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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。
OBJECTIVE: To homologously recombine the recombinant adenovirus vector of human IL-2 with adenovirus DNA terminal peptide complex of Ad5, and to produce replication-defective recombinant adenovirus of human IL-2 efficiently and to detect the expression and activity in vitro. Methods The human IL-2 cDNA containing all coding sequences was placed downstream of the CMV promoter of the eukaryotic expression vector pCIcc. The ClaI fragment containing the CMV promoter, human IL-2 cDNA and the SV40 polyA signal peptide sequence was cut and inserted into the E1-substituted gland The viral vector, pAxlow, was selected for left transcribed human IL-2 recombinant adenovirus vector pAxlcw. 293 cells were cotransfected with CIhIL-2 and Ad5 adenovirus DNA-terminal peptide complex digested with EcoT22I, and the recombinant defective recombinant adenovirus of human IL-2 was obtained by homologous recombination. HeLa cells were transfected with human IL- Human T-cell lymphoma Hut78 cells and primary human dermal fibroblasts. Results The titer of virus was 2.1 × 109 PFU / ml. The expression of IL-2 was detected in all the human tumor cells transfected in vitro (400-2600U / 106 cells / 24 hours). Conclusion The replication-deficient recombinant adenovirus can effectively mediate the gene transfer of human IL-2 and is expected to be used in the clinical study of gene therapy for cancer.