Immunoprotection Of Nucleic Acid Vaccine Of Toxoplasma Gondii GRA1 Gene

Immunoprotection of nucleic acid vaccine of Toxoplasma gondii GRA1 geneToxoplasma gondii is an important opportunity protozoan which parasitic in human and animal nucleated cells. The Toxoplasma gondii can cause zoonotic diseases. The mammals and birds are susceptible to infect. Toxoplasma gondii infect adults in asymptomatic parasite status. But Toxoplasma gondii infect pregnant women can cause miscarriage, premature birth, stillbirth, and teratogenic, etc. For the immune suppressed or defective persons (such as organ transplants, cancer and AIDS) is one major cause of death. With the emergence of organ transplantation technology, as well as the increasing prevalence of AIDS, the incidence of toxoplasmosis has risen every year. In addition, Toxoplasma gondii infect pregnancy is also a serious threat to livestock, can cause serious economic losses.The harm of Toxoplasma gondii on human health and animal can not be ignored. There is no effective drugs to treat the disease. Its prevention is far greater than the significance of the treatment. Therefore, to take effective preventive measures and combat the incidence of toxoplasmosis is imperative. Development of safe, effective and inexpensive vaccine of Toxoplasma gondii is becoming the current research focus. Different antigens of Toxoplasma gondii immunogenic components of the immune protection and its genetic characteristics of the study, to lay the foundation for vaccine research for the genetic engineering and the rapid development of new nucleic acid together. The tachyzoites secrete antigen excretion (excreted-secreted antigen, ESA) in the acute and chronic infection has a strong immunogenicity, is Toxoplasma gondii diagnosis and vaccine development, an important molecule. Toxoplasma gondii GRA1 gene encoding the dense granule protein (granule dense antigen 1, GRA1)is one of secreted Toxoplasma gondii tachyzoites excretion (excreted-secreted antigen, ESA),1.69kb, encoding GRA1 protein molecular weight of 24kD, is a Ca2+ -binding protein. In human and animal infection experiments show strong immunogenicity. Duquesne etc reported that GRA1 gene T-cell epitopes of the research to prove that GRA1 of Toxoplasma gondii gene is an excellent vaccine candidate genes. In addition, Toxoplasma gondii in the general population was infected with latent or chronic state. Therefore exist in tachyzoites mild Masuko dense granules in the GRA1 protein as diagnostic antigen of chronic infection rather have research value. In this study, clone GRA1 gene into the eukaryotic expression vector pVAXl and construct pVAX1-GRA1 DNA vaccine. This vaccine immunology and functional analysis in vivo study, explore the GRA1 gene for gene vaccine effectiveness and practicality, As the basis for Toxoplasma gondii vaccine for clinical applications and the prevention of toxoplasmosis.Extract the RNA reverse transcription into cDNA by RT-PCR, according to Genbank reported designed and synthesized a pair primers of GRA1 gene sequences and introduced BamHâ… arid EcoRâ… restriction site, amplified GRA1 gene cDNA fragment in vitro. The purified fragment was connected with the pMD18-T vectors. Double restriction enzyme digestion were sequenced after plasmid-positive. Amplifie 573bp GRA1 by PCR, The open reading frame blast with the original sequence by homology compared to 98%. Clone GRA1 gene fragment into the eukaryotic expression vector pVAX1,and then transferred into E.coli DH5a competent cells, screening positive clones on ampicillin LB plate. Wiht BamH I and EcoR I restriction enzyme digestion and PCR identification. The results show that, RT-PCR products by 1% agarose gel electrophoretic analysis, occurring amplified a specific band in the 573bp, consistent with the expected value of the size. Blast sequence with the published sequence in Genbank, nucleotide homology results is 97%.Note from the Toxoplasma gondii genome in total RNA was amplified by encoding protective antigen GRA1 gene.GRAl gene was successfully constructed the eukaryotic expression recombinant plasmid pVAX1-GRA1.The second experiment, Culture the Hela cells, transfected pVAXl-GRA1 plasmid DNA into the logarithmic phase of Hela cells by LipofectamineTM 2000,And detected expression product activity by SDS-PAGE and Western blot.The results showed that,in pVAX1-GRA1 transfected samples with a size of approximately 24kD electrophoretic bands,Section with mouse anti-Toxoplasma gondii can be recognized by sera.Display of foreign genes in cells to express and have a certain degree of immunogenicity.The third test, Use pVAX1-GRA1 recombinant eukaryotic expression plasmid immunized on BALB/c mice and detect immune protection on Toxoplasma gondii,The protocoal of immunization is once every two weeks and a total is three times. Detected the dynamic changes of humoral and cellular immunity,the results showed CD4+ and CD8+ values and antibody titers by recombinant plasmid immunized group mice were higher, compared with the control group show significant difference (P<0.01).But the CD4+ /CD8+ ratio is no significant difference (P> 0.05).The changllenged trials showed the survival time of in test group is more longer than the control group (P<0.05).