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.
出处
《现代免疫学》
CAS
CSCD
北大核心
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)
