人粒-巨噬细胞集落刺激因子和结核杆菌特异性抗原CFP10嵌合基因重组卡介苗的构建及鉴定

Construction and identification of recombinant BCG containing a shuttle plasmid with mosaic gene GM-CSF and CFP10

目的构建能共同表达人粒-巨噬细胞集落刺激因子(GM-CSF)和结核分枝杆菌培养滤液蛋白10(CFP10)的重组卡介苗(recombinant BCG,rBCG)。方法运用分子克隆技术,通过SOE法(重叠延伸,Gene splicing by overlap exten-sion),扩增GMCSF-CFP10嵌合基因,将该基因定向克隆到穿梭表达质粒pMV361中,构建重组pMV GMCSF-CFP10质粒。用电穿孔法将重组质粒导入BCG菌构建rBCG,经热诱导后对rBCG表达产物作SDS-PAGE及免疫印迹分析。结果重组质粒pMVGMCSF-CFP10经PCR、酶切及测序证实构建成功,并在BCG中经热诱导成功表达了具有GMCSF-CFP10的嵌合蛋白。结论成功构建能表达GMCSF-CFP10蛋白的重组BCG,为发展新型结核病疫苗奠定基础。

This study was conducted to construct a recombinant shuttle plasmid containing human granulocyte macrophage colony-stimulating factor（GM-CSF） and 10kD culture filtrate protein（CFP10） mosaic gene to analyze its coexpression in BCG.GMCSF-CFP10 mosaic gene was amplified by SOE and inserted into shuttle plasmid pMV361 to construct the recombinant plasmid pMV GMCSF-CFP10.The pMV GMCSF-CFP10 was transformed into BCG by electroporation,and target protein was identified by SDS-PAGE and immunoblotting after heating induced.Results showed that construction of recombinant plasmid pMV GMCSF-CFP10 was confirmed successfully by Polymerase Chain Reaction,restriction endonuclease digestion analysis,and sequencing.And GMCSF-CFP10 mosaic proteins were expressed in BCG by heat induction.In conclusion,the recombinant shuttle plasmid containing GM-CSF and CFP10 mosaic gene is constructed successfully,which provides an experimental basis for developing new Mycobacterium tuberculosis vaccine.