真核双基因表达载体pBud-TRAIL-IL-24的构建及其在宫颈癌细胞中的表达

Construction of eukaryotic co expression plasmid pBud-TRAIL-IL-24 and its expression in cervical cancer cells

摘要:

目的：构建肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor related apoptosis inducing ligand,TRAIL)基因和白细胞介素24(Interleutin-24,IL-24)基因的真核双基因表达载体，并观察其对宫颈癌细胞生长的影响情况。方法:采用分子克隆技术，将IL-24基因插入真核表达载体 pBud-TRAIL中，构建双表达质粒 pBud-TRAIL-IL-24，经酶切和测序鉴定后，转染宫颈癌HeLa细胞。荧光显微镜和免疫荧光双标技术检测TRAIL和IL-24在HeLa细胞中的表达。MTT法检测重组质粒对HeLa细胞生长状况的影响。结果:成功构建了真核双基因表达载体pBud-TRAIL-IL-24，并在宫颈癌细胞中表达；转染48 h后可见重组质粒对宫颈癌细胞生长有明显抑制作用(P<0.05)。结论:构建的双表达载体pBud-TRAIL-IL-24能在宫颈癌细胞中表达，并可有效抑制宫颈癌HeLa细胞增殖，为进一步研究TRAIL和IL-24联合应用于宫颈癌的基因治疗奠定了基础。

Abstract:

Objective:To construct the eukaryotic co expression plasmid encoding TNF related apoptosis inducing ligand(TRAIL)gene and interleukin 24(IL-24)gene and to study its inhibitory effect on cervical cancer cell lines.Methods:IL-24 gene was inserted into the expression vector of pBud-TRAIL to obtain co-expression plasmid pBud-TRAIL-IL-24 by molecular cloning technology.The constructed co-expression plasmid was identified by restriction endoenzyme digestion and nucleotides sequencing.The recombinant plasmid of pBud-TRAIL-IL-24 was transfected into HeLa cells.The expressions of TRAIL and IL-24 were detected by fluorescence microscopy and immunofluorescence double labeling staining.The proliferation of cervical cancer cells was assessed by MTT assay．Results:The eukaryotic co-expression plasmid pBud-TRAIL-IL-24 could be successfully constructed and be efficiently co-expressed in HeLa cells.The recombinant plasmid significantly inhibited the growth of cervical cancer cells(P<0.05).Conclusion:The plasmid pBud-TRAIL-IL-24 can be successfully constructed and efficiently co-expressed in HeLa cells,and it can effectively inhibit the proliferation of HeLa cells,which lays the foundation for further study of the joint application of TRAIL and IL-24 in cervical cancer gene therapy.