目的:构建含有6个编码结核杆菌抗原HLA—A*0201限制性CD8+CTL表位基因序列的重组质粒,并在大肠杆菌中诱导表达。方法:选取结核杆菌抗原Rv0309、Rv0173、Ag85A、Ag85C的6个HLA-A*0201限制性CD8+CTL表位,引入Th表位PADRE及木马肽序列,并以RVKR序列作为接头,合成全基因序列,经PCR扩增后插入融合表达载体pGEX-4T-1,将重组质粒转入大肠杆菌BL21并以IPTG诱导表达,经SDS-PAGE和Western印迹鉴定重组表达蛋白。结果:成功构建了结核杆菌多表位蛋白的表达质粒,并经IPTG诱导表达了大小约为40000的融合蛋白。结论:多表位融合蛋白的重组表达为进一步开发新型结核疫苗打下了基础。
Objective: To construct a recombinant multiepitope fusion polypeptide containing 6 HLA-A * 0201 restricted CTL epitopes derived from Mycobacterium tuberculosis(Mtb. ) antigens and express it in E. coll. Methods: The full-length sequence encoding 6 HLA-A* 0201 restricted CTL epitopes were derived from Mtb. antigens Rv0309, Rv0173, and Ag85A, Ag85C; pan DR Th epitope (PADRE), Trojan peptide, and furin-sensitive linker RVKR were synthesized, amplified by polymerase chain reaction(PCR), inserted into the expression vector pGEX-4T-1, and transformed into E. coli BL21 (DE3). After induced by isopropyhhio-β-D-galactoside (IPTG), the recombinant protein expression was examined by SDS-PAGE and Western blot. Results: The recombinant multiepitope fusion polypeptide plasmid was successfully constructed and expressed(about 40 000) in E. coli BL21 (DE3). C.ondusion.. The recombinant multiepitope fusion polypeptide may provide a basis for developing novel TB vaccine.