Der p2重组胞壁型E.coli-BCG穿梭表达载体的构建和鉴定

Construction and identification of the E.coli-BCG shuttle vector expressing lipoprotein Der p2 on cell wall of mycobacterium vaccae

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

AIM: To construct the E.coli BCG shuttle vector carrying and expressing dust mite antigen gene Der p2 on cell wall of mycobacterium vaccae . METHODS: The gene fragment encoding 19 kDa antigen and the upstream control element (19 ss) was amplified by PCR from the mycobacteria tuberculosis H37Rv. Subsequently, the 19 ss gene was cloned into the E.coli BCG shuttle vector pOLYG, which can schlepped and expressed exogenous antigen gene on cell wall of mycobacteria and containing the Der p2 gene. The expression of Der p2 gene in mycobacterium vaccae determined by indirect immunofluorescence staining.RESULTS: Sequencing proved that the cloned sequence of 19 ss gene was correct.The constructed E.coli BCG shuttle vector (pCW) containing 19 ss gene had function of shuttle between E.coli and mycobacteria , and mediated the expression of antibiotic resistance gene.Indirect immunofluorescence staining indicated that the Der p2 gene was expressed in the form of lipoprotein on surface of the mycobacterium vaccae . CONCLUSION: E.coli BCG shuttle vector has been constructed successfully, which can express exogenous antigen gene as a chimeric protein on cell wall.