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目的 构建编码结核分支杆菌 (MTB)Ag85B分泌蛋白的重组真核表达质粒 ,并研究其免疫原性。方法 采用聚合酶链反应 (PCR)方法从结核分支杆菌H3 7Ra 基因组DNA中扩增出Ag85B分泌蛋白基因 ,用HindⅢ和EcoRⅠ消化后 ,与同样酶消化的pcDNA3连接 ,转化大肠杆菌JM10 9,阳性克隆用酶切鉴定 ;重组表达质粒肌注免疫小鼠 4周后 ,分别用dotblotting和ELISA方法检测抗体的产生和滴度。结果 酶切鉴定重组表达质粒pTB30s构建成功 ;dotblotting检测免疫小鼠血清抗Ag85B特异性抗体阳性 ,ELISA检测抗体几何平均滴度为 1∶12 0。结论 应进一步研究pTB30s刺激机体的细胞免疫应答 ,以用于结核病 (TB)的防治研究
Objective To construct recombinant eukaryotic expression plasmid encoding Mycobacterium tuberculosis (MTB) Ag85B and study its immunogenicity. Methods Ag85B secreted protein gene was amplified from the genomic DNA of Mycobacterium tuberculosis H3 7Ra by polymerase chain reaction (PCR), digested with Hind Ⅲ and EcoRI, ligated with pcDNA3 digested with the same enzymes and transformed into E. coli JM10 9. The positive clones The recombinant plasmids were identified by restriction enzyme digestion. Four weeks after the recombinant plasmids were immunized, the production and titer of antibody were detected by dot blotting and ELISA, respectively. Results The constructed recombinant plasmid pTB30s was identified by restriction enzyme digestion. Dotblotting was used to detect the anti-Ag85B specific antibody in the serum of the immunized mice. The geometric mean titer of the antibody was 1:12 0. Conclusions The cellular immune response to pTB30s stimulation should be further studied for the prevention and treatment of tuberculosis (TB)