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| 结核杆菌抗原多表位融合蛋白的重组表达与鉴定 |
| 吴传勇,娄加陶,蒋廷旺,钱琤,周晔,陈燕,谷明莉,邓安梅,仲人前 |
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| (第二军医大学长征医院实验诊断科,解放军临床免疫中心,200003) |
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| 摘要: |
| 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40 000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。 |
| 关键词: 分枝杆菌,结核 表位 疫苗,合成? |
| DOI：10.3724/SP.J.1008.2006.01281 |
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| 基金项目: |
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| Recombinant expression of multiepitope fusion polypeptide derived from Mycobacterium tuberculosis(Mtb.) antigens? |
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| Abstract: |
| 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.coli. 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 isopropylthio-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). Conclusion: The recombinant multiepitope fusion polypeptide may provide a basis for developing novel TB vaccine. |
| Key words: Mycobacterium tuberculosis epitope vaccines,synthetic? |
