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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 a lentiviral vector expressing TRADD gene after restenosis and esophageal stent implantation, and to investigate its effect on the proliferation of hypertrophic scar fibroblasts. Methods Using the purchased TRADD gene as a template, the target gene fragment was amplified by PCR. The PCR product was purified by recombination with linearized lentiviral vector pLVX-EGFP-3FLAG-Puro. Recombinant plasmids were transformed into DH5α competent cells. Transformants were identified by colony PCR, and positive clones were sequenced correctly. Plasmids were co-transfected into 293FT cells to prepare lentivirus. Real-time quantitative PCR detection of virus titer. Western blot was used to detect the expression of TRADD-GFP-FLAG fusion protein. Effect of TRADD gene lentivirus on the proliferation of hypertrophic scar fibroblasts by MTT assay. Results The colony PCR products were cloned by gel electrophoresis and cloned into a 1 200 bp fragment. The gene sequence showed that the TRADD sequence in the recombinant plasmid was identical with that in GenBank. After 48 hours of transfection of 293 FT cells with lentivirus, the green fluorescence was observed under a fluorescence microscope with a titer of 3.22 × 108 IU / ml. Western blot analysis showed that the fusion protein TRADD-GFP-FLAG was efficiently expressed in 293FT cells MTT assay confirmed that the fusion protein has biological activity, which can effectively inhibit the proliferation of scar fibroblasts. Conclusion The pLVX-TRADD-EGFP-3FLAG-Puro lentiviral vector was successfully constructed and packaged, which could significantly inhibit the proliferation of hypertrophic scar fibroblasts.