结核杆菌抗原多表位融合蛋白的重组表达与鉴定

Recombinant expression of multiepitope fusion polypeptide derived from Mycobacterium tuberculosis(Mtb.) antigens

目的构建含有6个编码结核杆菌抗原HLA-A0201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-A0201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.