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目的 :构建能够携带外源蛋白屋尘螨抗原 (Derp2 )基因并能在胞壁表达的E .coli BCG穿梭载体。方法 :用PCR技术 ,从结核分支菌毒株H37Rv基因中 ,扩增结核分支杆菌 19kDa抗原的胞壁区及其上游调控元件 (19 ss)基因 ,并克隆入含有Derp2基因的E .coli BCG穿梭载体 pOLYG中。用间接免疫荧光染色法 ,检测该载体能够携带并表达的Derp2基因在牡牛分支杆菌宿主中表达。结果 :经测序证实 ,所克隆的 19 ss基因序列正确。所构建的含有 19 ss基因的E .coli BCG穿梭载体 (pCW )能完成在大肠杆菌和牡牛分支杆菌细胞之间的穿梭 ,并介导抗生素抗性基因表达。经免疫荧光检查 ,外源基因Derp2以脂蛋白的形式表达于分支杆菌宿主的表面。 结论 :成功地构建了以胞壁嵌合形式表达外源蛋白的E .coli BCG穿梭载体
OBJECTIVE: To construct E.coli BCG shuttle vector which can carry Derp2 gene and express in cell wall. Methods: The mtDNA of the 19kDa antigen of Mycobacterium tuberculosis and its upstream regulatory element (19 ss) were amplified by PCR from Mycobacterium tuberculosis H37Rv gene and cloned into E. coli BCG shuttle containing Derp2 gene Vector pOLYG. Indirect immunofluorescence staining was used to detect the expression of Derp2 gene, which the vector can carry and express, in Mycobacterium bovis. Results: The sequencing confirmed that the cloned 19 ss gene was correct. The constructed E. coli BCG shuttle vector (pCW) containing the 19 ss gene completes the shuttle between E. coli and M. bovis cells and mediates antibiotic resistance gene expression. After immunofluorescence, the foreign gene Derp2 was expressed as a lipoprotein on the surface of mycobacterial host. CONCLUSION: E. Coli BCG shuttle vector expressing the foreign protein in the form of cell wall chimera was successfully constructed