复制缺陷型人IL-2重组腺病毒的制备与鉴定  

Preparation and identification of replicationdeficient human interleukin2 recombinant adenoviruses

作　　者：章卫平[1,2] 曹雪涛[1,2] 滨田洋文[1,2] 

机构地区：[1]第二军医大学免疫学教研室 [2]日本癌研究会肿瘤研究所

出　　处：《中华微生物学和免疫学杂志》1998年第2期152-156,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家九五科技攻关项目

摘　　要：目的将人ＩＬ－２的重组腺病毒载体与Ａｄ５腺病毒ＤＮＡ末端肽复合物同源重组，高效制备人ＩＬ－２的复制缺陷型重组腺病毒，并进行体外表达和活性检测。方法将含全部编码序列的人ＩＬ－２ｃＤＮＡ置于真核表达载体ｐＣＩｃｃ的ＣＭＶ启动子下游，切出含ＣＭＶ启动子、人ＩＬ－２ｃＤＮＡ和ＳＶ４０ｐｏｌｙＡ信号肽序列的ＣｌａＩ片段，插入Ｅ１区替代的腺病毒载体ｐＡｘｌｏｗ，选择左向转录的人ＩＬ－２重组腺病毒载体ｐＡｘｌｃｗ．ＣＩｈＩＬ－２与经ＥｃｏＴ２２Ⅰ酶切的Ａｄ５腺病毒ＤＮＡ－末端肽复合物共转染２９３细胞；通过同源重组获得人ＩＬ－２的复制缺陷型重组腺病毒；体外转染人宫颈癌ＨｅＬａ细胞、人Ｔ细胞淋巴瘤Ｈｕｔ７８细胞和原代人皮肤成纤维细胞。结果扩增到的病毒滴度达２．１×１０９ＰＦＵ／ｍｌ；体外转染的人肿瘤细胞均检测到ＩＬ－２的表达（４００～２６００Ｕ／１０６细胞／２４小时）。结论所制备的复制缺陷型重组腺病毒能有效介导人ＩＬ－２的基因转移，可望用于肿瘤基因治疗的临床研究。The fulllength human IL2 encoding cDNA was placed at the downstream of CMV promoter of the expressing vector pCIcc, and the resultant ClaI fragment containing CMV promoter,hIL2 cDNA and SV40 polyA signals was inserted into E1substituted adenovirus vector pAx1cw.Subsequently,the hIL2 recombinant adenovirus vector was cotransfected into 293 cells together with EcoT22Idigested Ad5TPC,and by homologous recombination the replicationdeficient hIL2 recombinant adenovirus was generated efficiently with a titer of 2.1×109 pfu/ml.Infected with prepared hIL2 recombinant adenovirus in vitro,HeLa cells, Hut78 T lymphoma cells and human skin fibroblasts expresed high levels of hIL2(400～2600U/106cells /24h)48 hours after infection.These suggest that COS/TPC is an efficient method to prepare recombinant adenoviruses and the prepared hIL2 adenovirus could be used in cancer gene therapy.

关 键 词：白细胞介素2 腺病毒 载体 制备 肿瘤 免疫疗法 

分 类 号：R730.51[医药卫生—肿瘤] Q78[医药卫生—临床医学]