Leishmania mexicana recombinant filamentous acid phosphatase as carrier for Toxoplasma gondii surface antigen 1 expression in Leishmania tarentolae

10.21203/rs.3.rs-303993/v1 ◽

2021 ◽

Author(s):

Dalia Ahmed Kalaf

Keyword(s):

Acid Phosphatase ◽

Toxoplasma Gondii ◽

Fusion Protein ◽

Protein Complex ◽

Ribosomal Subunit ◽

Surface Protein ◽

Host Cells ◽

Leishmania Mexicana ◽

Leishmania Tarentolae ◽

Fusion Construct

Abstract Leishmania tarentolae has been used to produce recombinant intracellular and secreted proteins for their easy handling and posttranslational modifications. Filamentous acid phosphatase is a multimeric protein complex composed of many subunits assembled in a linear highly glycosylated filament, which is secreted in vast amounts into the culture supernatant via the flagellar pocket of Leishmania mexicana promastigotes. This suggested that the protein could be used as a carrier for other proteins for easy purification to generate a protein complex decorated with multiple SAG1 subunits suitable for immunisation. Surface Antigen1 protein of a Toxoplasma gondii has an immunodominant structure that is involved in binding to host cells. Previous studies used this surface protein for vaccination for its immunological importance for triggering a type 1 immune response in the host. This study aims to determine the production of recombinant filamentous protein carried subunits of the surface protein of Toxoplasma gondii for vaccination purposes. Leishmania codon-optimised SAG1 was cloned as a fusion construct into pLEXSY-ble2.1 and introduced into Leishmani. tarentolae to generate recombinant cell lines expressing of a filamentous fusion protein called SAP2SAG1. PCR confirmed the correct integration into the small ribosomal subunit RNA gene locus of Leishmania tarentolae. Immunofluorescences and Immunoblot analyses were used to detect the fusion protein in the sediment of culture supernatants of recombinant L. tarentolae promastigotes after purification by ultracentrifugation. The yield of purified protein was low that suggested further investigations of other methods for scaling large production of secreted protein.

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Analysis of Structures and Epitopes of Surface Antigen Glycoproteins Expressed in Bradyzoites ofToxoplasma gondii

BioMed Research International ◽

10.1155/2013/165342 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Hua Cong ◽

Min Zhang ◽

Qingli Zhang ◽

Jing Gong ◽

Haizi Cong ◽

...

Keyword(s):

Immune Response ◽

Toxoplasma Gondii ◽

Immune Responses ◽

Surface Antigen ◽

Protein Sequence ◽

Protozoan Parasite ◽

3D Models ◽

Chronic Infections ◽

Homologous Proteins ◽

Protein Sequence Alignment

Toxoplasma gondiiis a protozoan parasite capable of infecting humans and animals. Surface antigen glycoproteins, SAG2C, -2D, -2X, and -2Y, are expressed on the surface of bradyzoites. These antigens have been shown to protect bradyzoites against immune responses during chronic infections. We studied structures of SAG2C, -2D, -2X, and -2Y proteins using bioinformatics methods. The protein sequence alignment was performed by T-Coffee method. Secondary structural and functional domains were predicted using software PSIPRED v3.0 and SMART software, and 3D models of proteins were constructed and compared using the I-TASSER server, VMD, and SWISS-spdbv. Our results showed that SAG2C, -2D, -2X, and -2Y are highly homologous proteins. They share the same conserved peptides and HLA-I restricted epitopes. The similarity in structure and domains indicated putative common functions that might stimulate similar immune response in hosts. The conserved peptides and HLA-restricted epitopes could provide important insights on vaccine study and the diagnosis of this disease.

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