Construction And Identification Of Leishmania Tarentolae Expressing SAG1 Gene From Toxoplasma Gondii

Toxoplasma gondii (T. gondii) is a kind of opportunistic pathogenic protozoa which is widely ranged in nucleated cells of humans and animals. The parasites can invade a variety of human and animal organs, causing toxoplasmosis, which has a high infection rate and caused tremendous threat to public health and livestock production. So far there are no effective drugs against toxoplasmosis. Therefore, effective screening and expression of the antigen molecules and the development of safe and effective vaccine has important theoretical and practical value.Leishmania tarentolae (Lizard Leishmania) was originally isolated from reptiles, which is susceptible to the lizards, but does not infect mammals. Due to its parasitic lifestyle, Leishmania tarentolae has a similar way with mammals in post-translational processing of the target protein, such as protein glycosylation, phosphorylation, prenylation and so on. In addition, Leishmania tarentolae has advantages as a eukaryotic expression system such as easy to culture and broke, and pollution of its expressed protein can be easily avoided. Hence, Leishmania tarentolae as a kind of exogenous expression vector systems receive more attention.In this study, the antigen gene SAG1 of T. gondii was amplified from T. gondii RH strain genome, and cloned into the eukaryotic expression vector pLEXSY-neo2 to construct eukaryotic expression vector pLEXSY-neo2-SAG1, which was transfected by electroporation into the Leishmania tarentolae genome, and positive clones were screened by G418. The expressed proteins were identified by SDS-PAGE and Western blotting, which was proved to be expressed in the Leishmania tarentolae. The construction of eukaryotic expression vector pLEXSY-neo2-SAGlUsing PCR to amplify major surface antigen SAG1 gene fragment and the truncated fragment through Toxoplasma gondii RH strain genome, respectively, and cloned into the eukaryotic expression vector pLEXSY-neo2. Antigen gene sequence analysis of SAG1 showed that the long fragment was 957bp and the short was 650bp, the homology was 99% compared with the RH strain SAG1 gene published in Genbank. By restriction enzyme analysis, we have constructed eukaryotic expression vector pLEXSY-neo2-SAG1. The transfection and screening of eukaryotic expression vector pLEXSY-neo2-SAG1 in Leishmania tarentolaeAfter the electroporation of the vector into the Leishmania tarentolae, which was later integrated by homologous recombination into the host genome, the positive clones that integrated the G418 resistance gene neo into the genome could grow in selection medium, no integration and negative control parasites are killed by G418 drugs. In two weeks we have obtained positive clones. Identification of transgenic lizard LeishmaniaExtract positive and negative control parasites' genome, which was used to amplify SAG1 fragment using specific primers, the results showed specific target band could be detected in the positive parasites. Parasites in fresh selection medium after 72h of static were used to extract expressed protein, SDS-PAGE of the secreted and intracellular proteins expressed were approximately 33ku and no target band in negative control was detected, the expressed protein could be recognized by anti-histidine antibody using Western blotting.The successful cloning and expression of major antigen gene of Toxoplasma gondii SAG1 in the Leishmania tarentolae has laid the foundation in the molecular diagnosis of toxoplasmosis, meanwhile with prevention and treatment, and has broadened the field of eukaryotic expression research of Toxoplasma gondii antigen.