小鼠白介素18与白介素1β转化酶共表达载体的构建及真核表达  

Construction and expression of converting enzyme coexpression vector for murine interleukin-18 and interleukin-1β

作　　者：徐玢[1] 马小彤[1] 董成亚[1] 石洋[1] 林永敏[1] 宋玉华[1] 吴克复[1] 

机构地区：[1]中国医学科学院中国协和医科大学血液学研究所血液病医院实验血液学国家重点实验室,天津300020

出　　处：《现代免疫学》2005年第3期177-181,共5页Current Immunology

基　　金：国家自然科学基金资助项目(30471964)

摘　　要：为了制备白介素18前体(proIL-18)和白介素1β转化酶(ICE)共表达肿瘤疫苗,从小鼠脾细胞克隆proIL-18和ICE基因,构建小鼠proIL-18和ICE共表达质粒(pIRES-proIL-18-ICE),经测序证实序列正确后,脂质体法转染小鼠淋巴细胞白血病细胞系L1210,筛选稳定表达细胞株。转染细胞RT-PCR及转染细胞培养上清IL-18ELISA检测结果表明,肿瘤细胞可以表达并分泌成熟的IL-18。培养上清的IL-18可显著诱导ConA刺激的小鼠脾细胞产生IFN-γ,表明其具有功能活性。生长曲线和细胞周期实验证实与未转染的肿瘤细胞相比,转染空载体pIRES、pIRES-proIL-18、pIRES-ICE、pIRES-proIL-18-ICE基因对肿瘤细胞生长增殖无明显影响。IL-18白血病瘤苗的成功制备表明,利用pIRES载体在同一肿瘤细胞中共表达的proIL-18和ICE能够完全模拟生理过程,在真核细胞内有效酶切,分泌出成熟并有功能活性的IL-18;构建的IL-18瘤苗为进一步评价其在小鼠体内的抗白血病效果奠定基础。To prepare the tumor vaccine coexpressing precursor interleukin-18 (proIL-18) and IL-1β converting enzyme (ICE) gene, proIL-18 and ICE genes were cloned from murine splenocytes, and their coexpression vector pIRES-proIL-18-ICE was constructed. After confirming the sequence of the insert to be correct, the recombinant vector was transfected into L1210, a murine lymphoid leukemia cell line, by using the Lipofectamine reagent. The stable expression transfectants were selected by G418. RT-PCR analysis of transfectant cells and ELISA assay for IL-18 in the culture supernatants indicated that the transfectant tumor cells could express and secret mature IL-18. The IL-18 secreted to culture supernatants apparently stimulated IFN-γ production in ConA primed murine splenocytes, suggesting IL-18 secreted from transfectants retained its bioactivity. In addition, the cell growth curve and cell cycle test showed that transfection of pIRES, pIRES-proIL-18, pIRES-ICE and pIRES-proIL-18-ICE have little influence on the growth and proliferation of L1210 cells. The successful production of IL-18 leukemia vaccine indicates that, similar to physiological state, coexpression of proIL-18 and ICE in the same tumor cell using pIRES vector can result in efficient cleavage and secretion of bioactive mature IL-18. The IL-18 leukemia vaccine can be used in future study to assess its efficacy of anti-leukemic immunity in a murine model.

关 键 词：小鼠白细胞介素18 真核表达载体pIRES L1210白血病细胞 肿瘤疫苗 

分 类 号：R392.11[医药卫生—免疫学]