人颗粒溶素活性肽重组卡介苗的构建与表达

Construction and expression of recombinant Bacille Calmette-Guerin carrying human granulysin active peptide

目的构建携带人颗粒溶素(GLS)活性肽的重组卡介苗(BCG),并鉴定该重组菌向真核细胞递呈质粒的能力。方法以分子生物学方法 构建携带GLS活性肽基因和分支杆菌复制子OriM的真核穿梭共表达质粒pBMS,以电转化方式将pBMS质粒转入BCG构建重组BCG,抽提质粒进行PCR鉴定重组菌;将重组菌感染巨噬细胞,通过RT-PCR了解重组菌向真核细胞递呈质粒的能力。结果采用电转化方式可将pBMS导入BCG,抽提质粒鉴定出与GLS基因相符的目的条带。以该重组BCG感染小鼠巨噬细胞,96h后用RT-PCR法可扩增出相应的基因条带。结论成功构建含pBMS的重组BCG。该重组BCG能向小鼠巨噬细胞递呈携带基因。

Objectives To construct recombinant Bacille Calmette Guerin（BCG） carrying human granulysin genes,and to identi- fy the ability of recombinant BCG for submitting plasmids to eukaryotic cells. Methods Co-expression shuttle plasmid of pBMS,including human granulysin genes and mycobacterial replicon of Orim,was built using molecular biological approach. The pBMS was transfected to BCG competence hy electroporation to construct recombinant BCG. The plasmid of recombinant BCG was extracted for PCR identification. The ability of submitting plasmids to eukaryotic cells was determined by RT-PCR. Results The plasmid pBMS may be transduced into BCG with electroporati.on. The specific bands for GLS of the recombinant BCG were detected by PCR. The specific bands for 267 bp GLS were amplified from transfected macrophages after 96 hours by RT-PCR. Conclusion The recombinant BCG carrying pBMS was constructed successfully. The recombinant BCG can submit the carried genes to mice macro- phages