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Molecular characterization of surface antigen 3 (SAG3) gene of Toxoplasma gondii RH-IVRI strain.

ABSTRACT: Toxoplasmosis, caused by Toxoplasma gondii, is a zoonotic disease and is responsible for abortions, stillbirth, and neonatal complications in livestock, especially in sheep, goats, and pigs. The molecular characterization of the parasite having global distribution is considered important to delineate the phylogenetic relationship among different isolates/strains of the parasite. The present communication deals with the molecular cloning and sequence analysis of the 1,158 bp entire open reading frame of surface antigen 3 (SAG3) of T. gondii RH strain being maintained at the Indian Veterinary Research Institute (IVRI). The sequence comparison analysis revealed 99.9 % homology with the published sequence of T. gondii RH strain, with a single substitution of guanine 'G' instead of adenine 'A' at the 397th position of SAG3 sequence. The substitution of single nucleotide consequently resulted in the change of one amino acid residue of aspartic acid (D) instead of asparagine (N) present in the published sequence of RH strain. This denotes that the SAG3 gene of this RH strain has not undergone a major change in its molecular conformation even after repeated passage in mice for more than a decade at IVRI. The finding is important from the molecular phylogeny point of view.

SUBMITTER: Vikrant Sudan

PROVIDER: S-EPMC3427684 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Molecular characterization of surface antigen 3 (SAG3) gene of Toxoplasma gondii RH-IVRI strain.

Vikrant Sudan Anup Kumar Tewari Singh Harkirat H Saravanan B C BC Sankar M M

Journal of parasitic diseases : official organ of the Indian Society for Parasitology 20120324 2

Toxoplasmosis, caused by Toxoplasma gondii, is a zoonotic disease and is responsible for abortions, stillbirth, and neonatal complications in livestock, especially in sheep, goats, and pigs. The molecular characterization of the parasite having global distribution is considered important to delineate the phylogenetic relationship among different isolates/strains of the parasite. The present communication deals with the molecular cloning and sequence analysis of the 1,158 bp entire open reading f ...[more]

PMID: 24082530

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Project description:Toxoplasma gondii is a globally distributed obligate intracellular parasite which can cause zoonotic toxoplasmosis with great harms. The average death time of mice that infected with Toxoplasma gondii RH strain tachyzoites recovered from the liquid nitrogen was shortened after multiple generations. It has been reported that the parasite is in a state of static virulence during cryopreservation and the virulence of the protozoan parasite can be enhanced after continuous passages in hosts under laboratory conditions. However, no research has been conducted to elucidate its biological mechanism. Herein, we sequenced the T. gondii transcriptome using RNA-Seq technology and performed de novo assembly to investigated the virulence factors expression changes by comparing gene expression profiles between incipiently recovered and completely resuscitated tachyzoites. Transcriptome analysis identified 1,951 differentially expressed transcripts in infected liver, of which 1,752 were significantly downregulated and 199 upregulated. We identified many differentially expressed proteins and genes, including serine/threonine kinase, calnexin, myosin and microtubule-associated protein which have previously been reported to be either involved in cell adhesion, parasite gliding or participate in cell invasion. The great majority of the virulence factors including microneme proteins, rhoptry proteins and dense granule proteins were upregulated in fully recovered tachyzoites. The enhanced virulence of recovered Toxoplasma gondii RH strain from the liquid nitrogen is associated with the up-regulated expression of MICs, ROPs and GRAs. Our data will facilitate future genomic research and in-depth annotation of Toxoplasma gondii RH strain genomes. This study provides a profile of the candidate genes that are suspected to be involved with virulence enhancement of recovered Toxoplasma gondii RH strain tachyzoites. Many further studies should be carried out to confirm the function of the candidate genes. Moreover, the preliminary identification of genes and pathways exhibiting differential expression in complete resuscitation stage may further our general understanding of virulence enhancement in this parasite.

Project description:BACKGROUND:Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. OBJECTIVES:The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. MATERIALS AND METHODS:Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB). RESULTS:The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. CONCLUSIONS:The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites and are promising approaches for antigen preparation in vaccine development.

Dataset Information

Molecular characterization of surface antigen 3 (SAG3) gene of Toxoplasma gondii RH-IVRI strain.

Publications

Molecular characterization of surface antigen 3 (SAG3) gene of Toxoplasma gondii RH-IVRI strain.

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