论文部分内容阅读
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 ,为自杀基因联合细胞因子基因疗法提供有利条件
OBJECTIVE: To construct pEGFP-TK-rhTNF-α expression vector and observe its transient expression in Hep-2 laryngeal carcinoma cells. Methods: The target genes were amplified by PCR and sequenced by sequencing vector pGEM-T-Easy. The fusion gene TK -rhTNF-α was fused with multiple cloning sites in the retroviral vector PLXSN The recombinant plasmid pEGFP -TK -rhTNF-α was constructed by subcloning into EcoRI and BamHI sites of pEGFP-N3. The expression of fusion protein was observed after Hep-2 was transfected into human laryngeal carcinoma cell line Hep-2. Results: The results of restriction enzyme digestion confirmed that TK-rhTNF-α fusion gene fragment was cloned into EcoRI and BamHI sites of pEGFP-N3 and TK-rhTNF-α fusion gene was transfected into Hep-2 cells Green fluorescent protein expression. CONCLUSION: Expression of pEGFP-TK-rhTNF-α facilitates the expression of TK-rhTNF-α fusion protein and protein localization in transfected cells, which provides a favorable condition for suicide gene combined with cytokine gene therapy