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 fulllength human IL2 encoding cDNA was placed at the downstream of CMV promoter of the expressing vector pCIcc, and the resultant ClaI fragment containing CMV promoter,hIL2 cDNA and SV40 polyA signals was inserted into E1substituted adenovirus vector pAx1cw.Subsequently,the hIL2 recombinant adenovirus vector was cotransfected into 293 cells together with EcoT22Idigested Ad5TPC,and by homologous recombination the replicationdeficient hIL2 recombinant adenovirus was generated efficiently with a titer of 2.1×109 pfu/ml.Infected with prepared hIL2 recombinant adenovirus in vitro,HeLa cells, Hut78 T lymphoma cells and human skin fibroblasts expresed high levels of hIL2(400～2600U/106cells /24h)48 hours after infection.These suggest that COS/TPC is an efficient method to prepare recombinant adenoviruses and the prepared hIL2 adenovirus could be used in cancer gene therapy.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
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
