Sreeninging And Identification Of New Genes Wx2 And Wx With Potential Value In The Research Of Vaccine Of Toxoplasma Gondii

Background:Toxoplasma gondii is an obligate intracellular protozoan parasite which could infect nearly all kinds of animals and birds and involve multiple host organs.The infection can result in toxoplasmosis.In humanbeings,severe disease in adults exists mainly in mmunosuppressed patients.Toxoplasmosis was regarded as an important recurring disease only second to tuberculosis which was seriously harmful to human heath.The infected pregnant woman could transmit the parasite to fetus through placenta,leading to abortion,stillbirth,neonatal deformety, mental symptoms and congenital toxoplasmosis of survival infants.It brought very serious effects on the eugenesis and family planning in our country.Recently,it was reported that Toxoplasma encephalitis is the most important manifestation and the main cause of death of in immunodeficiency individuals,such as AIDS patients et al.Furthermore, toxoplasmosis is also the main cause of death in the patients who received organ transplantation and is one of the main reasons of first-episode in schizophrenia and cryptogenic epilepsy patients.At present,there are still difficulties in prevention,diagnosis and treatment of the disease. Chemotherapy is not satisfactory because of the toxicity,need for long-term treatment and rapid re-infection by the parasite;moreover, there is no drug that can kill the encysted bradyzoites.As a result, toxoplasmosis has not been well controlled in the world.The development of cheap,safe and effective vaccines and new therapeutic drugs become priorities in the control of the disease.Therefore,the screening of good candidate molecules for the toxoplasmosis vaccine is not only a research bottleneck,but also a focus and hot point in the currently vaccine research.On the basis of the previous studies,we devoted ourselves to find and identify more effective vaccine candidate genes or drug target molecules,and then to produce DNA vaccines, illuminate the function and mechanism of the gene,as well as its potential value.Objective:(A)Through preparing new monoclonal antibodies that possessing protection effect against T.gondii,to find and identify new candidate genes for the vaccine of toxoplasmosis.(B)Constructing the DNA vaccines corresponding to the genes screened before,and surveying their protection effect in animals and the mechanism of the effect.(C)RNAi technique was used to silence the genes at the post-transcription level to make its expressed protein being loss or declined,so as to illuminate the function of the genes and evaluate the practical prospects of the genes and their DNA vaccine.Methods:(1)Monoclonal antibodies against Toxoplasma gondii were produced by hybridoma technology,and then their protective effect in vitro and in vivo experiments were evaluated.Using these monoclonal antibodies as probes to immunoscreen toxoplasmosis tachyzoite cDNA library,the corresponding gene sequences were obtained.Afterward the homology analysis was blast at websites:http://www.ncbi.nlm.nih.Gov /BLAST and(http://www.toxodb.Org/ToxoDB.html.)Login GenBank and accessible sequence were acquired.(2)The encoded proteins were identified by immurlofluorescence microcopy and the expressions in eukaryotic cells.The B cell epitope, amino acid sequence and the structure of the proteins encoded by the genes were predicted and homology analysis(including Phylograrn tree and the homology blast)was performed by bioinformation methods.(3)Using plasmid pBluescript 7C3-C3 in which wx2 gene was carried as the template in PCR to amplify the ORF of wx2,and plasmid pBluescript 2B9-G1 in which gene wx was carried as the template in PCR to amplify the ORF of wx,the DNA vaccines with eukaryotic expression vector of the two new genes were constructed.Protective experiments in animals were done and the survival time of challenged mice was observed.Lymphocyte transformation rate,CD₄⁺ and CD₈⁺ lymphocyte ratio of the spleen cells,IFN-γlevel and specific antibody in serum of the immunized animals were examined.