三重表达肺结核DNA疫苗在细胞水平的检测

目的 检测共表达siRNA、抗原基因与hIL-12的新型肺结核DNA疫苗在人胚肾293细胞中表达。方法 在已构建含有靶向抗调亡基因Mcl-1L的siRNA、结核分枝杆菌抗原基因Ag85B-ESAT6 (PVAE)、hIL-12的新型肺结核DNA疫苗pVAX-siRNA-PVAE-IL-12基础之上，将抗原基因与增强型绿色荧光蛋白（EGFP）基因融合，得到真核表达重组质粒pVAX1-siRNA-PVAE-EGFP-hIL12。并将siRNA单元用靶向EGFP基因的siEGFP代替，得到pVAX1-siEGFP-PVAE-EGFP-hIL12重组表达载体。用重组质粒转染人胚肾细胞293，分别观察融合抗原基因与siEGFP的表达。以ELISA测定培养细胞上清中hIL-12的表达。结果 经酶切鉴定和测序证实肺结核DNA疫苗改造成功。转染细胞中检测到绿色荧光，证实抗原表达。对照组检测到siEGFP的表达。转染48 h后检测出细胞上清中hIL-12的量为1571.63 pg/mL；72 h后检测出细胞上清中hIL-12的量为2392.25pg/mL。结论 已构建的肺结核质粒DNA疫苗能在真核细胞中有效表达siRNA、抗原基因与hIL-12。为进一步研究该DNA疫苗抗肺结核的免疫治疗和基因治疗效果打下基础。

The Detection of Triple Expression of Tuberculosis DNA Vaccines on the Cell Level

Objective To express a new tuberculosis DNA vaccine expressing siRNA, the Mycobacterium tuberculosis fusion antigen gene and the hIL-12 in human embryonic kidney 293 cells. Methods On the basis of the new tuberculosis DNA vaccine expressing siRNA targeted Mcl-1L, the Mycobacterium tuberculosis fusional antigen gene Ag85B and ESAT6(PVAE) and the hIL-12, it is fused with enhanced green fluorescent protein(EGFP) gene and get the eukaryotic expression vector pVAX1-siRNA-PVAE-EGFP-hIL12. Use siEGFP targeted EGFP instead of the siRNA and get the expression vector pVAX1-siEGFP-PVAE-EGFP-hIL12. Then the two recombinant plasmids was transfected 293 cells, observe the expression of Ag85B and ESAT6 fusion antigen gene through the expression of the green fluorescent protein and the siRNA. ELISA analyze the expression of hIL-12. Results Coexpression vector of MTB DNA vaccine was changed successfully by the restriction enzyme analysis and sequencing. Green fluorescent can be detected in the transfected cells which confirm the expression of the fusion antigen gene and the siRNA. The mount of hIL-12 excretion was 1571.63pg/ml supernatant 48h after transfection, 2392.25pg/ml supernatant 72h after transfection. Conclusion The DNA vaccine co-expressing the MTB fusion antigen gene and the hIL-12 was constructed and transiently expressed in 293 cells. It laid a foundation of further study on anti-MTB therapeutic effect of the new DNA vaccine.