Journal of Vaccines & Vaccination

Cloning and expression of immunogenic molecules of L. major promastigote by Patient?s immune sera

2^nd International Conference on Vaccines and Vaccination

August 20-22, 2012 Hilton Chicago/Northbrook, USA

Hemanta Koley, Soumik Barman, Dhira Rani Saha and Ranajit Kumar

Scientific Tracks Abstracts: J Vaccines Vaccin

Abstract:

L eishmaniasis is caused by parasitic protozoa transmitted by the bite of female sand fly and is currently endemic in 88 countries, affecting 12 million people worldwide and threatening 350 million more. The susceptibility of human to parasite has been attributed in part to the expansion of Th2 cells and production of their cytokines, IL-4 and IL-10 and down-regulation of Th1 cytokine, INF-γ. Leishmaniasis treatment involves pentavalent antimony or amphotericin B, but with diverse side-effects and generation of drug-resistant organisms. Therefore, it is necessary to discover new drugs and an effective vaccine. In this report we tried to identify the immunogenic leishmania major proteins using human immune sera that were infected and cured from the leishmania major infection. mRNA of L. major promastigotes of stationary phase was isolated and cDNA was made using Invitrogen CloneMiner kit. cDNA was inserted into kanamycin resistant pDONR222 plasmid and then transferred into DH5a competent E. coli. The positive plasmids were inserted into pDEST17 vector and transferred into BL-21 cells for expression. The colonies were monitored for expressed proteins by overlapping nitrocellulose filters impregnated with 10 mM IPTG. Filter was incubated with patient?s immune sera and after washing, HRP-conjugated goat anti-human Ig was added and finally expressed proteins were detected with DAB substrate. The immunopositive clones were picked, and stored for further expression and purification of proteins to be testes as candidate vaccine.