含hTERT基因片段重组逆转录病毒的构建  

Construction of recombnant retrovirus carrying fragment of human telomerase reverse transcriptase gene

作　　者：胡贵方[1] 孙莉莎[2] 欧程山[3] 蒋毅萍[2] 庞建新[2] 

机构地区：[1]南方医科大学公共卫生与热带医学学院流行病教研室,广东广州510515 [2]南方医科大学药学院药理学教研室,广东广州510515 [3]南方医科大学公共卫生与热带医学学院毒理学教研室,广东广州510515

出　　处：《中国热带医学》2005年第7期1405-1407,共3页China Tropical Medicine

基　　金：广东省自然科学基金(基金编号:32876)

摘　　要：目的构建含hTERT基因片段的重组逆转录病毒. 方法采用RT-PCR法从人白血病细胞株K-562中扩增hTERT基因片段(1590～2540bp),亚克隆至逆转录病毒表达载体pLXSN,构建重组质粒pLXSN-hTERT;用脂质体转染的方法将从重组质粒转入PT67细胞,G-418压力筛选获得含hTERT基因片段的重组逆转录病毒细胞克隆;0.22μm微孔过滤抗性克隆上清获得含表达hTERT基因的重组逆转录病毒;Western Blot检测G-418抗性克隆的hTERT产物的表达.结果PCR扩增的hTERT基因片段与GenBank公布的序列相比仅有2个核苷酸差异,氨基酸序列完全一致;重组病毒的滴度为2.85×105PFU/ml;Western Blot检测到含hTERT基因片段的重组病毒能够表达出一分子量为37kD的多肽. 结论成功构建了表达hTERT基因片段的重组逆转录病毒,为进一步探讨内源性表达hTERT基因产物的树突状细胞能否激发特异性CTL的产生奠定了坚实的基础.Objective To construct recombinant retrovirus carrying fragment of human telomerase reverse transcriptase（hTERT） gone. Methods A 951 bp eDNA fragment of hTERT（ 1590 - 2540bp） in K - 562 cell line was amplified by RT - PCR and inserted into pMD18T plasmid. After the recombinant plasmid was cleaved by restriction cndonuelcase Hind III and BamH I, the hTERT fragment was subeloned into rctroviral expression vector pLXSN to form pLXSN - hTERT, which was transfceted into PT67 packing cell line by lipofcetaminc. Using G- 418 selection, resistant cell clones which carried recombinant retrovirus was obtained. The recombinant rctrovirus carrying fragment of hTERT was purified by fidtcring supcmatc of cultured resistant cell clone. Expression of fragment of hTERT was detected by Western blot. Results Compared with hTERT eDNA reported in GcnBank, only two nuelcotidc aeids（2171bp, T- C, 2220bp, C - A） had changed in amplified fragment of hTERT, and the homology was 100% in amino acid. The liter of the recombinant rctrovirus carrying fragment of hTERT was 2.85 × 10^5PFU/ml. The recombinant rctrovirus could express one polypeptidc with the molecular mass of 37 kD. Conclusion The recombinant retrovirus for expressing fragment of hTERT has been successfully constructed. It will lay a solid foundation for the suceedent study on hTERT as a tumor associated antigen in denritie cell-based immunotherapy.

关 键 词：HTERT 基因表达 逆转录病毒 

分 类 号：R73[医药卫生—肿瘤]