首页> 中文期刊>南方医科大学学报 >抗肿瘤免疫基因治疗剂pVAX-IL-12-GB的构建及体内外表达验证

抗肿瘤免疫基因治疗剂pVAX-IL-12-GB的构建及体内外表达验证

     

摘要

目的 构建能够同时表达人免疫调节因子IL-12,GM-CSF和B7.1的新型抗肿瘤免疫基因治疗剂pVAX-1L- 12-GB,验证以上免疫调节因子基因在293T细胞的表达以及IL-12基因在活体内的表达.方法 将人1L-12基因克隆到早期构建好的pVAX-IRES-GM-CSF-B7.1真核表达质粒的上游,将其构建成为pVAX-IL-12-GB重组质粒:利用脂质体法瞬时转染293T细胞,RT-PCR检测IL-12和GM-CSF-B7,1基因的转录,ELISA法检测细胞上清中上游IL-12基因和下游GM-CSF基因的蛋白表达,流式细胞术及免疫荧光检测下游B7.1基因的表达;利用电穿孔方法递送质粒进入小鼠股四头肌,免疫组化法检测IL-12基因在小鼠活体内的表达情况.结果 双酶切以及PCR鉴定获得的片段均与预期的片段大小一致,测序分析证实IL-12基因序列完全正确;瞬时转染293T细胞后,分别验证了人IL-12,GM-CSF和B7.1基因的mRNA转录和蛋白表达;电穿孔递送质粒后,活体肌肉组织内同样可以检测到IL-12基因的表达.结论 成功构建了可以同时表达人IL-12,GM-CSF和B7.1的新型抗肿瘤免疫基因治疗剂,为下一步开展肿瘤免疫基因治疗研究奠定了坚实的基础.%Objective To construct a novel immunogene therapeutic plasmid that expresses human interleukin-12 (IL-12),gramilocyte-maerophage colony stimulating factor (GM-CSF) and B7.1 and observe its expression in vivo and in vitro.Methods Human IL-12 gene fragment was cloned into the upper stream of IRES gene in the previously constructed plasmid pVAX-IRES-GM-CSF-B7.1,and the positive recombinant plasmid pVAX-IL-12-GB was transfected into 293T cells via Lipofectamine 2000.The expressions of IL-12 and GM-CSF-B7.1 mRNA and proteins in the transfected cells were assayed by RT-PCR and ELISA,and B7.1 expression was tested by fluorescence-activated cell sorting and immunofluorescence assay.The plasmid pVAX-IL-12-GB was delivered into mouse muscle by electroporation,and the expression of IL-12 in the muscle tissue was identified by immunohistochemistry.Results Enzyme digestion,PCR and sequence analysis all confirmed successful construction of the recombinant plasmid pVAX-IL-12-GB.IL-12,GM-CSF and B7.1 expressions were all detected in transfected 293T cells,and the expression of IL-12 was also detected in the transfected mouse muscular tissues.Conclusion A novel anti-tumor immunogene vaccine constructed can be expressed both in vivo and in vitro,which facilitates further studies of rumor immunogene therapy.

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