Abstract:

Objective To investigate the resistance of E77.43 gene of Microtus fortis（MfE77.43） to Schistosoma japonicum infection. Methods MfE77.43 was constructed into the recombinant Adeno-associated virus AAV2. The AAV2-MfE77.43 was transfected into HEK293 cells by the calcium phosphate DNA coprecipitation method. The recombinant rAAV2-MfE77.43 was purified and total RNA was extracted from the transfected cells. The expression of E77.43 was examined by RT-PCR and the purity of rAAV2-MfE77.43 was analyzed by SDS-PAGE. Eighteen KM mice were divided into three groups （n=6 in each group）. Mice in the experiment group were intramuscularly injected on days 0, 3 and 7 with 400 μl recombinant AAV2-MfE77.43 virus which was 5-fold diluted in normal saline. Mice in negative control and blank control groups received same volume of pAAV or normal saline. Venous blood was collected through the tail before each injection, and E77.43 expression in plasma was detected by dot-ELISA method. After the last injection, each mouse was infected with 40 S. japonicum cercariae and sacrificed on day 41 after infection. Adult worms and liver eggs per gram（LEPG） were counted. Worm and egg reduction rate was calculated respectively. Egg granulomas were observed by HE staining. Results RT-PCR resulted in a 330 bp specific band. SDS-PAGE of virus shell protein revealed three protein bands with Mr of 87 000, 72 000 and 62 000, respectively. Dot-ELISA showed that E77.43 protein began to be expressed on day 3 after rAAV2-MfE77.43 injection, remaining stable till day 41. The adult worm number and LEPG were 20.16±3.93 and 19 800±2 715, respectively, with a worm and egg reduction rate of 27.3% and 26.2% in the experiment group. While the worm number and LEPG in the negative control group were 29.16±2.44 and 28 000±2 192（P<0.01）, respectively. HE staining and observation revealed fewer eosinophils and inflammatory cells around the liver eggs in the therapy group. Conclusion The E77.43 gene shows protective effects against S. japonicum infection, indicating that E77.43 may participate in the natural resistance of Microtus fortis to S. japonicum infection.

Key words: Microtus fortis, Schistosoma japonicum, E77.43 gene, Adeno-associated virus vector