Construction and in vitro expression analyses of a DNA plasmid encoding dense granule GRA5 antigen of Toxoplasma gondii

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Date: July-September 2011
Publisher: Avicenna Research Institute
Document Type: Report
Length: 3,005 words
Lexile Measure: 1470L

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Abstract

Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii (T.gondii) throughout the world. Although usually asymptomatic, the infection can cause serious medical problems in immunocompromised individuals and fetuses. Toxoplasmosis also causes considerable economic Doss because of abortion in livestock. DNA vaccination is a promising approach against intracellular parasites such as T.gondii. The goal of this study was to construct and evaluate functionality of a mammalian plasmid expressing GRA5 antigen of T.gondii as a possible DNA vaccine. GRA5 gene fragment devoid of the signal sequence, was amplified from genomic DNA of T.gondii RH strain, and cloned into pcDNA3.1 plasmid. The pcDNA3.1-GRA5 (pGRA5) was analyzed by restriction enzyme digestion followed by sequence determination. The pGRA5 was transfected into HER 239-T human kidney cells, and expression of GRA5 antigen was investigated by Western blotting and immunofluorescence staining. The sequence encoding GRA5 was cloned into pcDNA3.1 plasmid. Restriction digestion of pGRA5 with Pst 1 enzyme showed correct insertion of GRA5 DNA into the plasmid. Sequence analysis revealed 100% homology with the published sequence of gra5. immunofluorescence and Western blotting analyses of HER 293-T cells trcinsfected with pGRA5 showed specific expression of GRA5. Immunogeni city of pGRA5 will be evaluated in mice.

Avicenna I Med Biotech 2011; 3(3): 135-141

Keywords: Toxoplasma gondii DNA vaccine, GRA5 protein

Introduction

Toxoplasma gondii is a common, singlecelled parasitic agent which causes Toxoplasmosis worldwide. It is estimated that up to one-third of the world human Population are infected by the parasite (1). The infection is transmitted orally by ingesting parasite cysts in undercooked meat, or by accidental ingestion of oocysts shed in the feces of the feline (2-4). Toxoplasma infection is often benign in immunocompetent individuals, but in individuals with impaired immune system, such as AIDS patients, toxoplasmosis is an important Opportunistic infection. In these people, latent T.gondii infection can be reactivated to cause fatal Toxoplasmic encephalitis (5). Primary infection during pregnancy can result in permanent neurological and ocular complications in the fetus 13t In livestock, particularly in sheep and goats, infection can lead to abortion, causing significant loss to the farming industry.

Chemotherapy efficiently controls acute Toxoplasma infection and reduces rate of neurological and ocular problems in the congenitally infected newborns, however, it cannot prevent development of latent infection as represented by tissue cyst production in neural and muscular tissues (3-6). Therefore, it would be of great interest to produce an effective Toxoplasma vaccine to prevent the infection. So far, the only developed vaccine is the live, attenuated tachyzoite S48 strain known as Toxovax (7), however, it is not widely accepted because of its adverse effects, short shelf-life, and high cost (8), (9). Live vaccines also carry a risk of accidental infection of humans and unexpected harmful reverse mutations. Subunit vaccines, derived from native antigens of the parasite as recombinant protein or DNA, might overcome these hurdles (9), (10)

Vaccinations by DNA-based structures have been shown to elicit a potent, long lasting humoral and cell-mediated immunity, in addition to providing protection against viral, bacterial, and parasitic...

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Gale Document Number: GALE|A304727696