| Toxoplasma gondii(T.gondii)is a worldwide opportunistic pathogenic protozoa that can infect almost all warm-blooded animals and cause severe zoonotic diseases,with one third of the world’s population at risk.Dogs are human companions and pets.They are also an important intermediate host of T.gondii and can transmit it to humans through a variety of ways.Currently,there is no ideal vaccine against toxoplasmosis,so it is of great significance to develop an effective dog toxoplasmosis vaccine to prevent toxoplasmosis and reduce the risk of human infection.Currently,the genetic engineering vaccines of T.gondii mainly include subunit vaccine,gene deletion vaccine,recombinant live vector vaccine and DNA vaccine,etc.Subunit vaccine has the advantages of simple construction and high safety,and has attracted much attention.At present,there are few studies on T.gondii subunit vaccine.Therefore,in this study,the ENO2 and GRA7 genes of T.gondii and mouse IL-2 genes were connected in tandem to express the recombinant protein ENO2-GRA7-IL2,and immunized Balb/c mice to evaluate its immune protective effect.In order to evaluate the immunoprotective effect of the recombinant protein on dogs,the recombinant plasmid ENO2-GRA7-IL2(D)was constructed in tandem with the genes of T.gondii ENO2 and GRA7.The recombinant plasmid was induced and expressed to immunize Beagle dogs,and the immunoprotective effect was evaluated,which laid the foundation for the research of T.gondii subunit vaccine.Expression and identification of ENO2-GRA7-IL2 recombinant protein:The recombinant plasmid p ET-32a-ENO2,p ET-32a-GRA7 and p ET-32a-IL2 were constructed by PCR amplification of the ENO2 and GRA7 genes and mouse IL-2 genes,respectively.The recombinant plasmid p ET-32a-ENO2-GRA7-IL2 was synthesized by bioinformatics analysis of the three genes.After induction and purification by Escherichia coli expression system,the purification results were analyzed by SDSPAGE,and the reactivity of the recombinant protein was tested by Western blot.The results showed that target bands of Eno2-Gra7-IL2(2376bp),ENO2(1335bp),GRA7(447bp)and IL2(510bp)were obtained by double enzyme digestion,respectively.The recombinant proteins ENO2-GRA7-IL2(110k Da),r ENO2(70 k Da),r GRA7(45 k Da)and r IL-2(40k Da)were obtained after induction and purification,which were consistent with the size of the target proteins.Western blot indicated that the recombinant protein ENO2-GRA7-IL2 had good reactivity.Immune protection of ENO2-GRA7-IL2 recombinant protein in mice:A total of 105 Balb/c mice were randomly divided into negative control group,positive control group,r ENO2 group,r GRA7 group,r IL-2 group,mixed immunization group and ENO2-GRA7-IL2 group,with 15 mice in each group.Lymphocyte proportion and cytokine levels in peripheral blood of mice were detected after two weeks of immunization.Mice were infected with tachyzoites of T.gondii at the dose of 1×103per mouse,and the survival time was observed.The mice were dissected 6 days after infection,and the parasites burden of liver was detected and the pathological changes were observed by biopsy.The results showed that compared with the control group,the proportion of CD4+/CD8+ in all immune groups except the mixed immune group was significantly increased(p < 0.05),and serum IFN-γ in all immune groups was significantly increased(p < 0.05).IL-12 in ENO2-GRA7-IL2 group of r ENO2 and r GRA7 group was significantly increased(p < 0.05).The results of immune protection test showed that the mice in the ENO2-GRA7-IL2 group still survived 4/8 days after the 21-day test period,and all the other mice in the infected group died.The parasites burden of liver test results showed that all immune groups was significantly decreased compared with the positive control group(p < 0.05),and the parasites burden of liver of ENO2-GRA7-IL2 immune group was decreased by 92.71%.There was no obvious degeneration of hepatocytes in the ENO2-GRA7-IL2 group,and only a small amount of neutrophil infiltration,while pathological changes such as degeneration and necrosis of hepatocytes and large amount of neutrophil infiltration were observed in the other immune groups.Immune protection of ENO2-GRA7-IL2 recombinant protein in dogs:It was found that the recombinant protein ENO2-GRA7-IL2 had a good immunological protection effect on mice.Therefore,p ET-32a-ENO2-GRA7-IL2(D)recombinant plasmid was constructed by combining canine IL-2 gene with ENO2 and GRA7 in series.The reactivity was determined by SDS-PAGE and Western blot after the expression was induced by Escherichia coli expression system.Eight beagles were randomly divided into negative control group(2),positive control group(3)and immune group(3).The recombinant protein was injected subcutaneously and immunized twice.Serum samples were collected at 7 d intervals from 1 week before immunization to 2 weeks after immunization,and the antibody levels were detected by indirect ELISA.Two weeks after inoculation,the tachyzoites of the RH strain were infected,and the temperature changes and clinical manifestations were recorded.On the 8th day after infection,dogs were euthanized and their livers were collected,and the amount of charged parasites was detected by absolute fluorescence quantitative PCR and pathological tissue sections were prepared.The results showed that the recombinant protein ENO2-GRA7-IL2(D)with the size of 100 k Da was obtained successfully,and Western blot identification showed good reactivity.The antibody level of immunized dogs increased to 1:25 600.After infection,the body temperature of dogs in both the positive control group and the immune group increased,and the positive control group showed obvious clinical symptoms and slight ascites.Compared with positive control group,the average parasites burden of liver of immune group was decreased by 61.08%,and the difference was very significant(p < 0.01).In the immune group,there were no obvious pathological changes in hepatocytes and less neutrophil infiltration.These results indicate that recombinant protein ENO2-GRA7-IL2(D)plays a certain immune protective role against acute T.gondii infection in dogs.In conclusion,the recombinant protein ENO2-GRA7-IL2 expressed in this study proved that its immunoprotective effect on mice was superior to that of a single recombinant protein.The immunoprotective study of ENO2-GRA7-IL2(D)showed that the recombinant protein had certain immune protective effect against acute T.gondii infection in dogs,which provided a reference for the immunoprevention of toxoplasmosis in dogs in the future. |
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