Cloning and Expression of Leishmania infantum LPG3 Gene by the Lizard Leishmania Expression System

Abstract

Background: Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum (L.infantum).

Methods: The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining.

Results: Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed.

Conclusion: These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 (rLPG3) to further research in vaccine designing against leishmaniasis.

Keywords: Leishmania; Leishmania infantum; Recombinant proteins; Vaccines.

Figure 1

Detection of the recombinant plasmid pTZ57R vector-LPG3 by PCR and restriction enzyme digestion: amplified LPG3 gene (Lane 1), restriction analysis of pTZ57R-LPG3 vector (Lane 2), undigested vector (Lane 3), and 1 kb DNA size marker (Lane M). The products were electrophorased on 1% agarose gel

Figure 2

Detection of the recombinant pLEXSY-LPG3 vector by PCR and restriction enzyme digestion: amplified LPG3 gene (Lane 1), restriction analysis of pLEXSY-LPG3 vector (Lane 2), undigested vector (Lane 3), and 1 kb DNA size marker (Lane M). The products were electrophorased on 1% agarose gel

Figure 3

Sequence alignment of the deduced amino acid sequence of L.infantum LPG3 with other related proteins. The sequences were aligned with CLC protein workbench 5.5.1 software. The identical residues are indicated by dots. The boxes indicate the signal sequence at the N-terminus (box I), ATPase_c domain (box II), dimerization domain (box III), and the C-terminal ER retention signal (box IV). The GXXGXG motif (grey letters in box II) is also conserved in all Hsp90 family proteins. Potential N-linked glycosylation sites are indicated by gray underlines and protein kinase C phosphorylatin sites are marked by asterisks. Hyphens represent the introduced gaps for the optimum alignment.

Figure 4

Confirmation of genomic integration into the ssu locus of Lizard Leishmania by diagnostic PCR with the forward LPG3 and ssu reverse primers: transfected cells with pLEXSY-LPG3 vector (Lane 1), wild type cells (Lane 2), and 1 kb DNA size marker (Lane M). The products were electrophorased on 1% agarose gel

Figure 5

Western blot analysis of the transfected Leishmania cells lysate: lysate of wild type cells (Lane 1), lysate of cells transfected with pLEXSY-LPG3 expression vector blotted with anti-His tag monoclonal antibody (Lane 2), and molecular weight markers (Lane 3)

Figure 6

Indirect imunofluorescence analysis: (A, B, C), wild type cells; (D, E, F), transfected cells; (A, D), phase-contrast images; (B, E), DAPI staining, indicating the localization of DNA in the nucleus and kinetoplast in blue, and (C, F) FITC staining (green) using the anti-His tag monoclonal antibody diluted (1:40) for wild type and transfected cells (× 40).