结核分枝杆菌CFP10、ESAT6、Ag85A和Ag85B抗原真核共表达载体的构建与表达

Construction and expression of a eukaryotic vector co-expressing immunodominant antigens of CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis

摘要:

CFP10、ESAT6、Ag85A和Ag85B是结核分枝杆菌的主要免疫优势抗原。为了构建一种可同时表达这4种抗原的真核多基因共表达载体pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB)，并利用HEK 293T细胞对其体外表达进行检测。采用酶切、连接的方法，将CFP10和ESAT6编码基因以 (Gly4Ser) 3蛋白Linker连接，插入至质粒pcDNA3.1(+)多克隆位点CMV启动子与加尾信号BGH pA之间，使两者融合表达，将Ag85A和Ag85B编码基因以内部核糖体进入位点 (Internal ribosome entry site, IRES) 序列连接，并赋予RSV启动子和加尾信号BGH pA，使两者在RSV启动子作用下独立表达。重组质粒经酶切及测序验证后转染至HEK 293T细胞中进行体外表达实验，48 h后提取总蛋白，利用CFP10、ESAT6、Ag85A和Ag85B特异性抗体进行Western blotting检测。结果显示多基因共表达载体pcDNA-CEAB在真核细胞HEK 293T中得到表达，且CFP10、ESAT6、Ag85A和Ag85B抗原能被相应的特异性抗体所识别，表明质粒pcDNA-CEAB构建正确，这为进一步研究其免疫原性和免疫保护效果奠定了基础。

Abstract:

CFP10, ESAT6, Antigen 85A (Ag85A) and antigen 85B (Ag85B) are the key immunodominant antigens of Mycobacterium tuberculosis. In order to construct a eukaryotic vector able to co-express the four genes in one vector, we amplified the target gene fragments encoding the CFP10, ESAT6, Ag85A and Ag85B antigens and inserted them into the multicloning site of the shuttle plasmid vector pcDNA3.1 (+), of which the CFP10 and ESAT6 encoding genes were in frame fused with a linker encoding (Gly4Ser)3 residue, before the fused gene was inserted downstream of CMV promoter with a bovine growth hormone poly A(BGH pA) sequence at the 3’-end; Ag85A and Ag85B encoding genes were fused with a separation of internal ribosome entry site (IRES) sequence before the fused gene cassette was inserted downstream of RSV promoter with a BGH pA sequence at the 3’-end. The final plasmid containing all four genes was confirmed by sequence analysis and designated as pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB). In order to verify the ability of this construct to express target proteins, we then transfected the recombinant plasmid into Human embryonic kidney (HEK) 293T cells and harvested the cell lysates, and the cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis 48 h after transfection. All four of the target proteins were detected in the cell lysates against the respective specific antibodies, suggesting that we have successfully constructed a eukaryotic vector co-expressing the four immunodominant antigens of Mycobacterium tuberculosis, which lay a foundation for the further study of the immunogenicity and protective activity of the four antigens.