The Influence Of Toxoplasma Gondii In Vivo Induced Antigens In Pigs On NF-kB Signaling Pathway

Toxoplasma gondii (T.gondii) is an obligate intercellular protozoan infecting all of warm-blooded animals and humans worldwide, and can cause serious zoonotic toxoplasmosis. Toxoplasmosis is usually asymptomatic, because our immune system keeps the parasite from causing illness. But these parasites usually lead to serious consequences for some immune dysfunction or immunocompromised persons. Thus preventing toxoplasmosis is extremely important. The life cycle of T.gondii is quite complex, which includes numerous developmental stages, for example, tissue cysts, oocysts, gametocytes, tachyzoites, bradyzoite and so on. In addition, in the long-term parasitic life, T.gondii develops a lot of immune escape mechanisms to evade attacking of host immune system. In vivo induction of antigen of the host would be expressed during T.gondii invading. A hypothesis is that the host immune system releases immune factors and then triggers the expression of in vivo induced antigen genes.The transcription factor of NF-κB family is the original regulation for κB’light chain in B cells of mammals. It is also a key regulated factor in the process of active immunization of animals. The regulation of NF-κB family’s transcription has important relevance to inflammatory response, apoptosis, cell proliferation, differentiation and survival. In addition, the NF-κB signaling pathway is an important componentfor the regulation of host immune system.Therefore, in vivo induction of antigen and NF-κB signaling pathway is likely to have potential interactions. In this study, I firstly verified the differential expression between vivo and vitro in part of Toxoplasma gondii pigs’ in vivo induced antigen-positive results, which had been acquired by a previous study by a person in our lab. I also did a PCR amplification of full-length ORF genes. Then dual luciferase assay was used to detect the activated effects to NF-κB signaling pathway by these antigens and also to detect the inhibitory effect on this signaling pathway under the stimulation of TNF-a. Specific work includes the following aspects:(1) Verified differential expression between vivo and vitro of Toxoplasma gondii pigs’in vivo induced antigen (IVIA) and PCR amplificated full-length ORF genes.Five functional proteins and threehypothetical proteins were selected from among the in vivo induced antigen-positive results which had been acquired by a previous study by a person in our lab. These were leucine repeat sequence (LRL), calmodulin (CaM), D-3-phosphate dehydrogenase (D3), dense granule protein5(GRA5), TGGT1-098800 (tentatively called G1), TGME49-212160(M4), and TGGT1-059670(JK) on TOX.DB. Primers were designed to detect relative expression of these eight genes’mRNA level between Toxoplasma gondii inoculated in BALB/c mice for72h andToxoplasma gondii cultured in BHK cells for72h. The results showed that the six genes’amount of relative expression in BALB/c were significantly higher than in vitro expression of BHK, and statistical analysis were significant difference (P<0.05). Differential expression of LRL gene and M4gene in vivo and in vitro is not obvious. Eukaryotic primers were designed according to the full-length CDS of gene sequences at database and six genes’ORF fragments were cloned and connected to pCMV-tag2B to construct eukaryotic expression plasmids with Flag label. The pCMV-tag2B/G1plasmid can express in293T cell.(2) Detect the activate effects to NF-κB signaling pathway by these antigens.The dual luciferase assay was used to detect the impacting on NF-κB signaling pathway of the in vivo induced antigen. The luciferase reporter plasmidwas constructed using prebuilt NF-κB transcription regulatory elements inserting into the upstream of the firefly luciferase gene (promoter or3’UTR). Then Renilla luciferase gene also exists as an internal control. These two plasmids were co-transfected to detected activation of NF-κB signaling pathway. The results showed that in vivo induced antigen GRA15(RH strain) had a significant effect activated NF-κB signaling pathway usingGRA15gene of Toxoplasma gondii ME49strains as a positive control. But subsequently nuclear transfer experiments of the p65protein of the NF-κB family failed to verify this result.(3) Detect the inhibition effects to NF-κB signaling pathway by these antigens.The inhibitory effect to NF-κB signaling pathway was observed bythe TNF-a-stimulation in both vector group and IVIA genes group using dual luciferase assay. These IVIA could not inhibit NF-κB signaling pathway.(4) Immunogenicity of dense granule protein GRA15(Ⅰ/Ⅱ).T. gondii GRA15(Ⅰ/Ⅱ) has a certain influence on NF-κB signaling pathway which associated with the immune system of the host. To evaluate the protective efficacy of T. gondii GRA15(Ⅰ/Ⅱ), in this study, a new DNA vaccine-encoding T. gondii GRA15(Ⅰ/Ⅱ) was constructed using the pcDNA3.1vector. Expression of GRA15(Ⅰ/Ⅱ) from this vector was confirmed by indirect immunofluorescence assay following transfection into baby hamster kidney (BHK) cells. Intramuscular immunization of BALB/c mice with pcDNA/GRA15(Ⅰ/Ⅱ) was carried out to evaluate the immune responses by serum antibodies titers, lymphoproliferation assay, and cytokines assay. The protective efficacy was evaluated by survival rate in mice after challenging with highly virulent strain of T. gondii. The results demonstrated that this vaccination can not stimulate humoral responses and can not increase the survival rate of immunized mice when they were challenged with a lethal dose of tachyzoites of T. gondii RH strain. These data suggest that T. gondii GRA15(Ⅰ/Ⅱ) can’t be an efficient candidate vaccine. Why T. gondii GRA15(Ⅰ/Ⅱ) influence NF-κB signaling pathway and haven’t efficient protective immunity needs to further study.