Construction, Expression and Identification of a Recombinant BCG Vaccine Encoding Human Mycobacterium Tuberculosis Heat Shock Protein 65

• Published in: Current medical science Vol. 24; no. 2; pp. 107 - 111
• Main Author: 载五星 梁靓 高红 黄海浪 陈智浩 程继忠 皇甫永穆
• Format: Journal Article
• Language: English
• Published: China Department of Biochemistry and Molecular Biology, the School of Basic Medical Science, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China 2004
• Subjects: Bacterial Proteins - biosynthesis; Bacterial Proteins - immunology; BCG Vaccine - biosynthesis; BCG Vaccine - immunology; Chaperonin 60; Chaperonins - biosynthesis; Chaperonins - immunology; Cloning, Molecular; Escherichia coli - metabolism; Genetic Vectors; Humans; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - immunology; Plasmids - genetics; Sequence Analysis, DNA; Vaccines, Synthetic - biosynthesis; Vaccines, Synthetic - immunology; 免疫原; 基因表达; 热休克蛋白65; 结核分支杆菌; 重组BCG疫苗; genetic vectors; Mycobacterium tuberculosis; heat shock proteins; BCG vaccine; gene expression
• ISSN: 1672-0733; 2096-5230; 1993-1352; 2523-899X
• DOI: 10.1007/BF02885404

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA se-quence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two se-quences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plas-mid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA se-quencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M.tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.