Construction And Immunoprotection Of DNA Vaccine Based On P43and P53Gene Of Trichinella Spiralis In Mice

Trichinellosis is caused by Trichinella spiralis, a severe zoonotic risk of food-borne parasiticdisease. It has a great threat to the health status of the human, and has a higher rate of infectionworldwide which can cause the infected host to produce complications, such as myocarditis, thetoxemia and even death. Therefore, it is very important to study the DNA vaccine ofanti-Trichinella and the immune mechanism of Trichinellosis prevention.The DNA vaccine was constructed via cloning Trichinella spiralis p43and p53genes into theeukaryotic expression vector pcDNA3.1(+). Intramscular injection of the obtained recombinantplasmids was performed in mice three times in two weeks. After the third set of vaccination, eachof mice was infected perorally with100T. spiralis muscle larvae. To access the effects of immuneprotection, the reduction rates of adult worms and muscle larvae and the index of reproductivecapacity were calculated on the7thday and the35thday after inoculation. In addition, serum frommice was prepared for the detection of antibody levels and mouse heart-type fatty acid bindingprotein (H-FABP) at different times. The results showed that the reduction rates of adult wormswas significant difference(P＜0.05) compared with the PBS and the control group. Group I, II andIII inducted reduction rates of adult worms were37.31%,34.33%and41.79%. The resultsindicated that the reduction rates of muscle larvae and the index of reproductive capacity wasextremely significant difference(P＜0.01)compared with the PBS and the control group. Group I, IIand III inducted reduction rates of muscle larvae were70.16%,65.60%and72.07%. They inductedreduction rates of the RCI were71.33%,76.60%and70.38%. The results indicated that asignificantly higher antibody level was found in the test group than in the PBS group and theplasmid group. The IgG antibody level was increased and significantly different with the PBSgroup(P＜0.01). H-FABP test in each group showed the index was lower than70pg/ml. TheCD4+/CD8+ratio was detected for mouse lymphocytes using Flow cytometry. The results showedthat each group was immune-enhancing in the immune process compared with the PBS group.There was immunosuppressive at7thpost infection of T. spiralis and then rising stage at21thand35th. There were still immune-enhancing at the end of the test.The TLR2and TLR4expression levels of the mouse small intestine were detected bySYBR-Green I of Q-PCR. The results showed that each group was low expression level of TLR2at each point-in-time, except for21thpost infection of T. spiralis. The expression level was increasedduring immunization and reached the maximum at7thpost infection in TLR4test. Then theexpression level was declined. The research indicated, the protein of p43and p53may be theligand for TLR2and TLR4, but not obvious.In this experiment, it is important to discuss the effect of immune protection and mechanismsthrough DNA vaccine of p43and p53gene, and the TLR2and TLR4expression levels of themouse small intestine. And it laid the theoretical foundation for further to study T. spiralis immunemechanism and pathogenic mechanism.