结核分枝杆菌Ag85B+Rv3407融合基因自杀性DNA疫苗的构建及鉴定  

Construction and identification of a suicidal DNA vaccine containing the Mycobacterium tuberculosis Ag85B + Rv3407 fusion gene

作　　者：邵丽军[1] 李猛[2] 伊正君[2] SHAO Li-jun;LI Meng;YI Zheng-jun(School of Public Health and Management, Weifang Medical University, Wei fang , Shandong 261053, China;Department of Laboratory Medicine, Wei fang Medical Univer.~i ty)

机构地区：[1]潍坊医学院公共卫生与管理学院,山东潍坊261053 [2]潍坊医学院医学检验学系

出　　处：《中国病原生物学杂志》2018年第4期368-371,共4页Journal of Pathogen Biology

基　　金：山东省医药卫生科技发展计划项目(No.2013WS0288)

摘　　要：目的构建表达结核分枝杆菌生长期抗原Ag85B和休眠期蛋白Rv3407的自杀性DNA疫苗融合基因表达载体,并检测其在BHK-21细胞中的表达。方法采用PCR方法从结核分枝杆菌基因组中扩增Ag85B基因和Rv3407蛋白基因,再以二者的混合物为模板扩增Ag85B+Rv3407融合基因,构建真核表达载体pSCA1/Ag85B+Rv3407,以ELISA法检测其在BHK-21细胞中的瞬时表达。结果从结核分枝杆菌基因组中扩增得到Ag85B+Rv3407融合基因,片段大小为1 323bp,与预期相符。成功构建重组质粒pSCA1/Ag85B+Rv3407,该重组质粒能在BHK-21细胞中表达目的蛋白(实验组P/N≥2.1)。结论成功构建结核分枝杆菌自杀性DNA疫苗融合基因表达载体,该重新载体能在真核细胞中表达目的蛋白。Objectives To construct a suicidal DNA vaccine using a vector expressing a fusion gene consisting of the Mycobacterium tuberculosis growth antigen Ag85 Band the dormant protein Rv3407 and to detect its expression in BHK-21 cells. Methods The Ag85 Bgene and Rv3407 protein gene were amplified from the M.tuberculosis genome using PCR,and the Ag85 B＋Rv3407 fusion gene was amplified using a mixture of the two as a template to construct the eukaryotic expression vector pSCA1/Ag85 B＋Rv3407.After the vector was identified as correct with enzyme digestion and PCR,transient expression of the vector in BHK-21 cells was detected with ELISA. Results The Ag85 B＋Rv3407 fusion gene was amplified from the M.tuberculosis genome,and the fragment was 1 323 bp in size,which accorded with expectations.The recombinant plasmid pSCA1/Ag85 B＋Rv3407 was verified as correct with enzyme digestion and PCR.The average absorbance（A）of the supernatant of BHK-21 cells transformed with the recombinant plasmid pSCA1/Ag85 B＋Rv3407 was 0.715 according to ELISA,A of the supernatant of BHK-21 cells transformed with the negative control was 0.297,and A of the supernatant of BHK-21 cells transformed with the blank control was 0.146.P/N ≥2.1 for cells transformed with the recombinant plasmid. Conclusion A suicidal DNA vaccine was successfully constructed using a vector expressing a fusion gene from M.tuberculosis.The recombinant vector expressed the target protein in eukaryotic cells.

关 键 词：结核分枝杆菌 自杀性DNA疫苗 生长期抗原Ag85B 休眠期蛋白Rv3407 pSCAl载体 

分 类 号：R378.911[医药卫生—病原生物学]