| Schistosomiasis is a zoonotic disease that is widespread and harmful.The development of vaccines against schistosomiasis plays an important role in the prevention of schistosomiasis.The development of vaccines against schistosomiasis plays an important role in preventing schistosomiasis.Some scholars discovered that the lung-stage schistosomula is the main target of host immune attack and that its antigen molecules are regarded as the best vaccine candidates.The preliminary research of our laboratory has screened 105 genes such as adenylate kinase 1(AK1),cathepsin L3(CL3),glyceraldehyde-3-phosphate dehydrogenase(GAPDH)from the Schistosomula japonicum(S.japonicum)lung-stage schistosomula that are differentially expressed under the influence of host factors,in which the recombinant protein expressed by SjAK1 gene could induce a 50% reduction in worm burden and a40% reduction in egg load,but the immune function of SjCL3 and SjGAPDH genes remains unclear.Therefore,this study cloned and expressed S.japonicum CL3(SjCL3)and GAPDH(SjGAPDH),observed their expression and level and histological location in different developmental stages of S.japonicum,and studied the effects of immunization with SjGAPDH alone or combined with SjCL3 on mice against S.japonicum infection,exploring their role in immune evasion and the treatment of type I diabetes,which could add new content to the exploration of the interaction between schistosome and its host,anti-insect targets,and schistosomiasis vaccine research with not only theoretical significance but also practical significance.In this study,recombinant SjCL3(rSjCL3)and rSjGAPDH were cloned and expressed and results showed that the rSjCL3 and rSjGAPDH were both active proteins,and the enzyme activity of them was tested to be 26822 U/g and 3220 U/g,respectively.The results of immunofluorescence histochemistry showed that SjCL3 was expressed in eggs shell,tegument,the partial muscle layer,and alimentary canal and that SjGAPDH was expressed in eggs shell,tegument,and most of the muscle layer.The results of fluorescence quantitative PCR showed that SjCL3 and SjGAPDH were expressed in various periods of S.japonicum,in which SjCL3 had the highest m RNA level expressed in the eggs,and the lowest level in the 10-day old schistosomula,and that SjGAPDH had the highest m RNA level expressed in the male worms and the lowest level in the 18-day old schistosomula.After immunization with rSjCL3,rSjGAPDH,and their combination,mice got a reduction of 30.5%,37.8%,and 65.6% in worm burden,a reduction of 31.7%,38.1%,and 70.9% in egg load,a reduction of 29.5%,41.1%,and 74.6% in granuloma area,and a reduction of 35.4%,52.4%,and 76% in fibrosis area,respectively.However,the immunization of adjuvant-free combination can only induce a 12.1% reduction in worms and a 14.2% reduction in eggs without the effect of Freund's adjuvant,which presented that SjCL3 had no adjuvant-like function.The results of flow cytometry showed that the percentages of Tregs in mice immunized with rSjCL3,rSjGAPDH,and their combination were 3.94%,3.68%,and 2.61%,respectively,and the percentage of Tregs in the combination group decreased significantly(P <0.05)compared with Freund's adjuvant.ELISA results showed that the value of IgG1/IgG2 a in mice serum immunized with rSjCL3,rSjGAPDH,and their combination were all<1,and the value of it was 0.8,0.71,and 0.48,respectively,indicating that the mice after immunization all had Th1-type shifted immune responses;in vitro studies have shown that immune serum-mediated macrophages have insecticidal rates of 12.05%,14.08%,and 18.86%,respectively,and compared with the negative serum group,the difference was statistically significant(P<0.05).The insecticidal mechanism of destructive effects for rSjCL3 and rSjGAPDH in vitro was observed using antibody-dependent cell-mediated cytotoxicity(ADCC),and it was found that rSjCL3 and rSjGAPDH can destroy the insecticidal effect of ADCC by reducing the adhesion of macrophages to schistosomula,and when the corresponding inhibitors were added,the insecticidal rate increased from 8.17% to15.46% and 6.1% to 19.49%,respectively,and the number of macrophages attached to the surface of the worm increased from 2.45 to 5.75 and 1.43 to 6.57,respectively,and the differences were statistically significant(P<0.05).When the target genes genes were inhibited by dsRNA of SjCL3 and SjGAPDH alone and in combination,the survival rates of mechanically transformed young worms were 72.7%,62.5%,and56.5%,respectively.Compared with the EGFP group,there was no significant difference in survival rate forSjCL3 group,yet the other two groups have significant differences(P<0.05).The survival rates of 21-old day young worms were 92.77%,91.83%,90.56%,respectively,and the difference was not statistically significant(P>0.05).The survival rates of 42-old day adult worms were 100%,96.15%,and93.33%,respectively,and the difference was not statistically significant(P>0.05).Besides,the therapeutic effects of immunization of rSjCL3,rSjGAPDH,and their combination on type I diabetic mice were also explored,and the results showed no effect on the blood glucose levels of mice(P>0.05).The value of IgG1/IgG2 a for serum in type I diabetic mice after immunized with rSjCL3,rSjGAPDH,and their combination were 0.65,1.06,and 0.51,respectively.In summary,rSjCL3 and rSjGAPDH from lung-stage schistosomula that were cloned and expressed this study were active that play an important role in evading immunity of host.SjCL3 was expressed in the eggs shell,tegument,partial muscle layer,and alimentary canal,and expressed at the highest levels in eggs.SjGAPDH was expressed in the eggs shell,tegument,and most of the muscle layer,and expressed at the highest levels in eggs and males,respectively.Moreover,rSjCL3 could not play the role of adjuvant without the action of Freund's adjuvant,yet combined with rSjGAPDH in Freund's adjuvant background can induce mice to produce a 65.6% reduction rate in worm burden and 70.9% reduction rate in egg load,which is worthy of further study. |
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