摘要
目的克隆人可溶性TRAIL(sTRAIL)基因并构建肿瘤细胞特异性真核表达载体,为鼻咽癌基因治疗的靶向性奠定实验基础。方法提取人外周血淋巴细胞总RNA,采用RT-PCR扩增含有IL-2信号肽和TRAIL凋亡诱导功能区的融合基因,克隆入真核表达载体pGL3-181hTERT肿瘤特异性端粒酶启动子的下游,构建可分泌表达TRAIL基因的真核表达载体pGL3-181hTERT/TRAIL。用Western blot鉴定鼻咽癌细胞株CNE-2中表达产物。结果RT-PCR扩增得到了613 bp的cDNA片断。经酶切及测序鉴定,与GenBank中报道的IL-2信号肽和TRAIL凋亡诱导功能区cDNA序列完全一致,成功构建了肿瘤特异性高效分泌表达可溶性人TRAIL的重组真核表达载体pGL3-181hTERT/TRAIL。Western blot结果显示,获得的瞬时转染TRAIL在鼻咽癌CNE-2肿瘤细胞中能有效表达。结论重组真核表达载体pGL3/TRAIL的成功构建,为鼻咽癌基因治疗的靶向性提供了可能性。
Objective To clone human soluble TRAIL cDNA and construct its eukaryotic expression vector for establishing experimental foundation for targeted gene therapy of nasopharyngeal carcinoma（NPC）.Methods The total RNA was extracted from human peripheral blood lymphocytes.TRAIL gene with interleukin 2 signal peptide was amplified by RT-PCR and cloned into the downstream of vector pGL3-181hTERT promoter to construct an eukaryotic vector,pGL3-181hTERT/TRAIL.The protein expressed in NPC cell line CNE-2 was identified by Western blot.Results Sequence of 613bp cDNA was obtained.Identified by enzyme digestion and sequence analysis,the recombinant eukaryotic expression vector for TRAIL gene was successfully constructed.The results of the Western blot showed that the TRAIL protein could be expressed in transfected CNE-2 cell.Conclusion Successful construction of eukaryotic expression vector pGL3-181hTERT/TRAIL provides the possibility for gene therapy of nasopharyngeal carcinoma.
出处
《中国耳鼻咽喉颅底外科杂志》
CAS
2007年第6期407-410,共4页
Chinese Journal of Otorhinolaryngology-skull Base Surgery
