Cloning, Expression And Initial Assessment Of Immunoprection Of TOM34Genes Of Schistosoma Japonicum

Schistosomiasis is a serious threat, widespread zoonotic disease. Half century, schistosomiasis control in China has made remarkable achievements, but because of repeated severe, medication alone can not control the prevalence of schistosomiasis, schistosomiasis vaccine candidate molecules to enhance screening and vaccine development is undoubtedly the schistosome disease prevention and control of important initiatives. Schistosoma japonicum has a complex life history, parasites large antigen complex, separation, identification and growth-related molecular schistosomiasis, the development and maturation of Schistosoma mechanisms, screening of new anti-schistosome vaccine candidate molecules are of great significance. Author from the schistosome proteomics research laboratory on the basis, from mice, rats, voles East10d schistosomula sources of information in the differentially expressed genes, screening out of TOM34, to carry out the following studies.In this study, first cloning gene SjTOM34of schistosomiasis japonicum, GenBank accession number is226473300, by blasting obtained the full-length cDNA sequence of SjTOM34.Through bioinformatics analysis, The full length cDNA of SjTOM34were1122bp, and the Open Reading Frame for SjTOM34were1083bp, encoding360amino acid, theoretical molecular weight of40843.38Daltons, theoretical isoelectric point of4.94. Real-time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of SjTOM34was highest in the35days schistosomula, while in female and male worms, SjTOM34expression did not differ significantly, were highly expressed in the susceptible host, mice compared to the unsusceptible host, rats or the resistant host, M. fortisand.Successfully constructed pET28a (+)-SjTOM34recombinant prokaryotic expression plasmid, recombinant protein in E.coli (DE3) in soluble expression, molecular weight of44kD, Western blotting analysis showed that the fusion protein with good antigenicity. Purified recombinant protein SjTOM34/HIS immunized BALB/c mice, compared with the control group, recombinant proteins were induced in mice SjTOM34a worm reduction rate of15.9%and30.41%of the liver egg reduction rate, a significant difference (p <0.05). The change of the specific IgG and subtypes IgG1,IgG2a was measured by ELISA; the induced specific IgG and subtypes IgG1, IgG2a was maintained in a high level. It is show that SjTOM34had the potential for the anti-schistosomal drugs.Successfully constructed the recombinant eukaryotic expression plasmid pVAX1-SjTOM34, recombinant pVAX1-SjTOM34in BALB/c mice induced a35.4%worm reduction rate of30.22%of the liver and egg reduction rate, significant difference (p<0.05), after recombinant pVAX1-SjTOM34immunizing mice three times, high titers of specific IgG antibodies was induced.Flow cytometry (FCM) detection of immune and non-immune mice CD4+and CD8+cells, IL-4, IFN-y cytokines.The change of the specific IgG and subtypes IgG1, IgG2a was measured by ELISA. preliminary results show that, compared with the control group, immunized mice CD4+, CD8+cells and CD4+/CD8+ratio, cytokines IL-4, IFN-y levels have differet degrees of change, immunohistochemistry of mouse body to induce a mixed Thl/Th2cellular immune response.All the results will assist us to understand more functions about SjTOM34and provide the basis for screening new Schistosomiasis vaccine candidate molecules and drug targets.