Korean J Parasitol.  2007 Dec;45(4):287-293. 10.3347/kjp.2007.45.4.287.

Purification and biochemical characterization of two novel antigens from Leishmania major promastigotes

Affiliations
  • 1Immunology Laboratory, Institute of Biochemistry and Biophysics, University of Tehran, Iran. ardestani@ibb.ut.ac.ir
  • 2Department of Immunology, Pasteur Institute of Iran, Iran.

Abstract

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.

Keyword

Leishmania major; soluble Leishmania antigen; purification; IgG2a; vaccine

MeSH Terms

Animals
Antigens, Protozoan/*chemistry/*isolation & purification
Blotting, Western
Chromatography, High Pressure Liquid
Electrophoresis, Polyacrylamide Gel/methods
Humans
Leishmania major/growth & development/*immunology
Protein Isoforms/chemistry/isolation & purification
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