Cloning and expression of recombinant protein CFP21 from Mycobacterium tuberculosis as a tuberculosis vaccine candidate

Abstract

Abstract Increased resistance to TB drugs, may render vaccine development a more effective approach to stop or reduce TB epidemics. The antigen Culture Filtrat Protein Filtrat 21 (CPF21) is an immudominant protein encoded in RD 2 region of the Mycobacterium tuberculosis genome, capable of obtaining a strong hypersensitivity reaction and to induce very high interferon-gamma (IFN-γ) responses in patients with tuberculosis. In order to construct the recombinant plasmid pGEM-T Easy-CFP21 and express it in E. coli BL21, the CFP21 gene was amplified from M. tuberculosis H37Rv genomic DNA using PCR in vitro , and inserted into the pGEM-T Easy cloning vector. The recombinant plasmid was then transformed into E. coli JM109, followed by plasmid extraction, PCR amplification, and DNA sequencing. The correct recombinant CFP21 gene was subcloned into expression vector pGEX-2TK and transformed into E. coli BL21 strain. The white recombinant colony was selected, cultured, induced with 50 µM IPTG, and identified using SDS-PAGE electrophoresis method. These results demonstrated that CFP21 gene has been constructed and expressed successfully. The molecular weight was about 47 kDa as the fusion protein GST-CFP21 and expressed as insoluble protein. In conclusion, the target gene CFP21 has been cloned into host E. coli BL-21 strain and expressed successfully. In the future, the purified recombinant fusion protein GST-CFP21 paves the way for TB diagnosis and vaccine development.

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