小鼠白细胞介素12双顺反子真核表达载体的构建及其在COS-7细胞中的表达

Construction of Bicistronic Eukaryotic Expression Vector Containing Murine Interleukin 12 and its Expression in COS 7 Cells

克隆小鼠白细胞介素１２（ＩＬ－１２）ｐ４０及ｐ３５ｃＤＮＡ，并构建同时含ｍＩＬ－１２ｐ４０和ｐ３５ｃＤＮＡ的双顺反子真核表达载体及其在哺乳动物细胞中的表达．白细胞介素１２是由巨噬细胞，树突状细胞等抗原提呈细胞产生的一种异二聚体细胞因子，对机体的细胞免疫功能起着重要的调节作用．利用脂多糖（１００ｐｇ／ｍｌ）和小鼠重组干扰素（ＩＦＮ－γ５００Ｕ／ｍｌ）体外联合刺激小鼠腹腔巨噬细胞，从中提取总ＲＮＡ，经ＲＴ－ＰＣＲ扩增出含信号肽的小鼠白细胞介素１２（ｍＩＬ－１２）ｐ４０及ｐ３５全长ｃＤ－ＮＡ．ＰＣＲ产物经酶切后，分别克隆至ｐＢｌｕｅｓｃｒｉｐｔⅡＳＫ载体中，序列测定结果与文献报道序列一致．然后利用脊髓灰质炎（Ｐｏｌｉｏ）病毒内核糖体进入位点（ＩＲＥＳ）连接ｍＩＬ－１２ｐ４０及ｐ３５ｃＤＮＡ，亚克隆至ｐｃＤＮＡ３载体中，构建成含ｍＩＬ－１２ｐ４０及ｐ３５ｃＤＮＡ双顺反子真核表达载体，即ｐｃＤＮＡ３／ｍＩＬ－１２，ｐ４０及ｐ３５ｃＤＮＡ同时受ｐｃＤＮＡ３中ｈＣＭＶ启动子驱动，将ｐ４０及ｐ３５转录至同一ｍＲ－ＮＡ上．通过ＬｉｐｏｆｅｃｔＡＭＩＮＥ将ｐｃＤＮＡ３／ｍＩＬ－１２转染ＣＯＳ－７细胞，７２ｈ收集培养上清，测定ｍ?

Murine interleukin 12 p40 and p35 cDNA,were cloned and bicistronic eukaryotic expression vector containing murine IL 12 p40 and p35 cDNA was constructed.Both p40 and p35 cDNAs of murine interleukin 12 were amplified by means of reverse transcription and polymerase chain reaction from total RNA of murine peritoneal macrophage stimulated synergistically by 500 U/ml IFN γ and 100 pg/ml LPS.The amplified cDNA fragments were inserted into pBluescriptSK plasmid respectively.The nucleotide sequence of both p40 and p35 cDNAs was determined by dideoxy termination and showed that it was the same sequence as that reported previously.Both p40 and p35 cDNAs of mIL 12 were linked by internal ribosome entry site (IRES) of 5′ nontranslation region from poliovirus and then cloned into the eukaryotic expression vector pcDNA3,thus constructing the eukaryotic expression vector pcDNA3/mIL 12 containing both p40 and p35 cDNAs.pcDNA3/mIL 12 was transfected into COS 7 cells by LipofectAMINE.RT PCR and murine IL 12 ELISA demonstrated expression of mIL 12 in COS 7 cells and the cultural supernatant of the COS 7 cells transduced with pcDNA3/mIL 12 could induce proliferation of murine spleen cells activated by ConA.Results suggest that cloning of mIL 12 can provide basis for further study of its immunoregulatory mechanism and antitumor immune response.