Preliminary Study On The Functional Identification Of Kazal Serine Protease Inhibitor Gene Of Trichinella Spiralis Using DsRNA Interference

Trichinellosis is an important foodborne parasitic disease in the world.Serine protease inhibitors secreted by Trichinella spiralis play a key role in larval development.Serine protease inhibitor is also a target molecular antigen for early host immune response and can be used as a new antigen for the early diagnosis of Trichinella spiralis infection.This experiment uses dsRNA interference technology to silence the expression of Trichinella spiralis Kazal serine protease inhibitor gene,and further analyzes from the aspects of survival in vitro,invasion of intestinal epithelial cells,in vivo development,female fertility and the impact,So as to lay a certain research foundation for studying the function of Kazal serine protease inhibitor gene.In this experiment,specific dsRNA-TsKaSPI was introduced into muscle larvae and adults of Trichinella spiralis by soaking and electrophoretic transfer,and then the changes of TsKaSPI gene transcription and TsKaSPI gene protein levels in muscle larvae and adults were detected by real-time fluorescence quantitative PCR and Western-blot.The results showed that compared with the control group,the gene transcription level of muscle larvae and adults decreased by 59.36%and 67.80%(P<0.001)by soaking method,and 63.36%and 68.91%by electrophoretic method(P<0.001).Compared with the control group,the protein level of muscle larvae decreased by 70.67%and 69.24%respectively(P<0.001),and the silencing efficiency of electrophoretic method was better than the immersion method.The optimal interference concentration of dsRNA-TsKaSPI is 30?g/ml.At the same time,a real-time fluorescent primer for Tsp03044 gene was designed to verify that dsRNA interference is gene-specificity.The results showed that the transcriptional level of TsKaSPI gene was significantly lower than that the control gene Tsp03044,that is,dsRNA-TsKaSPI could only cause the silencing of its homologous genes.After that the dsRNA-TsKaSPI was treated by two methods of soaking and electrophoretic,and the transcription level was changed every Id after continuous culture in vitro for 6d.The results showed that the transcriptional level of soaking method decreased on the 1st day of culture,and then decreased significantly by 63.21%(P<0.001)on the 3rd day compared with the control group,and achieved the best silencing effect.With the increase of days,the transcriptional level of electrophoretic method decreased significantly by 69.31%(P<0.001)compared with the control group,and the effect of silence was the best.The transfection efficiency of electrophoretic method was better than that of soaking method,it was still at a low level on the 2nd day and 3rd day,and gradually recovered in the next few days.Observing the effect of TsKaSPI gene silencing on the survival of muscle larvae in vitro and invasion of intestinal epithelial cells in vitro.The results showed that the muscle larvae were continuously cultured in vitro for 6 days after soaking and electro-transfer treatment.The survival rates of the immersion method were 96%,91%,87%,67%,55%,and 46%(P>0.05).96%,91%,82%,67%,61%,52%(P>0.05),no significant difference from the control group,speculating that the TsKaSPI gene has no effect on the viability of Trichinella spiralis in vitro.The results of muscle larvae infecting intestinal epithelial cells in vitro showed that compared with the control group,the invasion rates of immersion method and electroporation method in the TsKaSPI treatment group were 41%and 43%respectively at 5 h,which were significantly lower than those in the control group.At the same time,with the increase of culture time,the number of invaded muscle larvae gradually increased,and the invasion rate reached the best at the 5th hour.Speculating that TsKaSPI gene silencing has a certain inhibitory effect on muscle larvae invasion of intestinal epithelial cells in vitro.To observe the effect of TsKaSPI gene silencing on the development of muscle larvae,the mice were orally inoculated with muscle larvae after the introduction of dsRNA.The results showed that the worm reduction rates of 6-day intestinal adults and 35-day muscle larvae were 36.22%and 31.87%respectively compared with the control group.At the same time,it was found that the number of newborn larvae in the PBS group,eGFP control group and TsKaSPI treatment group was 49.20±5.93,49.40±4.62,50.80±4.32 respectively.There was no significant difference in the number of female larvae between the treatment group and the control group.TsKaSPI gene has an important effect on the development of muscle larvae in the host,but it has little to do with the fecundity of female.The number of cells and the level of antibodies in the serum to further analyze the impact of TsKaSPI gene silencing on host immunity.The results of flow cytometry(FCM)showed that the number of CD3+CD4+ and CD3+CD8+ cells in the TsKaSPI treatment group increased significantly compared with the PBS control group,and the CD4+/CD8+ ratio also increased significantly.At the same time,the results of ELISA detection showed that the levels of IgG1,IgG2a and IgM antibodies in the TsKaSPI treatment group were significantly lower than those in the control group,and the IgG1/IgG2a ratio was also significantly lower.Therefore,we preliminarily believe that the silence of TsKaSPI gene enhances the cellular immune response of host Th1 type and weakens the humoral immunity of Th2 type.To sum up,this study confirmed that dsRNA could significantly inhibit the transcription and expression of Kazal serine protease inhibitor gene,the invasion of muscle larvae to intestinal epithelial cells and the development of larvae in mice,and promote the immune response of Th1 type fine cells,thus weakening the humoral immune effect of Th2 type.It is suggested that Kazal serine protease inhibitors are not only found in Trichinella spiralis.