Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012;7(3):48-53.

Expression and Purification of P43 Toxoplasma gondii Surface Antigen

K Khanaliha  1 , Mh Motazedian, B Sarkari, M Bandehpour, Z Sharifnia, B Kazemi
Affiliations

Expression and Purification of P43 Toxoplasma gondii Surface Antigen

K Khanaliha et al. Iran J Parasitol. 2012.

Abstract

Background: Toxoplasma gondii is an obligate intracellular protozoan parasite, capable of infecting all species of mammals including man. Congenital toxoplasmosis is more important during pregnancy for the first time. In this study we expressed and purified P43 Toxoplasma gondii tachyzoite and bradyzoite specific surface antigen.

Methods: The recombinant pGEMEX-1 contained Toxoplasma P43 coding sequence was transformed into E. coli and mass cultured in LB medium contained 100 μg/ml ampicillin at 37°C over night. The T7 promoter was induced by 1mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG. Recombinant protein was purified by affinity chromatography and confirmed by gel diffusion dot blot and western blot,-using specific anti Toxoplasma antibodies.

Results: Recombinant plasmid was induced by IPTG and analyzed by SDS-PAGE. Recombinant protein was confirmed by Western-blot and dot blot using anti human Toxoplasma antibody.

Conclusion: Recombinant Toxoplasma P43 was produced successfully.

Keywords: Bradyzoite; Recombinant P43; Tachyzoite; Toxoplasma.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Confirmation of recombinant pGEME43.A PCR was performed using Toxo43 primer and PCR product was separated by electrophoresis on a 1.5% agarose gel. Lane 1 pGEMEX-1 without the SAG3 insert:. Lane 2: pGEMEX-1 containing the SAG3 insert (1158bp). Lane 3: 100bp DNA ladder marker
Fig. 2
Fig. 2
Restriction analysis of the pGEMEX43. Lane 1: un digested pGEMEX-1. Lane2: recombinant pGEMEX43 digested by PstI. There is no restriction site for PstI on pGEMEXI plasmid but there is one restriction site for PstI at position 748-753 on P43 gene
Fig. 3
Fig. 3
SDS-PAGE analysis of rSAG3 expression using 12% SDS-PAGE. lane 1: protein marker. Lane 2: 65 kDa expressed SAG3 with fusion protein 3 hours after induction. Lane 3: expressed of SAG3 fusion protein 5hours after induction. Lane 4: lysate of non-induced bacterial cells
Fig. 4
Fig. 4
Dot blot analysis of rSAG3 protein reacted with Toxoplasma gondii- positive serum. Left: recombinant SAG3. Right: lysate of bacterial cells from non-induced culture
Fig. 5
Fig. 5
Western blot analysis of the rSAG3 protein using Toxoplasma gondii- positive serum. Lane 1: lysate of non-induced bacterial cells. Lane2: expresed SAG3 2 hours after induction. Lane3: expressed SAG3 3 hours after induction. Lane4: expressed SAG3 5 hours after induction. Lane 5: expressed SAG3 7 hours after induction. Lane 6: protein marker
Fig. 6
Fig. 6
Gel difiussion of the rSAG3 using Toxoplasma gondii-positive serum. Well 1: recombinant SAG3. Well 2: lysate of bacterial cells from non-induced culture
See this image and copyright information in PMC

References

    1. McCabe R, Remington JS. Toxoplasmosis:the time has come. N Engl J Med. 2000;318:313–315. - PubMed
    1. Santoro F, Afchain D, Pierce R, Cesbron JY, Ovlaque G, Capron A. Serodiagnosis of Toxoplasma infection using a purified parasite protein (P30) Clin Exp Immunol. 1985;62(2):262–269. - PMC - PubMed
    1. Kavakeb P, Kazemi B, Dorostkar MD. Cloning, expression and characterization of Toxoplasma gondii P35 protein in E. col . Yakhteh Medical Journal. 2006;8(3):190–195.
    1. Kazemi B, Maghen L, Bandehpour M, Shahabi S, Haghighi A. Gene cloning of p43 surface protein of Toxoplasma gondii tachyzoite and bradyzoite (SAG3) Gene Therapy and Molecular Biology. 2007;11:113–116.
    1. Nagel SD, Boothroyd JC. The major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid. J Biol Chem. 1989;264(10):5569–5574. - PubMed

LinkOut - more resources