XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:46-1

Poster (Painel)

46-1	Heterologous expression and preliminary antigenic characterization of the orthologous gp43 from Paracoccidioides lutzii
Authors:	Natanael Pinheiro Leitão Júnior (UNIFESP - Universidade Federal de São Paulo) ; Milene Carmes Vallejo (UNIFESP - Universidade Federal de São Paulo) ; Palloma Mendes Conceição (UNIFESP - Universidade Federal de São Paulo) ; Rosana Puccia (UNIFESP - Universidade Federal de São Paulo)

Abstract

Glycoprotein gp43 is the main diagnostic antigen of paracoccidiodomycosis (PCM). Our laboratory has produced soluble recombinant gp43 isoforms that are useful in the diagnosis of PCM produced by Paracoccidioides brasiliensis phylogenetic groups S1, PS2 and PS3. Pb01-like isolates have recently been proposed as a new species, P. lutzii. The gp43 ortholog from Pb01 (Plgp43), is only 80% identical to gp43, which might impact the gp43-based diagnosis of PCM. The aim of this work was to express the PlGP43 in Escherichia coli and to test the antigenicity of the recombinant protein. Total RNA was isolated from Pb01 and used to amplify the PlGP43 cDNA with specific primers, which was then cloned into pGEM-T for maintenance and sequencing. The fragment was subcloned in the Sal1 and Kpn1 sites of the expression plasmid pHIS1, which was used to transform Escherichia coli BL21pLys. One recombinant clone expressing PlGP43 bearing a His6 tag was selected for large scale production of the recombinant protein. Induction of the foreign protein was carried out in LB broth (Luria-Bertani) for 4h at 37ÂºC, under shaking, upon addition of 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside). Bacterial extracts were analyzed for protein expression in SDS-PAGE 10%, where we could observe that the protein was present as inclusion bodies. Therefore, the extracts were solubilized in urea for fractionation in nickel columns (Ni Sepharose 6 Fast Flow, GE Healthcare). The enriched His-containing proteins were subjected to SDS-PAGE and transferred by Western blot to nitrocellulose membranes. Reactivity of the recombinant protein was tested by immunoblotting with sera from PCM patients from Southern and Southeastern Brazil, and anti-gp43 polyclonal and monoclonal antibodies. Native gp43 from Pb339 was used as positive control. Preliminary tests showed that recombinant PlGP43 was weakly recognized by the PCM sera tested when compared with native gp43. The same was observed with anti-gp43 polyclonal and monoclonal Mab17c. Our preliminary results suggest that antigenic differences between gp43 from P. brasiliensis and P. lutzii exist. We are currently trying to express soluble recombinant protein in Pichia pastoris. Testing a larger number of PCM sera from different regions of the country must be performed. Support: FAPESP, CNPq and CAPES.

Keyword: gp43, diagnosis, Paracoccidioides lutzii

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