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

摘要

Background: Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to onethirdudof the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetusesudand also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effectiveudvaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts withudhost cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as theudleading candidate for development of an effective vaccine against toxoplasmosis.udObjectives: The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain intoudthe eukaryotic expression vector pVAX1 in order to use for a DNA vaccine.udMaterials and Methods: Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing theudspecific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrimeudplasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1udwas then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse TranscriptaseudPolymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB).udResults: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed byudenzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods.udConclusions: The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1udgene from T. gondii parasites and are promising approaches for antigen preparation in vaccine development.