结核分支杆菌Ag85 B分泌蛋白基因疫苗的构建和免疫原性的研究

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）方法从结构分支杆菌H37Ra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JM109,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dot blotting和ELISA方法检测抗体的产生和滴度,结果：酶切监定重组表达质粒pTB30s构建成功,dot blotting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1：120。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。

Construction and immunogenicity of DNA vaccine encoding secreted form of Ag85B protein of

OBJECTIVE: To construct the recombinant eukaryotic plasmid DNA expression vector encoding Mycobacterium tuberculosis antigen 85B (Ag85B) and to investigate its immunogenicity. METHODS: The gene encoding secreted form of Ag85B was amplified by polymerase chain reaction (PCR) from genome of Mycobacterium tuberculosis H37Ra strain, and was inserted into sites cut with Hind III plus EcoR I of eukaryotic expression vector pcDNA3 after restriction endonuclease digestion. The gene fragment encoding secreted form of Ag85B was inserted into the vector of E. coli JM109 strain and was confirmed by restriction endonuclease digestion. After 4 weeks since BALB/c mice were vaccinated by recombinant eukaryotic expressing vector, dot blotting and ELISA were used to detect the serum antibody against Ag85B and its titer. RESULTS: Recombinant eukaryotic expressing vector, namely pTB30s, constructed successfully based on the gene encoding secreted form of Mycobacterium tuberculosis Ag85B; pTB30s induced high titer specific antibody against Ag85B in immunized mice. CONCLUSION: pTB30s as DNA vaccine should be further studied to confirm its stimulating role in cell-mediated immune responses in TB prevention.