Hsv-tk、重组人TNF-α融合基因表达载体的构建及瞬时表达

CONSTRUCTION OF pEGFP-TK-rhTNF-α EXPRESSING VECTORS AND DETECTION OF TRANSIENT EXPRESSION

目的 :构建 pEGFP -TK -rhTNF -α表达载体 ,观察其在喉癌细胞Hep - 2中的瞬时表达。方法 :应用PCR方法对各目的基因进行扩增并经测序载体 pGEM -T -Easy测序 ,利用逆转录病毒载体PLXSN中的多克隆位点将二者融合为融合基因TK -rhTNF -α并将其亚克隆至pEGFP -N3的EcoRI和BamHI位点间 ,成功构建表达载体 pEGFP -TK -rhTNF -α ,并在该载体转染人喉癌细胞Hep - 2后观察融合蛋白表达情况。结果 :酶切鉴定证实TK -rhTNF -α融合基因片段已克隆到pEGFP -N3的EcoRI和BamHI位点间 ,并在转染人喉癌细胞Hep - 2中观察到TK -rhTNF -α融合基因及绿色荧光蛋白的表达。结论 :pEGFP -TK -rhTNF -α表达载体便于观察转染细胞中TK -rhTNF -α融合蛋白的表达情况及蛋白定位 。

Objective:To construct pEGFP-TK-rhTNF-α expressing vector and to detect its transient expression in laryngocarcinoma cells Hep-2.Methods:The target genes were amplified by PCR and sequenced in p EGM-T-Easy.We constructed the fusion gene TK-rhTNF-α in the retroviral expressing vector pLXSN.The fusion gene then were subcloned into EcoRI and BamHI sites of pEGFP-N3,and pEGFP-TK-rhTNF-α expressing vectors were generated successfully.We observed its transient expression after it was transfected into laryngocarcinoma cells Hep-2.Results:Identification of pEGFP-TK-rhTNF-α by enzyme digestion showed that TK-rhTNF-α fragment had been cloned into EcoRI and BamHI sites of pEGFP-N3 and the expression of TK-rhTNF-α and GFP was observed in transfected laryngocarcinoma cells.Conclusions:PEGFP-TK-rhTNF-a expressing vector make it easy to assess the expression of TK-rhTNF-a-GFP fusion protein and the protein localization in transfected cells.It also can be beneficial to the study of suicide gene therapy combined with cytokine gene therapy.