Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

Abstract

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.

Evaluation of the Protective Effect of Deoxyribonucleic Acid Vaccines Encoding Granule Antigen 2 and 5 Against Acute Toxoplasmosis in BALB/c Mice

“…Mammalian cells such as CHO cells have been widely used for expression of Toxoplasma recombinant protein, 26 functional validation of Toxoplasma recombinant DNA plasmid [27][28][29] as well as growth investigation of the parasite. 30 In this study, CHO cell was used to validate the expression of GRA2 and GRA5 by the recombinant DNA construct prior to DNA vaccination study.…”

Evaluation of protective efficacy of three novel H3N2 canine influenza vaccines

“…As previously reported, the plasmid pVAX1 serves as a promising approach for antigen preparation in vaccine development. This DNA vaccine vector system is ideal for high-level recombinant protein production in CHO cells [ 40 ]. Additionally, as a recombinant viral vector, CAV2 provides a safe and long-term protective effect in veterinary fields [ 41 ] that reduced the natural infection risk [ 42 ].…”

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

“…It can form homodimer structures, which are attached to the parasite surface by glycosylphosphatidylinositol (GPI) anchors (He et al, 2002). Previous studies have focused on SAG1 for vaccination because it has a critical importance for the immune response in the initial stage of infection involving both the humoral and cellular immune response (Abdizadeh et al, 2015;Lekutis et al, 2001). Vaccination of mice with SAG1 has provided encouraging results with signi cant protection as measured by a reduction of the number of cysts in vaccinated animals compared with control animals (Lekutis et al, 2001).…”