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目的 Ag85A和Ag85B是结核分枝杆菌的主要优势保护抗原,为提高编码Ag85A和Ag85BDNA疫苗的免疫性和免疫效果,特构建真核共表达Ag85A和Ag85B抗原的真核表达载体pcDNA-Ag85A-IRES-Ag85B,并利用293T细胞其表达进行检测。方法 PCR扩增Ag85A和Ag85B基因,逐步将Ag85A和Ag85B基因定向插入至质粒pcDNA3.1(+)克隆位点CMV启动子与加A信号BGH poly(A)之间,并将Ag85A和Ag85B基因以内部核糖体进入位点(internal riboome entry site,IRES)序列连接,酶切和测序验证后将重组质粒转染至293T细胞中进行真核表达,48h后提取细胞总蛋白,达产物经SDS-PAGE分离和Western blot分析。结果限制性内切酶酶切及测序结果表明,重组质粒pcDNA-Ag85ARES-Ag85B基因序列和阅读框架正确。将重组质粒转入293T细胞后,利用Ag85A和Ag85B特异性抗体Western blot检测实两种抗原可在同一载体中各自独立表达。结论成功构建了能够同时独立表达结核分枝杆菌Ag85A和Ag85B抗原基的真核表达载体pcDNA-Ag85A-IRES-Ag85B,为下一步研究它们的免疫原性和免疫效果奠定了基础。
Objective Ag85A and Ag85B are the main protective antigens of Mycobacterium tuberculosis. To improve the immunity and immune effect of Ag85A and Ag85B DNA vaccines, eukaryotic expression vector pcDNA-Ag85A-IRES- Ag85B, and the use of 293T cells to detect its expression. Methods The Ag85A and Ag85B genes were amplified by PCR. The Ag85A and Ag85B genes were inserted into the plasmid pcDNA3.1 (+) cloning site CMV promoter and A signal BGH poly (A) Then the recombinant plasmids were transfected into 293T cells for eukaryotic expression. After 48h, the total cellular proteins were extracted and the products were analyzed by SDS-PAGE Isolation and Western blot analysis. Results Restriction endonuclease digestion and sequencing results showed that the recombinant plasmid pcDNA-Ag85ARES-Ag85B gene sequence and reading frame is correct. The recombinant plasmid was transfected into 293T cells, using Ag85A and Ag85B specific antibodies Western blot detection of the two antigens can be independently expressed in the same vector. Conclusion The eukaryotic expression vector pcDNA-Ag85A-IRES-Ag85B capable of independent expression of Mycobacterium tuberculosis Ag85A and Ag85B antigens was successfully constructed, which laid the foundation for further study on their immunogenicity and immunological effects.