结核分枝杆菌Ag85B和ESAT-6基因重组产单核细胞李斯特菌的构建  

Construction of recombinant Listeria monocytogenes expressing Mycobacterium tuberculosis antigens Ag85B and ESAT-6

作　　者：董慧[1] 焦新安[1] 殷月兰[1] 田德斌[1] 潘志明[1] 刘松婷[1] 方丽君[1] 

机构地区：[1]扬州大学江苏省人兽共患病学重点实验室,江苏扬州225009

出　　处：《中国兽医科学》2009年第8期707-711,共5页Chinese Veterinary Science

基　　金：国家重点基础研究发展计划(973)项目(CB504404);国家自然科学基金项目(30425031);江苏省科技攻关项目(BE2007340;BK2008011)

摘　　要：对编码结核分枝杆菌抗原Ag85B和ESAT-6的基因片段进行了扩增,并将其克隆入穿梭载体pKSV7,利用基因同源重组技术构建了表达结核分枝杆菌抗原Ag85B和ESAT-6的重组产单核细胞李斯特菌(LM)。结果表明,外源基因成功整合入LM基因组并获得转录;Western-blot分析结果显示,重组菌携带的外源基因在动物体内能够表达;溶血试验与细胞感染试验表明,较之野生型李斯特菌,外源基因的插入破坏了重组菌LM(pKSV7-H85-6B)的溶血活性,显著降低了对细胞的侵袭能力。证实,携带结核分枝杆菌保护性抗原Ag85B和ESAT-6的重组李斯特菌对结核病新型疫苗的研究具有潜在应用价值。The genes encoding Ag85B and ESAT-6 antigens of Mycobacterium tuberculosis were amplified and cloned into shuttle plasmid pKSV7.By using homologous recombination technique,a recombinant Listeria monocytogenes strain LM-4（pKSV7-H85-6B）expressing Ag85B and ESAT-6 was constructed.The results of PCR confirmed that the heterologous genes were inserted into the genome of L.monocytogenes.RT-PCR results showed that foreign genes were transcribed normally.Western-blot analysis confirmed that these two genes were successfully expressed in mice after vaccination.Compared with the wild type bacte-ria,the hemolytic activity of LM（pKSV7-H85-6B） was dramatically decreased,and the recombinant strain showed the reduced invasiveness to the CaCo-2 cells.All these results showed that LM（pKSV7-H85-6B） had the potential practical value in the research of new vaccines against tuberculosis.

关 键 词：结核病 结核分枝杆菌 产单核细胞李斯特菌 同源重组 

分 类 号：S852.618[农业科学—基础兽医学]