Molecular cloning and expression of TB antigen protein in microalga <i style="mso-bidi-font-style:normal">Chlamydomonas reinhardtii</i>

Abstract

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 <i style="mso-bidi-font-style:normal">Agrobacterium tumefaciens</i>-mediated genetic transformation and regeneration of microalga <i style="mso-bidi-font-style:normal">Chlamydomonas reinhardtii </i>with <i style="mso-bidi-font-style:normal">esxH </i>gene of <i style="mso-bidi-font-style: normal">Mycobacterium tuberculosis </i>H37Rv under the control of<i style="mso-bidi-font-style:normal"> </i>CaMV 35S<i style="mso-bidi-font-style: normal"> </i>promoter has been developed. <i style="mso-bidi-font-style:normal">C. reinhardtii</i> strain CC-125 was transformed with <i style="mso-bidi-font-style: normal">A. tumefaciens</i> strain LBA 4404, harbouring the binary vector pCAMBIA 1304 containing the sequence coding for hygromycin phosphotransferase (<i style="mso-bidi-font-style:normal">hpt</i>) as the selectable marker gene, β-glucuronidase (GUS) as the reporter gene and the sequence encoding 10 kDa T-cell antigen (<i style="mso-bidi-font-style:normal">esxH)</i> of <i style="mso-bidi-font-style:normal">M. tuberculosis.</i> The transformation event was confirmed by PCR amplification with <i style="mso-bidi-font-style: normal">hpt</i>, GUS and <i style="mso-bidi-font-style:normal">esxH</i> gene-specific primers. Expression of <i style="mso-bidi-font-style:normal">esxH</i> gene in transgenic <i style="mso-bidi-font-style:normal">Chlamydomonas</i> 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.