癌胚抗原启动子正调控白细胞介素-15表达质粒载体的构建及其在肿瘤细胞中的靶向表达

目的 构建癌胚抗原(CEA)启动子正调控人白细胞介素-15(IL-15)真核表达质粒载体,观察其在肿瘤细胞内的靶向性表达.方法 基因克隆技术构建巨细胞病毒(CMV)启动子正调控质粒pHi2-IL-15-CMV-TAT和CEA启动子正调控质粒pHi2-IL-15-CEA-TAT;再以IL-2信号肽(SP)置换IL-15SP构建质粒pHi2-IL-2SP-IL-15-CMV-TAT和pHi2-IL-2SP-IL-15-CEA-TAT;阳离子脂质体介导法体外转染CEA阳性结肠癌SW480细胞和CEA阴性乳腺癌MCF-7细胞,酶联免疫吸附方法(ELISA)检测转染后48h细胞培养上清液中IL-15表达.结果 质粒均成功构建.ELISA检测结果:(1)以IL-2SP置换IL-15SP可提高转染细胞的IL-15表达4.8～5.9倍(P＜0.01);(2)转染SW480细胞后,CEA启动子正调控的两质粒(pHi2-IL-15-CEA-TAT和pHi2-IL-2SP-IL-15-CEA-TAT)与相对应的CMV启动子正调控质粒(pHi2-IL-15-CMV-TAT和pHi2-IL-2SP-IL-15-CMV-TAT)之间的IL-15表达无统计学意义(P＞0.05),其效应等同于CMV启动子正调控质粒;(3)转染MCF-7细胞后,CEA启动子正调控的两质粒IL-15表达显著低于CMV启动子正调控质粒(P＜0.01).结论 CEA启动子正调控IL-15质粒,尤其是质粒pHi2-IL-2SP-IL-15-CEA-TAT在CEA阳性细胞中高表达IL-15,CEA阴性细胞中低表达IL-15,实现了IL-15基因高效、靶向性表达.

Construction and targeted expression of CEA promoter positively controlled IL-15 expression pissmid vectors

Objective To construct carcincembryonic antigen(CEA)promoter positively controlled human interlukin-15(IL-15)eukaryotic expression plasmids and aim to achieve high level and targeted IL-15 expression in CEA positive tumor cells.Methods CMV promoter positively controlled gene expression plasmid vector pHi2-IL-15-CMV-TAT and CEA promoter positively controlled gene expression plasmid vector pHi2-IL-15-CEA-TAT were constructed through genetic cloning.The IL-15 signal peptide (SP)sequence was then replaced by IL-2SP sequence through site-directed mutagenesis,resulting plasmids pHi2-IL-2SP-IL-15-CMV-TAT and pHi2-IL-2SP-IL-15-CEA-TAT.These plasmids were transfected into CEA-positive colon cancer SW480 cells and CEA-negative breast carcinoma MCF-7 cells by eationic lipid mediated gene transfer.IL-15 expression in culture supernatants 48 h after transfection was detected by enzyme-linked immunosorbent assay(EUSA).Results All plasmids were successfully constructed.EUSA results showed:(1)Replacing IL-15SP with IL-2SP increased IL-15 expression for 4.8-5.9 fold by transfected cells(P＜0.01):(2)There was no significant difference in the IL-15 expression in transfected SW480 cells between the CEA promoter positively controlled plasmids.pHi2-IL-15-CEA-TAT and pHi2-IL-2SP-IL-15-CEA-TAT,and the CMV promoter positively controlled plasmids.pHi2-IL-15-CMV-TAT and pHi2-IL-2SP-IL-15-CMV-TAT,respectively(P＞0.05);(3)After being transfected into MCF-7 cells,CEA promoter positively controlled plasmids gave rise to significantly lower IL-15 expression than that of CMV promoter positively controlled plasmids(P＜0.01).Conclusion CEA promoter positively controlled IL-15 expression plasmids,especially plasmid pHi2-IL-2SP-IL-15-CEA-TAT,express high level of IL-15 in CEA positive cells.whereas low level of IL-15 in CEA negative cells.Efficient and targeted IL-15 expression is achieved.