Molecular cloning and expression of TB antigen protein in microalga Chlamydomonas reinhardtii

Microalgae based therapeutics has had great success over the last few years. Although biotechnological processes based on transgenic microalgae are still in its infancy, researchers and companies are considering their high potential as bioreactors for drug development. In the present study, an efficient and reproducible protocol for Agrobacterium tumefaciens-mediated genetic transformation and regeneration of microalga Chlamydomonas reinhardtii with esxH gene of Mycobacterium tuberculosis H37Rv under the control of CaMV 35S promoter has been developed. C. reinhardtii strain CC-125 was transformed with A. tumefaciens strain LBA 4404, harbouring the binary vector pCAMBIA 1304 containing the sequence coding for hygromycin phosphotransferase (hpt) as the selectable marker gene, β-glucuronidase (GUS) as the reporter gene and the sequence encoding 10 kDa T-cell antigen (esxH) of M. tuberculosis. The transformation event was confirmed by PCR amplification with hpt, GUS and esxH gene-specific primers. Expression of esxH gene in transgenic Chlamydomonas was confirmed through RT-PCR. In future, this microalgal expression system can be used to meet the ever growing need for therapeutic proteins by the pharmaceutical industries.