Study Of DNA Vaccine And RNA Interference Of Schistosoma Japonicum TOR

Schistosomiasis is a chronic devastating parasitic disease that seriously affects public health in developing countries.Schistosomiasis japonica is widespread in southern China,causing serious harm to people's health and animal husbandry production.Schistosome with immune escape characteristics can long-term survival in the mammalian host.Complement is an important mechanism for the body to against parasites.The previous evidence has shown that schistosome may have multiple mechanisms to regulate the complement activity of host.The study shows that the tetraspaning orphan receptor?TOR?protein is a complement C2 antigen receptor on the surface of tegument,which can inhibit complement activity by combining complement C2a molecules.In this paper,the 1245bp DNA fragment encoding Schistosoma japonicum TOR protein was amplified from c DNA templates by PCR,and then cloned into the vector pXJ40-FLAG using Hind??Xho?to construct the eukaryotic expression plasmid pXJ40-FLAG-TOR.The recombinant plasmid was verified by sequencing and transfected into 293T cells.The expression of the target protein in 293T cell was detected by indirect immunofluorescence,Western blot and Quantitative Real-time PCR.Successful expression of SjTOR protein was confirmed by the specific green fluorescence in 293T cells 48 h after transfection and no green fluorescence in the cell transfected with the control plasmid.Quantitative Real-time PCR results showed that the transcription of SjTOR gene in 293T cells could be detected at6h and came to a peak at 24h after transfection,and then began to decline.Western blot results demonstrated that SjTOR with the molecular weight of 53kDa was probed by Anti-FLAG monoclonal antibody and anti-SjTOR-ED1 antisera.Results suggested that the SjTOR proteins can be expressed in293T cells.DNA vaccines were prepared from a large amoun of purified plasmid of pXJ40-FLAG-TOR.The Kunming and BALB/c mice were immunized with the DNA vaccine using intramuscular injection?i.m.?or electroporationmethod?e.p.?to carry out the immunoprotection test for the evaluation of effect against schistosomiasis.Results showed that worm burden and liver egg count were induced to reduce by 25%and 28.87%in Kunming mice immunized through e.p.and by 0.12%and 42.38%through i.m.immunization respectively,compared with the mice of vector control group.In BALB/c mice,the reduction of worm burden and liver egg count of BALB/c mice vaccined by e.p.or i.m.were 22.58%and 32.6%or 20.58%and 51.34%respectively,compared with the plasmid control group.ELISA results showed that the higher levels of IgG and IgG1 antibodies were induced in Kunming and BALB/c mice of e.p.immunization,and maintained till the end of the trial,whereas those of mice in intramuscular injection group were not significantly increased.The specific IgG2a antibody levels of mice in each vaccine group were not significantly increased.Luminex liquid chip analysis indicated that the levels of IL-12 and IFN-?in sera of BALB/c and Kunming mice immunized through e.p.and i.m.were increased,but the levels of IL-4 and IL-10 were decreased compared with PBS control group.The results suggested that the response induced by DNA vaccine maybe Th1 type immunity.Four pairs of siRNA molecules specific to SjTOR were designed and synthesised for the screening.The293T cells were co-transfected with siRNA and p XJ40-FLAG-TOR plasmid,and then the interference effect of each siRNA were assessed.The results of qPCR analysis showed that S1,S2,S3 and S4 siRNA molecules induced the transcription level of SjTOR gene decreased by 70%,63%,3.63%and 43%respectively.RNAi test in vivo were performed by injecting S1,S2,S4 siRNA into BALB/c mice through tail vein.The results showed that administration of S1,S2,S4 siRNA reduced the transcription level of SjTOR gene by 84.7%,80.9%and 23%,and the worm burden and liver egg count by 6.07%and 10.06%,24.24%and 44.27%,14.02%and 63.18%respectively compared with the injection of DEPC H₂O.The results showed that the specific siRNA could interfere with the expression of SjTOR gene,and block the development of schistosome.In conclusion,the plasmid of pXJ40-FLAG-TOR can be expressed in eukaryotic cells,and can induce the trend of Th1 type immune response and immune protection effect against schistsomasis when mice were immunized.The results of RNAi test of specific siRNA suggested that SjTOR gene might play an important role in the immune escape of Schistosoma japonicum.