells,and then identified by PCR using colonies directly as templates.The positive recombinant clones were detected by DNA sequencing analysis.Consequently,recombinant lentiviruses were produced after the 293FT packing cells were co-transfected with pLVX-TRADD-EGFP-3FLAG-Puro and lentiviral packaging plasmids,while titer of virus was detected by real-time PCR and the expression level of TRADD-GFP-FLag fusion protein was analyzed by Western blotting.MTT assay was used to test the effect of recombinant lentiviruses on the proliferation of hypertrophic scar fibroblasts.Results A 1 200-bp strap was obtained from positive clone of colony PCR amplification production after gel electrophoresis,while DNA sequencing confirmed that the positive clone was consistent with that in GenBank.The green fluorescence was observed in 293FT cells under fluorescence microscopy after transfected with recombinat lentiviral vector.Real-time PCR showed the titer of the virus was 3.22×108 IU/ml,and Western blotting demonstrated that fusion protein was effectively expressed in 293FT cells.MTT assay indicated that recombinant lentiviruses exerted inhibitory effect on the proliferation of hypertrophic scar fibroblasts.Conclusion The recombinant lentiviral vector pLVX-TRADD-EGFP-3FLAG is constructed successfully,and it has inhibitory effect on the proliferation of hypertrophic scar fibroblasts.