结核分枝杆菌Ag85B-ESAT6融合蛋白稳定表达细胞系的建立

Establishment of the Stable Cell Line Expressed Mycobacterium tuberculosis Ag85B-ESAT6 Fusion Protein

建立了能够稳定表达结核分枝杆菌Ag85B-ESAT6融合蛋白的P815细胞系。将Ag85B和ESAT6基因分别克隆至真核表达载体pcDNA3,构建了Ag85B-ESAT6融合蛋白的真核表达质粒Ag85B-pcDNA3-ESAT6。在阳离子聚合物作用下,重组质粒转染与BALB／c遗传背景一致的P815(H-2d)细胞。通过G418压力筛选后,得到1株阳性克隆细胞。经过BT-PCR检测到该细胞中有 Ag85B-ESAT6融合蛋白mRNA表达,用间接免疫荧光可以在转染重组质粒的P815细胞膜上检测到较强的绿色荧光,证实P815细胞内有融合蛋白的表达。获得表达Ag85B-ESAT6融合蛋白的稳定细胞系。

To establish the stable cell line expressed Mycobacterium tuberculosis Ag85B-ESAT6 fusion protein, Ag85b and ESAT6 gene were cloned into the eukaryotic expression vector pcDNA3. This recombinant plasimd was transfected into P815 cells（H-2d） by citation lipids whose genetic was identified with BALB/c. One strains positive cell clone by G418 was selected, and specific mRNA of the fused protein was detected by RT- PCR. By indirect immunofluorescence technique （IFT） the P815 cell transfected with recombinant plasmid appeared strong green fluorescence. It proved that the fusion protein was expressed in the P815 cell. The stable expression cell line which could express Ag85B-ESAT6 fusion protein in P815 Cell is established.