Study On The Immune Responses Induced By Toxoplasma Gondii DNA Vaccine With Gene Encoding SAG1, GRA2 And SAG1-GRA2

Toxoplasma gondii, a kind of worldwide parasitic protozoan, occurs in a wide variety of mammals, including humans. Infections in pregnancy women may lead to dramatic sequences such as abortion, dead-born, immature labor, or malformation. In immunosuppressed and immunodeficient individuals, including patients with organ transplantation, malignant tumor, acquired immune deficiency syndrome (AIDS), toxoplasma can cause them to death. For the prepotency and healthy of human, the prevention and cure of toxoplasmosis is very important. The manufacture of Toxoplasma gondii's vaccine becomes urgent.With the molecular biology technology unceasing developing, Toxoplasma gondii's vaccine has made great progreses from the earliest dead vaccine to the late nucleic acid vaccine developed rapidly. Though Toxoplasma gondii is monocellular organism, it is different in complication of life cycle, diversity of form, extensity of invading host and pathogenic molecular mechanism. So in the course of studying Toxoplasma gondii's vaccine, it should become a common direction and sense in developing polyvalent vaccine and nucleic acid vaccine in connection with growth stage in different life cycle.SAG1, or SAG1, is the main surface antigen of the Toxoplasma tachyzoite covered 3 to 5 percent of the gross proteins of T. gondii. Being highly immunogenic and immuno-protective, it was considered as an important candidate for effective diagnostic reagents and vaccine antigen^[4,5].Dense granule protein is an excreted-secreted antigen which is diliveried by the dense granules of Toxoplasma cell. It is highly immunogenic, which expressed during the whole intermediate host life cycle of the parasite. It is a suitable candidate for vaccine of Toxoplasma development in human. The GRA2 protein appears to contain at lease three B-cell epitopes and one T-cell epitope and could induce IgG,IgM,IgA antibodies and active the special CD₄⁺ T cell of GRA2, which induce the protective immunity of the economy.As the reported abroad, SAG1 and GRA2 play an important role in the long term stimulation of Tg-specific helper T cells and expressed during the whole intermediate host life cycle of the parasite. CD₄⁺ Tg-specific T cell clones from chronically infected people react with both SAG1 and GRA2 support the hypothesis that a combination of these Ags or of appropriate derivative peptides represent suitable candidate for vaccine development in human.A pair of primers were respectively designed and synthesized according to the published gene sequence of the SAG1 gene and GRA2 gene. the products were inserted into pcDNA3.1(-) to generate eukaryotic expression plasmids pcDNA3.1-SAG1, pcDNA3.1-GRA2, pcDNA3.1-SAG1-GRA2. Then they were transiently introduced into HFF cells by liposomes respectively. The total RNA of the cell culture was extracted by use of Trizol after 48h of transfection. The eukaryotic expression vector pcDNA3.1 was verified to have the capability to transcript and translate the SAG1, GRA2 and the SAG1-GRA2 genes in mammal cells through RT-PCR methods. House-keeping geneβ—actin was used as an inner control. After that abundant plasmids pcDNA3.1-SAG1, pcDNA3.1-GRA2 and pcDNA3.1-SAG1-GRA2 were extracted by the means of alkaline lysis. BALB/c mice were intramuscularly immunized with the DNA vaccines (100μg per animal). Two booster injection were carried out at 2-week intervals. As negative controls, the empty vector pcDNA3.1 and PBS buffer were injected in the other two groups respectively. Mice were collected blood on time, to analyse the immune response induced by nucleic acid vaccine and the mice's survival ability.Our results showed that DNA vaccination could induced specific immune response against T. gondii, generate the specific antibody, extend survival time. Compound DNA vaccine of genes from different live stages of T. gondii elicit better immune protectivity in mice than vaccine of a single gene.