腺病毒伴随病毒载体介导的I型单纯疱疹病毒胸苷激酶基因转移及其杀肿瘤效应  

Adeno-associated virus vector mediated gene transfer of HSVI-TK and its effect on killing cancer cell

作　　者：姚二梅[1] 冯泽华[1] 吴小兵[1] 颜子颖[1] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室

出　　处：《中华实验和临床病毒学杂志》1998年第3期207-212,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：国家863高技术发展计划;国家攀登计划资助

摘　　要：目的研究腺病毒伴随病毒（ＡＡＶ）载体介导杀肿瘤基因的转移及其在肿瘤治疗方面的应用。方法应用作者构建的腺病毒伴随病毒载体，克隆Ｉ型单纯疱疹病毒胸苷激酶（ＨＳＶＩ－ＴＫ）基因，构建质粒ｐＡＣＴＫ－１９。用ｐＡＣＴＫ－１９转染５型腺病毒（Ａｄ５）感染的重组ＡＡＶ包装细胞系ＡＥ１２０１，获得重组病毒ｒＡＡＶ／ＡＣＴＫ。再用此重组病毒感染肺癌细胞系Ａ５４９，并联合丙氧鸟苷（ＧＣＶ）作用，研究其体外对肺癌细胞的杀伤作用。结果重组ｒＡＡＣ／ＡＣＴＫ的滴度为３４×１０５ＣＦＵ／ｍｌ，感染肺癌细胞系Ａ５４９，实现了ＴＫ基因的转移及与细胞染色体的整合和表达，对Ａ５４９细胞具有杀伤作用。The plasmid pACTK-19 was constructed by insering HSVI-TK gene into the multiple cloning sites of the general adeno-associated virus(AAV)vector pACR-Neo.When plasmid pACTK-19 was transfected to recombinant AAV's packaging cell line AE1201,which was exposed to Adenovirus 5 for two hours before transfection,we got rAAV/ACTK at the titer of 3.4×10 5 CFU/ml.After infecting human lung cancer cell A549 with rAAV/ACTK,we extracted host cell's chromosome cDNA and amplified part of the HSVI-TK sequence by a pair of HSVI-TK's primers and then hybridized to digoxin labelled HSVI-TK gene probe.We got corresponding positive band and it proved the integration of HSVI-TK into host cell's choromosome cDNA.By reverse transcripting rAAV/ACR-Neo infected A549 total cell RNA, we amplified part of HSVI-TK sequence and hybridized to Neo gene probe.The corresponding positive band demonstraed the expression of HSVI-TK.So through rAAV/ACTK,HSVI-TK was transfered into host cells and was expressed in them,the rAAV's genome was integrated into host cell's choromosome DNA.Connecting with Ganciclovir (GCV),the HSVI-TK gene transfer mediated by rAAV/ACTK,could inhibit the synthesis of choromosome DNA and lead to the killing of the lung cancer cell. This work proved that recombinant AAV could be used as vector for gene transduction to cancer cell,and also laid foundation for further research and application of AAV vector.

关 键 词：腺病毒伴随病毒 胸苷激酶基因 杀瘤效应 

分 类 号：R392.7[医药卫生—免疫学] Q782[医药卫生—基础医学]