Synthesis of recombinant KMP11 / LACK proteins of Leishmania major in prokaryotic host: an experimental candidate model as a vaccine

Introduction and Objectives: An attempt to build a vaccine against cutaneous leishmaniasis since 100 years ago. In order to reduce the side effects of dead and or weak live vaccines, studies on the second generation of vaccines, namely vaccine subunits, are ongoing. The aim of this study, construct and purify two KMP11 and LACK antigens of L. major in prokaryotic hosts. Methods: The gene sequences of LACK proteins (NC_007269.2) and KMP11 (NC_007284.2) were obtained from the Gene Bank. These genes synthesized for production of prokaryotic protein inside the pET28a-TEV vector. To extract of these proteins after expression, His-tag sequence for LACK protein and S-tag sequence for the KMP11 protein were inserted in the construct. The pET28a-TEV / LACK-KMP11 construct was transformed into E. coli XL1-Blue bacteria. To express of the protein, cells were induced with IPTG. After inducing, the bacteria disintegrated by ultrasound, freezing and thawing processes. Results: The collapsed body bacteria were separated from the medium by centrifuging. Extraction of LACK protein from His Trap HP affinity columns and KMP11 protein extraction from S-protein Agarose was performed. Concentration of extracted proteins was done by centrifugal filter unit with a limit of 10000 nominal molecular weight limit. Superdex 200 gel-filtration column (GE) columns were used for further purification of the solutions obtained. Finally, pure proteins were confirmed by Western Blot technique. Discussion: In this study, two LACK / KMP11 antigens were made and purified in E. coli. Two KMP11 and LACK antigens of l. major are effective immunogenic antigens. The simultaneous use of these two antigens as recombinant subunit vaccines can be considered as a new model in laboratory animals. Acknowledgments: The authors would like to thank the National Institute for Medical Research Development (NIMAD), for financial support (Grant number: 957777).

کلیدواژه ها:

Recombinant KMP11 / LACK proteins ، Leishmania major ، prokaryotic hosts ، vaccine.