Construction And Identification Of Recombinant Eukaryotic Expression Plasmid Containing SAG1 And MIC3 From Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular opportunistic protozoa, responsible for human and animal toxoplasmosis, it was first discovered by Nicolle and Manceaux, French scholar in 1908. There are 5 stages in its life cycle , they are trophozoite, cyst, shizont, gametocyte and oocyst, and, in that trophozoite, cyst, oocyst are due to transmission and pathogenesis. Most infecters are asymptomatic. however, in pregnant women and immunocompromised patients infection can cause severe complications. Surprisedly, toxoplasmosis can change infecters' character. It was reported by scholar that Toxoplasma-positive subjects have lower intelligence than Toxoplasma-negative subjects , statistical data also showed:there is some link between toxoplasmosis and schizophrenia. Livestock are susceptible to Toxoplasma gondii, toxoplasmosis is of great economic importance worldwide because it causes abortions, stillbirth, and neonatal loss in all types of livestock, Infected livestock are an important source of infection for humans . At present, vaccine shows great potentials on the prevention against toxoplasmosis. The devastating effect of toxoplasmosis in both humans and domestic animals makes it one of the diseases of great economic importance in many parts of the world. Therefore , a vaccine which will provide sterile immunity against Toxoplasma gondii is urgently required.Toxoplasma gondii's vaccine develops from dead vaccine to attenuation vaccine, from direct extracting surface antigen to purifying unit price subunit vaccine by genetic engineering, now to compound polyvalent vaccine. Tachyzoite is pathogenic stage , the surface of tachyzoite is the mainly attached target of host immune system, Among these surface antigens, P30 is one of the most promising vaccine candidates , which was focused on frequently .P30 is a stage-specific antigen ,only expressed in tachyzoite stage, covered 3-5 percent of the gross proteins of T. gondii, but, the antibody which it can induce cover 50 percent of total antibody, serum antibody responded to P30 were measured in acute and chronic infecters. The fact that polyclonal antibody and monoclonal antibody can prevent fibroblast from the infection of T. gondii showes their protective effect. The special CD₄⁺T cell of P30 can kill toxoplasm in vitro. The microneme protein is released during attachment and invasion of T. gondii. of all, MIC3 protein , coded by MIC3 gene could be involved in invasion by acting as a bridge between the parasite and the host cell.The MIC3 is a dimeric 90-kDa micronemal protein, looking particularly promising because it is expressed in all infectious stages of T. gondii (tachyzoites, bradyzoites, and sporozoites) and that elicits early and powerful immune responses in mice and humans, such as antigen-specific IgG antibodies;IFN-γ;IL—2;IL—4; L-10.Two pairs of primers were respectively designed, and synthesized according to the published gene sequence of the P30 gene and MIC3 gene . At the 5' ends of sens and antisens strand of primers, proper enzyme sites are added respectively, gene fragment was amplified by PCR identified and then subcloned into pMD18-T simple vector, then subcloned into pcDNA3.1 (-) togenerate eukaryotic expression plasmid pcDNA3. 1-SAG1-MIC3; pcDNA3. 1-SAG1; pcDNA3.1-MIC3. Ampicilin - resistant transformants were selected and identified by PCR, Enzyme digestion and DNA sequencing analysis. The recombinant plasmid was transfected into Hela cells by LipofectamineTM 2000. The total RNA of the cell culture was extracted by use of Trizol, the eukaryotic expression vector pcDNA3. 1 was verified to have the capability to transscript and translate the gene SAG1-MIC3;SAG1;MIC3 in anamal cells trough RT-PCR,used House-keeping geneβ-actin as an inner control .Our results showed that the recombinant eukaryotic expression plasmids of pcDNA3.1-SAG1-MIC3, pcDNA3.1-SAG1,pcDNA3.1-MIC3 have been successfully obtained, RT-PCR analysis of cells transfected gene fragment display positive bands, paving the way for further study on animals, and providing the candidate subunit compound gene for Toxoplasma gondii polyvalent vaccine.