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为构建膜表达糖基化磷脂酰肌醇(GPI)锚定的结核杆菌早期分泌靶抗原6 kD(ESAT-6)和分泌IL-21的B16F10瘤苗并鉴定其活性,利用重叠PCR法构建pIRES-ESAT-6-gpi/IL-21重组质粒,以脂质体转染重组质粒到B16F10细胞,G418筛选出阳性克隆,用RT-PCR、免疫荧光、FCM和Western blot检测瘤苗细胞靶抗原表达,用瘤苗细胞培养上清刺激小鼠CD8+T细胞,检测瘤苗所分泌IL-21的生物学活性。结果表明,pIRES-ESAT-6-gpi/IL-21重组质粒DNA测序正确,B16F10-ESAT-6-gpi/IL-21瘤苗细胞目的基因ESAT-6表达于瘤苗细胞表面,增殖能力未受外源基因导入影响,分泌的IL-21具有生物学活性,为研究膜表达ESAT-6和分泌表达IL-21瘤苗的抗瘤效应奠定了基础。
To construct and identify the early-secreting target antigen 6 kD (ESAT-6) and the IL-21-secreting B16F10 tumor vaccine by constructing Mycobacterium tuberculosis anchored by glycosylphosphatidylinositol (GPI), the recombinant pIRES -ESAT-6-gpi / IL-21 recombinant plasmid. The recombinant plasmid was transfected into B16F10 by lipofectamine. The positive clones were screened by G418. The expression of target antigen was detected by RT-PCR, immunofluorescence, FCM and Western blot The CD8 + T cells were stimulated with the supernatant of the tumor cells and the biological activity of IL-21 secreted by the tumor cells was detected. The results showed that the recombinant plasmid DNA of pIRES-ESAT-6-gpi / IL-21 was correctly sequenced and the target gene ESAT-6 of B16F10-ESAT-6-gpi / IL-21 cells was expressed on the surface of tumor cells without proliferation The secretion of IL-21 has the biological activity through the introduction of exogenous gene, which lays the foundation for studying the anti-tumor effect of membrane-expressed ESAT-6 and secreting IL-21.