Development Of Multi - Epitope Recombinant Antigen And Its Preliminary Application In The Diagnosis And Immunoprotection Of Schistosomiasis Japonicum In Japan

Schistosomiasis is still one of the major public health problems in C hina. The bovines infected Schistosoma japonicum are the most important source of infection and the role of sheep and goats in schistosomiasis transmission in some endemic areas can not be ignored. It can help to block the spread of schistosomiasis if the bovines, sheep and goats were effective controlled in C hina. The diagnosis of schistosomiasis is the central link in prevention and control the disease. The schistosome soluble egg antigen(SEA) is the most commonly used diagnostic antigen in serological diagnostic techniques. But when SEA was used as a diagnostic antigen, it usually possessed low specificity, high cross-reactivity and it can not distinguish between current infection and past infection. In addition, the SEA was not suitable for the efficacy assessment as a diagnostic antigen. Some schistosome recombinant antigens have been used as diagnostic antigens or vaccine candidate molecules; the sensitivity of most recombinant antigens was lower than that of SEA and the induced protective effect was unsatisfied and unstable. The multi-epitope recombinant antigens have the possible to enhance its antigenicity and immunogenicity for containing multiple epitopes. It is possible to increase the sensitivity and improve the immune protective effect using the multi-epitope antigens as diagnostic antigens or vaccine candidate molecules. Based on the above description, the paper launched the following research. 1 Epitope analysis of the candidate antigens SjPGM, SjRAD23 and the construction of single molecule and multi-epitope recombinant antigensThe online prediction software BepiPred 1.0、T-epitope Designer、PredictProtein and IEDB Analysis were used to predict the B, T cell epitopes, antigenicity and surface accessibility of SjPGM and Sj RAD23. The B-cell epitopes and antigenicity of the predicted proteins were considered first. Three epitopes enrichment polypeptide were selected, including one from SjPGM(aa 85-166) named BSjPGM, two from SjRAD23, named BSj RAD23-1(aa 46-123) and BSj RAD23-2(aa 166-230). The large hydrophilic region of Sj23(LHD-Sj23) was selected as another candidate fragment. A total of 9 recombinant prokaryotic expression plasmid were constructed which included 4 single molecule recombinant plasmids, BSjPGM/p ET-28a(+) 、 BSj RAD23-1/pET-28a(+)、 BSjRAD23-2/pET-28a(+)、 BSj23/p ET-32a(+) and 5 multi-epitope recombinant plasmids, BSjPGM-BSj23/p ET-28a(+) 、 BSjPGM-BSj RAD23-1/pET-28a(+) 、 BSjPGM-BSjRAD23-1-BSj23/pET-28a(+) 、BSj RAD23-2-BSjPGM-BSj23/p ET-28a(+) and BSj RAD23-2-BSjPGM/p ET-28a(+). The remaining six recombinant expression plasmids were successfully expressed in E. coli, and got the purified protein except BSjPGM/p ET-28a(+)、BSj RAD23-2/pET-28a(+) and BSj RAD23-2-BSjPGM/ p ET-28a(+). 2 Evaluation of the single molecule /muti-epitope recombinant antigens as the diagnostic antigens to detect goats schistomomiasisTo assess the potential of r LHD-Sj23, rSjPGM, rSj RAD23 and the newly constructed multi-epitope proteins as diagnostic antigens for goat schistosomiasis, sera from 91 schistosome- infected goats, 44 non- infected goats, 12 goats infected with H.contortus and 37 Orientobilharzia-infected goats were tested by ELISA using these recombinant antigens and SEA(control) as detecting antigen, the result show that, the sensitivity of the multi- epitope recombinant antigens were all higher than the four singe molecule recombinant antigens(rSj RAD23, rSjPGM, r BSj RAD23-1, rBSj23), except r BSjPGM-BSj RAD23-1 and the r BSjPGM-BSj RAD23-1-BSj23 possessed the highest sensitivity(97.8%, 89/91) of the all recombinant antigens, meanwhile keeping high specificity(100%, 44/44) and low cross-reactivity(H. contortus: 8.33%, 1/12; Orientobilharzia 13.51%, 5/37). When SEA was employed as diagnostic antigen, the sensitivity was 100%(91/91), but specificity was only 75%(33/44), and the cross-reactivity with H. contortus and Orientobilharzia was 25%(3 / 12) and 83.78%(31/37), respectively. The study presented here suggests that the application of recombinant multi-epitope proteins may increase the sensitivity of diagnostic techniques for schistosomiasis, and that the recombinant antigen rBSjPGM-BSj RAD23-1-BSj23 has the potential to be used as a diagnostic antigen for goat schistosomiasis. 3 Evaluation of the single molecule /muti-epitope recombinant antigens as the diagnostic antigens to detect water buffalo schistomomiasisFive recombinant antigens including two single-molecule epitope antigens(rBSjRAD23-1, r BSj23) and three multi-epitope recombinant antigens(rBSjPGM-BSj23, r BSjPGM- BSj RAD23-1-BSj23, r BSj RAD23-2-BSjPGM-BSj23), showing good effect in diagnosing goat schistosomiasis were chose as diagnostic antigens to detect water buffalo schistosomiasis. The SEA was also as reference antigen. A total of 114 sera from schistosome- infected water buffaloes, 92 sera fom uninfected water buffaloes and 14 sera from Paramphistomum- infected water buffaloes were chose to evaluate the sensitivity, specificity and cross-reactivity of the 6 diagnostic antigens. The ELISA test showed r BSjPGM-BSjRAD23-1-BSj23 got the highest sensitivity(95.61%, 109/114) among the five recombinant antigens and its specificity and cross-reactivity rate were 97.83%(90/92) and 7.14(2/14). Contrastly, the sensitivity, specificity and cross-reactivity of SEA were 100%(114/114), 82.61%(76/92) and 50%(7/14), respectively. We could concluded from this study that the application of multi-epitope antigen may increase the sensitivity of the method for water buffalo schistosomiasis and the multi-epitope recombinant antigen rBSjPGM-BSj RAD23-1-BSj23 were also preferred diagnostic antigen for buffalo schistosomiasis. 4 The immunoprotection of three epitope recombinant antigens in Schistosoma japonicumBased on the analysis of cell epitope of SjPGM and Sj RAD23, the new expression plasmid BSj RAD23-2-BSjPGM-BSj RAD23-1/p ET-28a(+) were constructed. The three recombinant antigens(r BSj RAD23-1,rBSjPGM-BSj RAD23-1,rBSj RAD23-2-BSjPGM-BSj RAD23-1) combined with 206 adjuvant were injected into BALB/C mice. The worm reduction rate(42.7%, 30.4 %) and egg reduction rate(40.3%, 30.3%) induced by the trivalent multi-epitope antigen rBSj RAD23-2-BSjPGM-BSj RAD23-1 was higher than the other two recombinant epitope antigens in two separate batches of animal experiments. This study suggested theconstruction of multi-epitope antigen might enhance the immune protective effect.The present study has developed a multi-epitope antigen named rBSjPGM-BSj RAD23-1-BSj23 possessing high sensitivity and specificity and established the ELISA test, providing an alternative new diagnostic technology for the prevention and control of livestock schistosomiasis in C hina.