Prokaryotic Expression and Analysis on the ag85b- mpb64 Fusion Gene of Mycobacterium bovis
For raising the antigenicity of Mycobacterium bovis single antigen, fusion protein of two genes was acquired. The DNA fragments of ag85b and mpb64 were fused by splicing by overlapping extension (SOE) polymerase chain reaction(PCR), and the fusion gene ag85b-mpb64 were cloned into pMD-18-T vector, then we got the recombinant plasmid pMD-85b-64. pMD-85b-64 and pET28a(+) were digested by BamH I and EcoR I double enzymes. The purified pMD-85b-64 fusion gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET-85b-64 was constructed. Plasmid containing pET-85b-64 was transformed into competence Escherichia coll BL21 (DE3). The bacterium was induced by isopropyl-β-Dthiogalactopyranoside(IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 58 ku exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western blotting, the results indicated that the protein was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of subunit vaccine and DNA vaccine against bovine tuberculosis.
Cloning Mycobacterium bovis ag85b-mpb64 fusion gene Prokaryotic expression
Xiuyun Jiang Tao Hu Yanru Zheng Darning Gao Chunfang Wang Lei Liu Yanhong Dong Xiaoai Zhu Hongxia Ma Bingjie Li Chengbo Xu
College of Life Sciences Jilin Agricultural University Changchun, China Key Laboratory of Animal Pro College of Animal Science and Technology Jilin Agricultural University Changchun, China Key Laborato College of Animal Science and Technology Jilin Agricultural University Changchun, China KeyLaborator College of Life Sciences Jilin Agricultural University Changchun, China Key Laboratory of AnimalProd
国际会议
上海
英文
1775-1778
2011-10-15（万方平台首次上网日期，不代表论文的发表时间）