| Schistosomiasis caused by infection with Schistosoma japonicum cercariae was one of the most prevalent tropical parasitic zoonosis, which is serious harm to health of people and animal in China. It is worthwhile to screen new drug target and develop the new candidate vaccine for further controlling and prevention of the disease. At present,Schistosomiasis japonicum, prevailing around12provinces (municipality, autonomous region) in South China, has been causing seriously threat to health of human and animals. Precise diagnosis is crucial to the prevention and treatment of a disease. However, there is no satisfaction for diagnosis of the disease by methods available. It is an urgent need to search for an approach with higher sensitivity and specificity as well as potential value on chemotherapeutic efficacy.Based on the study of tegument surface proteins at different development stages of Schistosoma japonicum by proteomic techniques. SjRAD23and SjMBLAC1were found at different stages.They may play important roles in Schistosoma japonicum’s life cycle and have the value of diagnosis of schistosomiasis japonica. In this study, SjRAD23and SjMBLACl were cloned, expressed and characterized, the potential of SjRAD23and SjMBLAC1F recombinant protein as a vaccine candidate were evaluated. 1Cloning expression characteristics and protective effect evaluation of Radiation sensitive protein23(RAD23) from Schistosoma japonicumRadiation sensitive protein23has nucleotide excision repair function and it also plays an important role in Ubiquitin-proteasome pathway (UPP). RAD23is indispensable to Schistosoma japonicum growth and development. In this study,using PCR technology and bioinformatics analysis,S.japonicum radiation sensitive protein23(SjRAD23) cDNA sequences was cloned.SjRAD23gene ORF is1053bp. The cDNA containing the open reading frame of SjRAD23was subcloned into PET28a (+) vector and transformed into competent Escherichia coli BL21for expression. The recombinant protein molecular weights about44kDa and is expressed in the supernatant and pellet. Using immunohistochemical technique to observe the distribution of the protein in the worm, the protein is widely distributed in the membrane of Schistosoma japonicum. ELISA test revealed that IgG, IgGl and IgG2a antibodies are significantly in the serum of rSjRAD23immunized mice. The results show that SjRAD23may play an important role in the growth and development of Schistosoma japonicum. Western blotting analysis showed that the recombinant SjRAD23could be recognized by anti-42d-old soluble protein serum and SjRAD23has good immunogenicity. The results of the immuno-protection experiments showed that the worm reduction was35.94%,the number of eggs in liver tissue was40.59%compared with206adjuvant control group. Experiments show that the rSjRAD23induced protective immunity in BALB/C mice 2Cloning expression characteristics and protective effect evaluation of Metallo beta lactamase domain protein1(MBLAC1) from Schistosoma japonicumMetallo beta lactamase (MBL) is a kind of dependence of zinc ions in the catalytic activity of the enzyme, its substrate covers various beta carbapenem, such as lactam antibiotics.In this study,using PCR technology and bioinformatics analysis, S.japonicum Metallo beta lactamase domain protein1(MBLAC1) cDNA sequences was cloned.SjMBLAC1gene ORF is711bp. The cDNA containing the open reading frame of SjMBLAC1was subcloned into PET28a (+) vector and transformed into competent Escherichia coli BL21for expression. The recombinant protein molecular weights about32kDa and is expressed in the supernatant and pellet. Using immunohistochemical technique to observe the distribution of the protein in the worm, the protein is widely distributed in the membrane of Schistosoma japonicum. ELISA test revealed that IgG, IgGl and IgG2a antibodies are significantly in the serum of SjMBLAC1immunized mice. The results show that SjMBLAC1may play an important role in the growth and development of Schistosoma japonicum. Western blotting analysis showed that the recombinant SjMBLAC1could be recognized by anti-42d-old soluble protein serum and SjMBLAC1has good immunogenicity. The results of the immuno-protection experiments showed that the worm reduction was33.79%,the number of eggs in liver tissue was37.79%compared with206adjuvant control group. Experiments show that the rSjMBLACl induced protective immunity in BALB/C mice.3Diagnosis study on Schistosomiasis japonicum in domestic animal with recombinant antigen rSjRAD23as diagnosis antigenThe conditions for ELISA were optimized through trail of cross-chess. By ELISA with recombinant antigen rSjRAD23and soluble egg antigen SjSEA as diagnosis antigen,60serum from water buffalo free of infections,75serum from these with infection of Schistosomiasis japonicum,14serum from these with infection of paramphistomum and6serum from these with infection of Fasciola gigantica were detected.The sensitivity, specificity of the two antigens were compared by ELISA with recombinant antigen rSjRAD23and soluble egg antigen SjSEA as diagnosis antigen. Levels of two specific antibodies were tested in rabbit serum prior to infection,2weeks,4weeks,6weeks after infection and every month after treatment. The variation of growth and decline of the two specific antibodies in rabbit serum were analyzed and the chemotherapeutic efficacies of the two methods were evaluated.The results showed that sensitivities and specificity of recombinant antigen rSjRAD23and soluble egg antigen SjSEA are equivalent in detection of Schistosomiasis japonicum in water buffalo.The cross reaction rate of recombinant antigen rSjRAD23is much lower than that of soluble egg antigen. Compared with soluble egg antigen,the antibody specific to recombinant antigen rSjRAD23disappears quickly after treatment, Therefore recombinant rSjRAD23is more valuable than SjSEA for efficacy assessment in the diagnosis Schistosomiasis japonicum. |
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