1. [Production of mycobacterial antigenes merged with cellulose binding protein domain in order to produce subunit vaccines against tuberculosis].
- Author
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Sergienko OV, Liashchuk AM, Aksenova EI, Galushkina ZM, Poletaeva NN, Sharapova NE, Semikhin AS, Kotnova AR, Veselov AM, Bashkirov VN, Kulikova NL, Khlebnikov VS, Kondrat'eva TK, Kariagina-Zhulina AS, Apt AS, Lunin VG, and Gintsburg AL
- Subjects
- Antigens, Bacterial chemistry, Antigens, Bacterial immunology, Base Sequence, Cloning, Molecular, Humans, Molecular Sequence Data, Protein Structure, Tertiary, Tuberculosis microbiology, Tuberculosis prevention & control, Antigens, Bacterial genetics, Genes, Bacterial genetics, Mycobacterium tuberculosis genetics, Recombinant Fusion Proteins genetics, Tuberculosis genetics, Tuberculosis Vaccines genetics, Tuberculosis Vaccines immunology, Vaccines, Subunit genetics, Vaccines, Subunit immunology
- Abstract
Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.
- Published
- 2012