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目的 :构建E .coli. BCG(BacilleCalmette Guerin)穿梭载体 ,在母牛分枝杆菌细胞壁融合表达结核分枝杆菌 (MTB)的分泌蛋白Ag85B ESAT 6 .方法 :用聚合酶链反应 (PCR)从MTB毒株H37Rv基因中扩增出结核分枝杆菌 190 0 0抗原 (19 ss)胞壁区及其上游调控元件基因 ,克隆入E .coli. BCG穿梭载体pOLYG中 ,构建表达蛋白能嵌入细胞壁中的E .coli. BCG穿梭载体 .用间接免疫荧光染色法观察该载体携带所构建的结核分枝杆菌分泌蛋白Ag85B和ESAT 6基因在母牛分枝杆菌宿主中的融合表达 .结果 :经测序证实所克隆的 19 ss基因序列正确 ,所构建的E .coli. BCG穿梭载体 (pCW)能完成在大肠杆菌和母牛分枝杆菌细胞之间的穿梭 ,经免疫荧光检查外源基因Ag85B ESAT 6可融合表达于分枝杆菌宿主表面 .结论 :本方法可成功构建能够在分枝杆菌胞壁融合表达MTB分泌蛋白Ag85B ESAT 6的E .coli. BCG穿梭质粒 .
OBJECTIVE: To construct the shuttle vector of BCG (Bacille Calmette Guerin) and express the secreted protein Ag85B ESAT 6 of mycobacterium tuberculosis (MTB) in the cell wall of Mycobacterium vaccae.Methods: Polymerase chain reaction (PCR) The H37Rv gene was amplified from the Mycobacterium tuberculosis 190 0 0 antigen (19 ss) cell wall region and its upstream regulatory element gene and cloned into E.coli BCG shuttle vector pOLYG to construct the expressed protein can be embedded in the cell wall E.coli BCG shuttle vector.Indirect immunofluorescent staining was used to observe the fusion expression of Mycobacterium tuberculosis secreted protein Ag85B and ESAT 6 in Mycobacterium vaccae host.Results: The cloned 19 ss gene was correctly sequenced. The constructed E. coli BCG shuttle vector (pCW) can complete shuttle between E. coli and Mycobacterium vaccae cells. After immunofluorescence, exogenous gene Ag85B ESAT 6 could be fused Which was expressed on mycobacterial host.Conclusion: This method can successfully construct E.coli BCG shuttle plasmid which can express MTB secretory protein Ag85B ESAT 6 in the cell wall of mycobacteria.