[Cloning, sequencing and expression of HSP70 c-terminal domain in Mycobacterium avium subsp. paratuberculosis]

ABSTRACT

Heat shock proteins [HSPs] have been shown to act as an adjuvant when co-administered with different antigens, especially tumor antigens or antigens from infectious agents. C-terminal domain of Mycobacterium tuberculosis heat shock protein 70 [Hsp70], when fused to peptide antigens, provides a unique structure that is able to induce potent immune responses. In this study, aneukaryotic expression vector pEGFP-N1, containing C-terminal domain of Mycobacterium paratuberculosis HSP 70, Green Fluorescent Protein [GFP] gene in the plasmid construct, was designed for use as a reporter. With GFP system, expression of the target protein was evaluated in the cell culture. The nucleotide sequence of the cloned gene was revealed by sequencing. The protein expression of designed plasmid was also proved by reverse transcriptase polymerase chain reaction [RT-PCR]. Our eukaryotic expression vector [pEGFP-N1 -hsp70 c-terminal] was successfully constructed and HSP70 c-terminal domain protein was expressed effectively. The current experiment, as a basis for a new DNAvaccine design, can be used for the future studies on reverse vaccinology