人颗粒溶素活性肽和小鼠单链白细胞介素-12重组卡介苗的构建及鉴定

Construction and Identification of Recombinant BCG Carrying Human Granulysin Active Peptide and Murine IL-12 Genes

目的构建含人颗粒溶素(GLS)活性肽和小鼠单链白细胞介素-12(mIL-12)基因的重组卡介苗(BCG),并鉴定其向真核细胞递呈质粒的能力。方法以分子生物学方法构建含相对分子质量为9000的颗粒溶素活性肽基因和小鼠IL-12基因及分枝杆菌复制子OriM的真核穿梭共表达质粒pBM9,以电转化的方式将pBM9质粒转入BCG,构建重组BCG,抽提质粒进行PCR鉴定;将重组菌感染小鼠巨噬细胞RAW264.7,经RT-PCR检测重组菌向真核细胞递呈质粒的能力。结果重组BCG可扩增出与GLS基因和mIL-12基因相符的目的条带。重组BCG感染RAW264.7细胞96h后,经RT-PCR可扩增出267bp的GLS基因条带和1632bp的mIL-12基因条带。结论已成功构建含GLS和mIL-12基因的重组BCG,该重组BCG能向小鼠巨噬细胞递呈携带的目的基因。

Objective To construct a recombinant BCG carrying human granulysin （GLS） active peptide and murine IL-12 （mIL-12） genes and determine its ability in delivery of plasmids to eukaryotic cells. Methods A eukaryotic shuttle co-expression vector pBM9 carrying the gene encoding GLS active peptide with a relative molecular mass of 9 000, mIL-12 gene and mycobacterial replicon OriM was constructed by molecular biological method, and transformed to BCG by electrotransformation. Plasmids were extracted from the constructed recombinant BCG（rBCG） and identified by PCR. The rBCG was infected to murine macrophages RAW264.7, and its ability in delivery of plasmids to eukaryotic cells was determined by RT-PCR. Results The target gene fragments consistent with GLS and mIL-12 genes were amplified from the constructed rBCG. The GLS and mlL-12 gene fragments, at lengths of 267 and 1 632 bp respectively, were amplified by RT-PCR from the RAW264.7 cells 96 h after infection with rBCG. Conclusion A rBCG carrying GLS and mIL-12 genes were successfully constructed, which showed an ability in delivery of the carried target genes to murine macrophages.