The Biological Characteristics And Immunogenicity Of Toxoplasma Gondii Microneme Protein 3 (MIC3)

Toxoplasma gondii, which is an intracellular protozoa, has a wide range of host groups. It can infect all kinds of animals in the world including pigs, cattle, sheep, horses, dogs and so on. The cat is the desnitive host. The outbreak of Toxoplasmosis in swine can get the whole pigs diseased, the resulting of which is that the motality is more than 60%. Accroding to the serological survey, about 25% of people in the world get infected on Toxoplasmosis. The reason why the people would have such a high T. gondii infection, in addition to the mostly inapparent infection, an important factor is the universality of its transmission. At present, in China, people are more likely to get infected from the pets, seafood, uncooked meat, eggs and livestock product. With the economic development, the increasing mobility of people makes the toxoplasmis spread in non-endemic areas. There is no infective medicine to cure Toxoplasmosis in people at present. The only way is still prevention. Although for the treatment of toxoplasmosis in livestock is the chemicals such as the sulfonamides, the problem of the increasing dug residues and drug resistance drive us develop new method to control toxoplasmosis. The vaccine which is a new and effective method for the disease prevention has a wide range of applications. Since T. gondii has a complex life style and many different antigens, there is no effective vaccine against toxoplasmosis. The MIC3 is secreted by the Microneme which plays an important role during the invasion. Because MIC3 can be expressed in all the stages of T. gondii, it is regarded as one of the most important vaccine candidates. In this study, we used the method of Bioinformatics, molecular biology, gene engineering, immunobiology to do the further research on the function of MIC3, attempting to know more about the function of MIC3 and related proteins, analyse the antigenicity, providing a novel strategy for vaccination against Toxoplasmosis.1. Cloning and bioinformatic analysis of MIC3 gene in Toxoplasma gondiiAccording to the sequence of MIC3 of T. gondii on the ToxoDB (www.ToxoDB.org), PCR was carried out to amplify the DNA of MIC3,5'flanking and 3' flanking from the four strains, RH (Type I), GT1 (Type I), PTG (Type II) and CTG (Type III), then the sequences were cloned to T vector. Based on this, we made an alignment from the four strains, analyzed their affinity and the evolutionary relationship. The transcriptional activity of MIC3 5'UTR, the basic parameter and the antigenicity of MIC3 protein were analyzed by the online software. Using the domain prediction software, the model prediction and building software online, the structure, location and function of the MIC3 domain were analyzed and predicted. From the information got from the software analyzing and the papers, we tried to imitate the protein senior structure, and built the model initially. The bioinformatic analysis of MIC3 gene provides us important information for our further research on the function of MIC3 protein.2. Analyzing the transcriptional activity of MIC35'flanking region and the cellular location of MIC3The MIC3 is screted by the Microneme during the parasite invades the host cell which plays an important role. To further analyze the transcriptional activity of MIC35' flanking region and the cellular location of MIC3, the transgenetic T. gondii which expressed the EGFP gene and MIC3 gene labled with EGFP was constructed. The MIC3 5'flanking region was amplified by PCR. With the help of the restriction enzyme, the MIC35'flanking region is linked with EGFP gene. To make sure the expression of EGFP, the EGFP gene is linked to the SAGl 3'UTR of T. gondii. The Ble gene with the SAGl promotor which is used as a selective marker is inserted after the egfp-SAG1 3' UTR gene. Then the plamid pBSK-5'flanking-egfp SAGl 3'UTR-SAG1 pro-ble is completed which can express EGFP and be selected by phleomycin. After the electrotransfection of the plasmid to T. gondii, the parasite expressed EGFP was selected. By the same method we can get the T. gondii which expresses MIC3 labled with EGFP. The method of RT-PCR, fluorescence electron microscopy were used to analyze the transcriptional activity of MIC3 5' flanking region and the cellular location of MIC3. According to the results, the MIC35'flanking region can start the EGFP gene to be expressed, MIC3 potein is expressed at the top of T. gondii during the invasion and attached to the cell to affiliate with the parasite to invade.3. The impact of overexpression of Toxoplasma microneme protein 3 We constructed the trans genetic parasite which overexpresses MIC3 to study the impact on the invasion, growth, multiplication. We got the MIC3 gene by PCR, with the help of restriction enzyme, it is linked with SAG13'UTR of T. gondii.The whole fragment is inserted to the plasmid pBSK by the enzyme restrction. To make sure the expression of MIC3, the MIC3 