中文摘要：

目的构建分枝杆菌膜锚定表达载体，并分析外源目标蛋白的表达情况及其亚细胞定位。方法在分枝杆菌胞内表达载体pMFA42的基础上进行改造，人工合成结核分枝杆菌（Myco—bacteriumtuberculosis，Mtb）相对分子质量（Mr）为19×10^3的脂蛋白膜分泌信号序列，将其插入高效的突变型furA基因启动子（pfurAma）下游构建新型的分枝杆菌膜锚定表达载体pMFA42M。PCR扩增增强型绿色荧光蛋白（EGFP）编码基因，亚克隆至上述两种载体中并电转化耻垢分枝杆菌（Mycobacte—riumsmegmatis，Ms），分别获得重组EGFP融合表达菌株和膜锚定表达菌株；接着将Mtb主要保护性抗原Ag85A及其嵌合抗原Ag856A2的编码基因亚克隆入膜锚定表达载体pMFA42M中构建Mtb抗原-EGFP融合表达菌株；通过Westernblot和流式细胞表面标记技术来分析目标抗原的表达水平及其亚细胞定位，并进一步通过荧光显微镜直接观察重组EGFP融合表达菌株在体外和感染巨噬细胞后的荧光强度。结果通过引入Mtb19×10^3脂蛋白膜分泌信号，在高效pfurAma启动子的驱动下，成功地构建了分枝杆菌膜锚定表达载体。利用EGFP作为标签抗原，通过Westernblot、荧光显微观察和流式细胞表面标记等技术均证实了外源目标抗原可在分枝杆菌中高水平表达并定位至细胞膜上。结论本研究为重组BCG和分枝杆菌膜蛋白功能研究提供了一种新型的膜锚定表达载体，所构建的EGFP重组表达菌株亦可作为一种模式示踪菌为细胞吞噬和动物免疫中的细菌定植和移位分析提供新思路。

英文摘要：

Objective To construct mycobacterial membrane-anchored expression vector and to analyze expression level and sub-cellualr localization of exogenous target protein. Methods Based on the mycobacterial intracellular expression vector pMFA42 which contained a strong promoter of pfurAma mutant, the signal sequence of Mycobacterium tuberculosis （Mtb） 19×10^3lipoprotein （19SS） was synthesized and was then cloned into the downstream ofpfurAma mutant to generate the mycobacterial membrane-anchored expres- sion vector pMFA42M. The coding gene of enhanced green fluorescent protein（EGFP） was amplified by PCR, and then sub-cloned into these two vectors described above to construct recombinant EGFP fused and membrane-anchored strains, respectively. The coding genes of Mtb immuno-dominant antigens Ag$5A and its chimera Ag856A2 were then sub-cloned intothe membrane-anchored construct pMFA42MG to produce re- combinant Mtb antigen EGFP fused-expression strains. After that, expression levels and sub-cellualr locali- zation of exogenous target protein were further analyzed by Western blot and flow cytometry sorting （ FCS）, and the fluorescence intensities of recombinant EGFP- expressed strains were observed in vitro directly and after transfection of marine macrophage cell line RAW264.7. Results The novel mycobacterial membrane- anchored expression vector was constructed successfully by introduction of signal sequence of Mtb 19×10^3 lip- oprotein. Using of EGFP as model antigen, exogenous target protein was demonstrated to be expressed with high level and could be anchored into cell membrane of recombinant mycobaterial strains. Conclusion A novel mycobacterial membrane-anchored expression vector was constructed successfully to research recombi- nant BCG and functions of mycobacterial membrane proteins, and the constructed EGFP-expressed recombi- nant strains could also be used to research cytophag＇y in cell model and mycobacterial colony and transloca- tion in animal immunization as model indicator bacteria.