人IL-12双亚基共表达载体的构建及其表达

CONSTRACTION AND EXPRESSION OF HUMAN IL-12 BICISTRONIC EXPRESSION VECTOR

目的构建人ＩＬ－１２的高效表达载体。方法采用ｐＣＤＮＡ３．１从已克隆的ｐ４０ｃＤＮＡ基因与国外提供的ｐ３５ｃＤＮＡ基因上获得相应的编码基因，分别构建表达载体进行转染。此后，又利用ｐＬＸＰＸＳＮ构建ＩＬ－１２双亚基共表达的逆转录病毒载体转导人肝癌细胞株，并经Ｇ４１８筛选。结果有功能活性的ＩＬ－１２的产生需要ｐ３５和ｐ４０基因的等量共转染。为此，人ＩＬ－１２双亚基共表达载体在转染肝癌细胞株并经选择后，上清液中查到了较共转染更高的具有刺激淋巴母细胞增殖活性的ＩＬ－１２。结论这种人ＩＬ－１２双亚基共表达载体是进行ＩＬ－１２基因转染的有效表达载体。

Objetive To construct human IL 12 efficient expression vector.Method Human IL 12 p40 cDNA (phDCp40, which was cloned by ourselves from human dendritic cells) and p35 cDNA (p35 NKSF 14 1 1,supplied by ATCC) coding region were cloned into mammalian cells expression vector pCDNA3.1 respectively to construct p40 and p35 expression vectors. And then bicistronic expression vector pL35P40SN was constructed by using pLXPXSN.Result The production of human IL 12, which has biological activities, needs the p40 and p35 expression vector to cotransfect the same cells in equal quantity. Therefore the hIL 12 bicistronic expression vector pL35P40SN was constructed to transduce human hepatocellular carcinoma cell line and selected by G418. This kind of bi cistronic expression vector was more effective in producing recombinant Hil 12. By detecting its abilities to stimulating human lymphoblasts it was found that the bi cistronic vector transduced cells supernatant had higher level of hIL 12 than the cotransfection.Coclusion hIL 12 bi cistronic expression vector is an efficient vector that can be used to transfect human cells.