结核杆菌免疫保护性抗原Ag85A的基因克隆与表达研究

Molecular Cloning and Expression of the Immunodominant Protein Ag85A from Mycobacterium tuberculosis H37Rv Strain

目的 为结核病新型疫苗研制提供靶基因和靶抗原。方法 根据结核杆菌 H 37Rv株免疫保护性抗原 Ag85 A的基因序列自行设计一对寡核苷酸引物 ,以结核杆菌 H37Rv株基因组 DNA为模板 ,通过 PCR扩增获得具有完整开架读码框 (ORF)的 Ag85 A抗原基因 ,并将其正向插入真核和原核穿梭表达型载体 p BK- CMV构建重组质粒 ,重组质粒转入大肠杆菌后 IPTG诱导表达 ,并对其表达产物进行 Western- blotting分析。结果 PCR扩增获得了具有完整开架读码框的 Ag85 A抗原基因 ;构建了 Ag85 A抗原基因真核和原核穿梭表达型载体 ;Ag85 A抗原基因在大肠杆菌中实现了稳定表达。结论 成功地对结核杆菌免疫保护性抗原 Ag85 A进行了基因克隆与表达 。

Objective To provide the target gene and target antigen for the development of new vaccine against tuberculosis. Methods According to the gene sequence encoding protein Ag85A from Mycobacterium tuberculosis H37Rv strain, we designed a pair of oligonucleotide primers, obtained the gene by using polymerase chain reaction, and inserted the gene into the BamHⅠand EcoRⅠsite of plasmid pBK-CMV to construct recombinant plasmid, and after that, the recombinant plasmid was transferred into E.coli XL1-Blue MRF′and induced with IPTG. The expression product of the gene was analyzed by using SDS-PAGE and western-blotting. Results The gene encoding the protein Ag85A of Mycobacterium tuberculosis H37Rv strain was successfully amplified by using PCR.A recombinant shuttle plasmid was constructed. The recombinant plasmid stably expressed recombinant Ag85A protein relative molecular mass 32×10 3 in E.coli XL1-Blue MRF ′. Conclusion A recombinant plasmid which contains the gene encoding the protein Ag85A of Mycobacterium tuberculosis H37Rv strain has been successfully constructed.The recombinant plasmid can stably express recombinant protein relative molecular mass 32×10 3 in E.coli XL1-Blue MRF′. These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of new vaccine against tuberculosis.