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目的:构建人4-1BB配体(h4-1BBL)全长基因的真核表达载体,并在肿瘤细胞HT-29中转染表达;探讨人4-1BBL基因转染的肿瘤细胞体外诱导的抗肿瘤活性。方法:用RT-PCR从Raji细胞中克隆h4-1BBL全长基因,测序后,构建重组真核表达载体pcDNA3.1(-)-h4-1BBL。通过脂质体法以重组载体转染HT-29细胞,用RT-PCR检测转染细胞中h4-1BBLmRNA的表达;用流式细胞术检测转染细胞表面h4-1BBL分子的表达。分离外周血单个核细胞(PBMC),用抗CD3mAb扩增T细胞,并与h4-1BBL基因转染及未转染的HT-29细胞混合培养。用MTT比色法检测CTL的增殖及杀伤活性;用流式细胞术检测分泌IFN-γ的T细胞。结果:从Raji细胞中克隆到h4-1BBL全长cDNA,测序完全正确。构建的h4-1BBL基因真核表达载体在HT-29中获得稳定表达。与未转染的细胞相比较,h4-1BBL基因转染的肿瘤细胞HT-29能更有效地刺激T细胞活化、增殖,促进IFN-γ分泌,并能有效地诱导CTL产生针对野生型HT-29细胞的特异性杀伤。结论:成功地构建pcDNA3.1(-)h4-1BBL重组真核表达载体。4-1BBL基因转染的肿瘤细胞介导的协同刺激信号,能增强野生型肿瘤细胞的免疫原性,诱导T细胞产生有效的抗肿瘤免疫应答。
OBJECTIVE: To construct an eukaryotic expression vector of full length human 4-1BB ligand (h4-1BBL) gene and transfect it in HT-29 tumor cells. To investigate the in vitro anti-tumor effect of 4-1BBL gene transfected tumor cells Tumor activity. Methods: The full length h4-1BBL gene was cloned from Raji cells by RT-PCR. After sequencing, the recombinant eukaryotic expression vector pcDNA3.1 (-) - h4-1BBL was constructed. The recombinant vector was transfected into HT-29 cells by lipofectamine. The expression of h4-1BBL mRNA in transfected cells was detected by RT-PCR. The expression of h4-1BBL on transfected cells was detected by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated, T cells were expanded with anti-CD3 mAb and mixed with h4-1BBL gene transfected and untransfected HT-29 cells. The proliferation and cytotoxicity of CTL were detected by MTT colorimetric assay. IFN-γ secreting T cells were detected by flow cytometry. Results: The h4-1BBL full-length cDNA was cloned from Raji cells and sequenced completely. The constructed h4-1BBL gene eukaryotic expression vector was stably expressed in HT-29. Compared with untransfected cells, HT-29 cells transfected with h4-1BBL gene can stimulate T cell activation, proliferation and IFN-γ secretion more effectively, and can effectively induce CTL production of wild type HT- 29 cell specific killing. Conclusion: The pcDNA3.1 (-) h4-1BBL recombinant eukaryotic expression vector was successfully constructed. 4-1BBL gene-transfected tumor cell-mediated costimulatory signals can enhance the immunogenicity of wild-type tumor cells and induce T cells to produce an effective anti-tumor immune response.