gene is linked to the MIC35'UTR of T. gondii which is identified as the promoter before. The Ble gene with the SAG1 promotor which is used as an selective marker is inserted after the MIC3-SAG13'UTR gene. Then the plasmid pBSK-5'flanking-MIC3 SAG13'UTR is completed which can express MIC3 and be selected by phleomycin. We got the positive parasite by electrotransfecting the plasmid to T. gondii. By the growth assay and replication assay, overexpression of MIC3 has little effct on the invasion, growth and multiplication, which maybe related to the combined actions of different proteins. This study is the foundation for further research of the function of MIC3.4. The immunogenicity of recombinant protein MIC3 of T. gondiiGinsenoside, the most important component isolated from Panax ginseng, exhibits a variety of biological activities. Particularly, ginsenoside Rgl is known to have various immune-modulating activities such as increase of immune activity of T helper (Th) cells. In the present study, we evaluated the immunomodulatory potentials of the recombinant protein MIC3 (rMIC3) expressed by E.coli and Rgl as the adjuvant on the cellular and humoral immune responses of ICR mice. ICR mice were immunized subcutaneously with saline alone,100μg rMIC3 alone,100μg rMIC3 dissolved in saline containing ginsenoside 50μg Rgl,100μg r MIC3 with freund's adjuvant on days 1 and 15. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that the groups immunized with rMIC3 and 50μg Rgl developed a high level of specific antibody responses against T. gondii rMIC3, a strong lymphoproliferative response, and significant levels of cytokine production, compared with the other groups. After lethal challenge, the mice immunized with the rMIC3 and 50μg showed a significantly increased survival time compared with control mice which died within 6 days of challenge. Our data demonstrate that the recombinant protein rMIC3 can elicit a strong humoral and cellular response. By addition of ginsenoside Rgl, the rMIC3 triggered a stronger humoral and cellular response against T. gondii, and that Rg1 is a promising vaccine adjuvant against toxoplasmosis, worth further development.5. The immunogenicity of Toxoplasma gondii MIC3/P30 gene carried by attenuated Salmonella typhimuriumInfection with the intracellular protozoan parasite T. gondii causes serious public health problems and is of great economic importance worldwide. The T. gondii microneme protein MIC3 is a significant candidate vaccine against toxoplasmosis. In this study, we evaluated safety, stability of ZJ111/pcDNA3.1-MIC3, a DNA vaccine delivered in attenuated Salmonella typhimurium, and immune responses induced by immunizing ICR mice orally with ZJ111/pcDNA3.1-MIC3, ZJ111/pcDNA3.1-P30 and ZJ111/pcDNA3.1-MIC3/P30. Mice had no significant differences in body weight between the groups before immunization and at week 4 after the booster immunization. The ZJ111/pcDNA3.1-MIC3 was eventually eliminated from the spleen and liver on week 6 post-immunization. The plasmid pcDNA3.1-MIC3 was stably maintained over 90% of the attenuated S. typhimurium population after 100 generations of growth in antibiotic-free media. Oral immunization of mice with ZJ111/pcDNA3.1-MIC3 elicited specific humoral responses and stimulated proliferation of splenocytes (P< 0.05). The cellular immune response was associated with the production of IFN-γ, indicating that a Th-1 type response was elicited, which was confirmed by the production of large amounts of IgG2a (P< 0.01). Mice immunized with ZJ111/pcDNA3.1-MIC3 displayed significant protection against an intraperitoneally challenge with 500 tachyzoite forms of T. gondii RH strain. Vaccination with ZJ111/pcDNA3.1-MIC3/P30 provided a 20% survival rate comparing 100% mortality of the nonimmunized mice at day 12, exhibiting longer living time and better survival rate. These results confirmed a DNA vaccine delivered in attenuated S. typhimurium, ZJ111/pcDNA3.1-MIC3, can elicit specific immune response as well as provide effective protection against T. gondii infection, and the multiple antigens vaccine ZJ111/pcDNA3.1-MIC3/P30 can elicite a higher immune response. In summary, the MIC3 gene of T. gondii was analyzed by the bioinformatics method. We compared the difference of gene expression and the relationship of evolution, predicted the domains and structure of MIC3 protein. The method of expressing EGFP gene in T. gondii and labling the target protein is developed, with which we observed the situation of secretion and the cellular location of MIC3 protein, also assessed the affection to the parasite of the MIC3 overexpression. The DNA vaccine delivered in attenuated S. typhimurium against T. gondii was constructed. We analyzed the possibility and the prospect of MIC3 as the potent vaccine by the aspects of antigenicity, the vaccine auxiliary of Genseng saponins, multiantigenic and the vaccine deliverd by attenuated S. typhimurium, hoping to provide a new method to develop a vaccine for prevent Toxoplasmosis.