(4)RNAi expressed vectors with the new gene wx2 and wx as their targets were constructed.The effect of interference was observed by lipid-mediated transfection of eukaryotic cells in vitro.Plasmid was transfected into the toxoplasma cells by electroporation,so that the silent strains of toxoplasma in which siRNA molecules could be expressed continuously were produced.By Western-blotting,RT-PCR,Northern blotting et al,the interference effect was characterized,and these artificially produced toxoplasma can be used for further study.Result(1)Two monoclonal antibodies against Toxoplasma gondii 7C3-C3, 2B9-G1 were obtained.The protective tests in vitro showed that these proteins could inhibit the invasion and proliferation of T.gondii RH strain in HeLa cells.The cell infection rate was 28%and 32%respectively,and that of the control was 85.2%.The average number of T.gondii tachyzoite of in 50 HeLa cells was 5.18±3.34 and 5.50±2.36,while that of the control was 11.12±4.29.The passive transfer test indicated that these proteins significantly prolonged the survival time of the challenged mice(7.2±0.42days and 7.0±1.41days,while that of the control was 5.0 days).It was also shown that the two McAbs had certain protection ability against T.gondii infection.(2)Through immunoscreening toxoplasma tachyzoite cDNA library, the gene sequences corresponding to these two monoclonal antibodies were obtained.The accessible numbers were AY238892 and AY208994 in GenBank.By immunofluorescence localization test,expression in vitro and bioinformatics analysis,it was demonstrated that WX2 was a new functional molecule on the membrane of T.gondii of which molecular weight was 49kDa,and WX was a 60S ribosomal L7 protein of T.gondii, a molecule in cytoplasm of which the molecular weight was 47kDa.(3)The DNA vaccines of new genes wx2 and wx were constructed successfully.Protective experiments of DNA vaccines pcDNA3-wx2 and pcDNA3-wx in animals showed that the vaccines could significantly prolong the survival time of the mice challenged by virulent RH strains of Toxoplasma.Comparing with other toxoplasma vaccines against the virulent strains reported currently,the survival time is the longest.The average survival time was 289.14±46.81hours and 284.29±47.30hours respectively.Moreover,30 days after challenge,there were still four mice survived in each group.The study on the impact to the immune system indicated that,pcDNA3-wx2 vaccine could stimulate the animal to produce high level IFN-γ(149.8±24.53ng),both pcDNA3-wx and pcDNA3-wx2 could induce the decrease of the CD4⁺/CD8⁺ ratio and the production of the specific antibody in the experimental mice.However, the level of antibody titers did not parallel with the degree of protection. It indicated that these two DNA vaccines could stimulate the host resisting Toxoplasma mainly by the way of CD8⁺CTL cells.(4)Five interference expression vectors against Gene wx2,wx were constructed,and two partially silent strains T.gondii wx2b-i and wxB-i, were obtained.At the same time the virulence of wx2b-i partially reduced. It was confirmed that the mRNA and protein expression of wx2 and wx genes were partly reduced which were identified by the in vitro expression,RT-PCR and Northern blotting.Conclusion:(1)Monoclonal antibodies 7C3-C3 and 2B9-G1 have some protective effect against Toxoplasma gondii.They can inhibit the invasion of the parasite to host cells and the propagation of it in the host cell.The passive transfer experiments suggested that they could prolong survival time of the mice challenged by RH strain Toxoplasma gondii.(2)WX2 is a new functional membrane molecule,with 49kDa molecular weight.WX is toxoplasmoa 60S ribosomal L7 protein,with 47kDa molecular weight in cytoplasm.So the corresponding genes wx2 and wx are good new candidates for DNA vaccine of T.gondii.(3)The DNA vaccines of new genes wx2 and wx were constructed successfully.Protective experiments in animals showed that the vaccine could significantly prolong the survival time of the mice challenged with virulent RH strains of Toxoplasma,The time is longer than that acquired by the other DNA vaccines on current reports,which indicated their potential value.The immue mechanism is mainly by the way of CD8⁺ CTL cell.(4)Five interference expression vectors against Gene wx2,wx were constructed,and two partial silent strains wx2b-iand wxB-i were obtained. All these formed a foundation for further study on the function and mechanism of gene wx2,wx.