Preliminary Studies On The Effects And Mechanism Of Related Gene Of Anti-Schistosoma Japonicum Schistosomula In Microtus Fortis

:Worldwide schistosomiasis, a serious public health problem, is an important endemic parasite disease. Schistosomiasis japonica remains a major public-health concern in China. Experimental and epidemiological evidence strongly implicates that Microtus fortis is a naturally resistant vertebrate host of Schistosoma japonicum. On the basis of preliminary experiments, in this study we isolated and purified M. fortis serum proteins by Blue Sepharose chromatography to test their anti-schistosomula effect. Then the killing effect of the albumin to schistosomula was tested in vitro. Our results showed that Mf-albumin provides potent resistance against S. japonicum. At a protein concentration of2.5mg/ml, it induced a schistosomula mortality rate of37.94%(P<0.05) in vitro, significant higher than that of negative control. To test the schistosomula-killing effect we constructed the eukaryotic expression vector of pcDNA3.1-Mf-albumin (+). Conditioned medium of pcDNA3.1-Mf-albumin (+) was added to cultured schistosomula at a concentration of50%. We also found that conditioned medium of pcDNA3.1-Mf-albumin (+) caused a38.7%of schistosomula death in96 h, considerably higher than that of the negative control (P<0.05). To characterize the mechanisms involved in clearance, schistosomula were incubated with FITC-labeled Mf-albumin, and fluorescent enrichment effect can be found in the gut lumen of schistosomula after48h incubation. Then albumin was digested by Legumain.In previous study, ILKAP was isolated and identified from M. fortis cDNA library which could significantly inhibit schistosomula. In this study a total of8ILKAP-interacting proteins from schistosomula were identified by the methods of coimmunoprecipitation and MALDI-TOF-MS. Then we constructed the eukaryotic expression vector of Myc-TA-pcDNA3.1/(-)b and Flag-ILKAP-pCMV-2b and then transiently transfected into HEK293T cells using Lipofectamine2000. Finally coimmunoprecipitation coupled with Western blotting were done to confirm that tegument antigen (TA) was interacted with ILKAP.Chapter1Isolating and purifying the albumin of M. fortis serum and identifying inhibitory effect on schistosomula in vitroM. fortis serum was applied to the Pre-swollen Blue Sepharose FF affinity chromatography column. Then the solution of the elution peak was lyophilized, desalted and lyophilized. Finally SDS-PAGE analysis was used to ensure the purity of the Mf-albumin. In order to confirm anti-schistosomula effect of Mf-albumin,Mf-albumin was added to cultured schistosomula at a final concentration of2.5,5.0and10.0mg/ml with Ms-albumin as a negative control and M. fortis serum as a positive control. We observed and calculated the death rate of schistosomula after96h incubation. Our results showed that when the concentration of Mf-albumin was2.5mg/ml, the death rate of schistosomula was37.94%(P<0.05), significant higher than that of the negative control.Chapter2Preparation of conditioned medium of pcDNA3.1/Mf-albumin and effect on schistosomulaTo further verify the anti-schistosome effect of Mf-albumin, conditioned medium containing Mf-albumin or Ms-albumin was added to schistosomula culture at a concentration of50%. We observed and calculated the death rate of schistosomula in96h. Our results showed the average schistosomula-killing rate of pcDNA3.1/Mf-albumin conditioned medium was38.7%, which was significantly higher than that of the negative control.Chapter3Detection of the deposite site of Mf-albumin in schistosomula and hydrolysis of Mf-albumin by LegumainSchistosomula culture was incubated with FITC labeled proteins or other control proteins. The results clearly showed that fluorescent enrichment effect can be found in the gut lumen of schistosomula after incubation with FITC-Mf-albumin for24h. After48h of incubation, the fluorescent enrichment effect became even stronger in the gut of schistosomula through fluorescence microscopy. However, there is no distinct fluorescence enrichment in schistosomula that were treated with the FITC-labeled Ms-albumin, fluorescence and the blank control.In order to address how proteases might function in the schistosomula gut, we focused on the endopeptidases, cathepsin B, D and L1, and the asparaginyl endopeptidase (Legumain). After sequence analysis the result showed there is difference when Legumain was used to evaluate the ability of the digestion process to M. fortis and mice albumins.The action of legumain on albumin was investigated by incubation at37℃in a mixture for2h and4h. The reaction was terminated by the addition of30μl of SDS loading buffer. Hydrolysate was used to examine Legumain activity to M. fortis and BALB/c albumins in the native states as described in the Experimental section by SDS-PAGE. The result showed that M. fortis and BALB/c serum albumins were very sensitive to Legumain, the two albumins were degradated after legumain was added and incubated for2h and4h. Although M. fortis and BALB/c serum albumins both can be cleaved by Legumain, there is still some difference after hydrolysis.Chapter4ILKAP-interacting proteins from schistosomulaIn previous study, we constructed bone marrow cDNA library of M. fortis, screened and identified gE77.43clone from the sub-library by expression cloning techniques. We found the produce of gE77.43could be integrin-linked kinase-associated serine/threonine phosphatase (ILKAP). Previous experiments showed that ILKAP provides potent resistance against S. japonicum. So a total of8ILKAP-interacting proteins from schistosomula were identified by the methods of coimmunoprecipitation and MALDI-TOF-MS.Chapter5Verification on ILKAP-interacting proteins from schistosomulaIn order to further verify the interaction of schistosomula proteins with ILKAP we used the cell coimmunoprecipitation and Western blotting. Then we constructed the eukaryotic expression vector of Myc-TA-pcDNA3.1/(-)b and Flag-ILKAP-pCMV-2b and then transiently transfected into293T cells using Lipofectamine2000. Finally coimmunoprecipitation coupled with Western blotting were done to confirm that tegument antigen (TA) was interacted with ILKAP. The obtaining of ILKAP-interacting proteins from schistosomula laid a strong basis for the further research.