双亚基共表达人白细胞介素12重组腺病毒载体的构建与应用

目的构建双亚基共表达人白细胞介素１２（ＩＬ－１２）重组腺病毒载体,为其临床研究提供实验基础。方法利用ＲＴ－ＰＣＲ分别克隆出人ＩＬ－１２两个亚基ｐ４０和ｐ３５的全长编码ｃＤＮＡ,经脑心肌炎病毒的内部核糖体结合位点（ＩＲＥＳ）序列连接后置于ＣＭＶ启动子下游,将其插入Ｅ１区替代的腺病毒载体ｐＡｘ１ｃｗ,构建成人ＩＬ－１２的双顺反子共表达重组腺病毒载体;与ＥｃｏＴ２２Ｉ酶切的Ａｄ５腺病毒ＤＮＡ－末端肽复合物共转染２９３细胞,经同源重组制备人ＩＬ－１２的重组腺病毒。结果扩增到的人白介素１２重组腺病毒滴度为２．１×１０９ｐｆｕ／ｍｌ,体外感染２９３细胞、ＨｅｐＧ２细胞和人原代皮肤成纤维细胞后,经ＥＬＩＳＡ检测证实人ＩＬ－１２的表达（３０～５０ｎｇ／１０６细胞／２４小时）,所表达的ＩＬ－１２体外能刺激人淋巴母细胞的增殖和ＩＦＮ－γ的产生。结论所制备的人ＩＬ－１２双亚基共表达重组腺病毒载体能有效表达具有生物学活性的人ＩＬ－１２,可望进一步用于临床研究。

The construction and application of adenovirus vector coexpressing the heterodimer of human IL-12

OBJECTIVE: To construct the recombinant adenovirus vector co-expressing the heterodimer of human interkeukin-12. METHODS: The full-length cDNA encoding human IL-12 subunits p40 or p35 was cloned by RT-PCR separately. The cDNA was ligated with encephalomyocarditis internal ribosome entry site (IRES), placed under the control of CMV promoter, and inserted into E1-substituted adenovirus vector pAx1cw to produce the bicistronic coexpression vector. Subsequently, the hIL-12 recombinant adenovirus vector was cotransfected into 293 cells together with EcoT22I-digested Ad5 DNA-TPC, and the replication-deficient hIL-12 recombinant adenoviruses was generated efficiently by homologous recombination. RESULTS: The human IL-12 recombinant adenoviruses were obtained with the titers of 2.1 x 10(9) pfu/ml. 48 hours after the infection of the 293 cells, HepG2 cells and human primary skin fibroblasts with hIL-12 recombinant adenoviruses, the hIL-12 expressions were detected by ELISA (30-50 ng/10(6) cells/24 h). The expressed hIL-12 could stimulate the in vitro proliferation and IFN-gamma production of human PMNC. CONCLUSION: Prepared hIL-12 recombinant adenovirus vector can express biologically active hIL-12 and can be potentially used in cancer gene therapy.