分枝杆菌胞壁融合表达Ag85B-ESAT-6穿梭质粒的构建

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H3 7Rv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

Constructing shuttle plasmid fusion expressing Ag85B-ESAT-6 on the surface of Mycobacterium

Objective To construct the E coli BCG (Bacille Calmette Guerin) shuttle vector expressing Mycobacterium tuberculosis secreted protein Ag85B ESAT 6 on the surface of Mycobacterium vaccae Methods The gene fragment containing 19 000 antigen (19 ss) were amplified by polymerase chain reaction (PCR)from the Mycobacterium tuberculosis H 37 Ra We cloned the 19ss gene into the E coli BCG shuttle vector pOLYG and named the pCW, which can shuttle and express exogenous antigen gene on cell wall of Mycobacterium Then Mycobacterium tuberculosis secret protein Ag85B and ESAT 6 gene were cloned into the vector and determined by indirect immunofluorescence Results The sequence of 19 ss gene was identified with Genbank reported by sequencing The constructed E coli BCG shuttle vector using 19ss gene had the function of shuttle between E coli and Mycobacteria By indirect immunofluorescence technique the secreted protein Ag85B ESAT 6 can be fused and expressed on surface of Mycobacterium vaccae Conclusion The E coli BCG shuttle vector is constructed successfully which could express exogenous antigen gene as a chimeric exported membrane