【摘要】 目的体外转录结核分枝杆菌Ag85B-mRNA并评价其免疫原性。方法结合密码子偏好性、GC含量和mRNA二级结构的自由能,优化Ag85B编码区序列,并在其两端插入β球蛋白3’和5’UTR序列。合成目的序列,进行体外转录。经琼脂糖凝胶电泳鉴定正确,体外转染细胞验证其表达后,联合鱼精蛋白免疫Balb/c小鼠,检测特异性细胞免疫应答。结果 Ag85B优化序列二级结构分析显示,其具有更高的稳定性。成功体外合成稳定的Ag85B-mRNA。Western blot检测Ag85B-mRNA转染293T细胞24、48 h,均有清晰特异性的目的蛋白条带。小鼠免疫试验结果显示,Ag85B-mRNA可诱导针对结核分枝杆菌Ag85B分泌高水平IFN-γ的Th1型免疫应答。结论成功体外合成稳定的Ag85B-mRNA疫苗,具有较好的免疫原性,为新型结核疫苗的研制提供新思路。更多还原

【Abstract】 This study was performed to transcript Mycobacterium tuberculosis Ag85 B-mRNA and evaluate its immunogenicity in vitro. The Ag85 B coding sequence was optimized by combining codon preference, GC content and free energy of mRNA secondary structure, while the β globin 3’and 5’UTR sequences were inserted at both ends.The sequence of interest is synthesized and subjected to in vitro transcription, then identified by agarose gel electrophoresis. After cell transfection and expression confirmation in vitro, the Ag85 B-mRNA was used to immunize Balb/c mice with protamine for evaluating specific cellular immune responses. Secondary structure analysis showed that optimized sequence of Ag85 B has higher stability. The stable Ag85 B-mRNA was constructed in vitro. Western blot showed after Ag85 B-mRNA transfection into 293 T cells for 24 and 48 hours, there were clear and specific target protein bands. The results of mouse immunoassay showed that Ag85 B-mRNA could induce high level of IFN-γ against mycobacterial Ag85 B which was responsible for Th1-type immune response. In conclusion,the synthesized stable Ag85 B-mRNA vaccine in vitro has good immunogenicity, which lays a foundation for the improvement of mRNA vaccine research and provides new ideas for the development of tuberculosis vaccine.更多还原