Primary Studies On Functions Of 22.6kDa Membrane-associated Protein Of Schistosoma Japonicum

Schistosomiasis is the most serious disease caused by parasites in China and the government has to spend a lot of money in preventing and controlling the disease. However, a large number of residents and livestocks in epidemic areas are still suffering from the disease because of the complex life cycle of the blood fluke. As one of basic things on controlling the disease it's urgent to develop a high sensible and accuracy way of diagnosis. To discover novel targets for diagnosis of schistosomiasis japonicum, we immunoscreened Juvenile S. japonicum cDNA library with sera from rabbits infected by cercarie. We obtained 34 positive clones in the end, 24 of which were chosed and sequenced and subsequent sequences were searched against the Genbank. We found that 13 of 24 positve clones were Schistosoma japonicum 22.6kDa tegument associated antigen gene which implicated that the antigen coded by Sj22.6 gene might be aboundant in the tegument and was supposed to be a potent candidate of diagnosis for Schistosomiasis japonicum accordingly. The gene was amplified by PCR methods with a pair of primers paired to the positive clone DNA and the PCR products were ligated into the expressing plasimid pET28a and the recombinant was transformed into E.coli BL21. The correct recombinant was confirmed by DNA sequencing and the recombinant protein Sj22.6 (rSj22.6) was expressed when induced by 1mmol/L IPTG (isopropylthio-β-D-galactopyranoside) at 37℃and when OD600 is 0.6 and production of rSj22.6 protein peaks at 4h after induction. The protein existed in soluble form in our experiment condition .Western blotting was performed and we found that rSj22.6 protein was recognized strongly by sera from rabbits (diluted in 1:100) infected by the cercarie but not from the nomal rabbits which showed its good immunoactivity. We also immunized four BABL/c mice with rSj22.6 protein and all mouse produced high titers of antibodies against the protein (over 1:16 000) which was tested by ELISA method, which means that the protein produced good immunogenicity against BABL/c mice. Additionally the protein is also recognized slightly by sera from both acute and chronic patients of schistosomiasis japonicum but not sera from normal people by Western Blotting. It implicated that the protein was a potent candidate of diagnosis. Concentration of rSj22.6 protein and adult worm antigen (AWA) for ELISA and dilution of sera were confirmed when ratio of ODs of positive and negative (P/N) peaked.When rSj22.6 protein was used to differentiate positive sera (18 sera from rabbits infected by cercarie ) from control sera (sera from 18 healthy rabbits), sensitivity and false positive rate was 77.8% (14/18)and 22.2% (4/18)respectively. When two batches of sera were tested over and over again by both of rSj22.6-ELISA and AWA-ELISA methods, sensitivity and false positve rate of the first batch was 5.6%(1/18) and 5.6%(1/18) by rSj22.6-ELISA and 61.1%(11/18) and 5.6%(1/18) by AWA-ELISA respectively, while that of the second batch was 26.7%(4/15) and 6.7%(1/15) by rSj22.6-ELISA and 93.3%(14/15) and 6.7%(1/15) by AWA-ELISA respectively. The result denied the potent role of rSj22.6 protein as a diagnostic candidate for Schistosomiasis japonicum. At last we tested antithrombotic role of the protein. When concentration of rSj22.6 protein in plasma was over 200μg/ml, both of PT and APTT were delayed a little and when the protein reached its maximal concentration in plasma PT and APTT were prolonged by 4.5s and 12s respectively and the effect was in a dose-dependent manner. We incubated rSj22.6 protein with bovis thrombin in different ratios of gram-molecule at 23℃for 30 min respectively and all the sample were spun at 12 000g for 15min at 4℃and the supernatants were detected by mice sera against rSj22.6 protein. We found that all samples incubated with bovis thrombin produced new proteins with molecule weight lower than rSj22.6 protein according to Western blotting, which means that bovis thrombin could degrade the rSj22.6 protein.In a conclusion, the rSj22.6 protein showed good immunogenicity and immunoactivity but was not an effective candidate for diagnosis of schistosmiasis japonicum according to our results. The protein has a slightly activity of anticoagulation and could bind bovis thrombin and be degraded by the latter. The reaction between the two proteins may be the reason why rSj22.6 protein could delay both of PT and APTT.