人颗粒溶素与小鼠单链IL-12真核共表达质粒的构建及其在RAW264.7细胞中的表达

Construction of the eukaryotic coexpression plasmid encoding human granulysin and murine single chain IL-12 and its expression in RAW264.7 cells

目的构建人天然颗粒溶素(Granulysin,GLS)和小鼠单链IL-12基因的真核共表达载体pZM02并在RAW264.7细胞中进行表达。方法将人GLS和小鼠单链IL-12基因同时克隆进多启动子真核共表达载体pBudCE4.1中,得到真核共表达质粒pZM02,以pZM02转染小鼠巨噬细胞株RAW264.7,通过RT-PCR、免疫细胞化学和ELISA的方法检测目的基因的表达。结果在转染后的RAW264.7细胞中同时检测到了人GLS和小鼠IL-12的表达。结论人GLS能够与小鼠IL-12在小鼠巨噬细胞株RAW264.7中实现共表达,pZM02的成功构建为进一步研究GLS与IL-12的协同细胞免疫作用机制和免疫治疗作用奠定了基础。

To construct an eukaryotic coexpression plasmid encoding human granulysin and murine single chain IL-12 and to observe its expression in RAW264.7 cells, human granulysin gene and murine single chain IL-12 gene were cloned into pBudCFA. 1 which has multiple promoters to construct recombinant plasmid pBudCE4.1/GLS/IL-12 （simply named pZM02）. The recombinant plasmid was transfected into RAW264.7 cells and the expression of target genes was detected by RT-PCR, immunocytochemistry and ELISA. It was found that the coexpression of human GLS and murine IL-12 could be detected in RAW264.7 cells simultaneously. It is concluded that human GLS and murine IL-12 can coexpress in murine macrophage strain RAW264.7. The successful construction of the coexpression plasmid pZM02 would establish the foundation for further researching on the mechanism of cellular immunity and immune therapeutic effect of GLS cooperating with IL-12.