| Neospora caninum is an intracellular apicomplexan protozoan parasite,which mainly causes the nervous system dysfunction of dogs,the motor nervous system disease of cattle abortion,stillbirth and newborn calves,causing huge losses to the cattle industry.In recent years,studies on the gene function of N.caninum have found that rhoptry proteins,microneme proteins and dense granule proteins are involved in the invasion and replication of N.caninum and other pathogenic processes.However,many gene functions of N.caninum,especially the pathogenic genes,are still unknown.c-Myc regulation proteins(MYRs)regulate the expression of host cell c-Myc.Recent studies have reported that Plasmodium,Toxoplasma gondii and Trypanosoma can significantly increase the expression of c-Myc in host cells.Toxoplasma gondii MYR is associated with host cell c-Myc,parasitophorous vacuoles and parasite pathogenicity,but the functions of MYR genes in N.caninum remain unknown.Therefore,the subcellular localization of NcMYR1 and NcMYR2 proteins were observed by immunofluorescence.CRISPR-cas9 genome editing system was used to generate NcMYR1 gene knockout strain(?NcMYR1)and NcMYR2 knockout strain(?NcMYR2),functions of ?NcMYR1 and ?NcMYR2 strains in invasion,growth,replication,virulence and pathogenicity were studied,and investigate the protective effect of recombinant NcMYR1 protein and ?NcMYR1 strain against infection of N.caninum wild-type strain in mice,in order to provide a theoretical basis for studying the pathogenicity of N.caninum to the host and the prevention and treatment of neosporosis.1.Subcellular localization of NcMYR1 and NcMYR2 proteinsThe sequences of NcMYR1 and NcMYR2 genes were obtained by Bioinformatics analysis,and the recombinant NcMYR1 and NcMYR2 proteins were prepared and purified by prokaryotic expression technique,then the anti-NcMYR1 and anti-NcMYR2 polyclonal antibodies were prepared.First,the expression vectors of pET-32a-NcMYR1 and pET-32a-NcMYR2 were successfully constructed,and the recombinant pET-32a-NcMYR1 and pET-32a-NcMYR2 proteins were successfully expressed.It was found that the recombinant pET-32a-NcMYR1 and pET-32a-NcMYR2 proteins were both supernatant,then the proteins were purified and the mice were immunized by them.After three times of immunization,blood samples were collected from the eyeballs of mice and serum was centrifugally collected as polyclonal antibodies.The subcellular localization of NcMYR1 and NcMYR2 proteins in N.caninum was observed by intracellular and extracellular immunofluorescence.The results showed that the NcMYR1 and NcMYR2 proteins were localized in the cytoplasm.2.Generation of ?NcMYR1 and ?NcMYR2 strainsCRISPR-cas9 genome editing system was used to generate NcMYR1 gene knockout strain(?NcMYR1)and NcMYR2 gene knockout strain(?NcMYR2).PCR,western blot and immunofluorescence were used to identify these knockout strains.First,Nc-pSAG1-Cas9:U6-SgUPRT-NcMYR1 and Nc-pSAG1-Cas9:U6-SgUPRT-NcMYR2 vectors were successfully synthesized and the homologous recombinant DHFR gene fragment was amplified.Subsequently,the monoclones were cultured and screened by electrorotation,and the successful construction was identified by PCR,western blot and immunofluorescence.The results showed that ?NcMYR1 and ?NcMYR2 strains were successfully inserted into DHFR homologous recombinant gene fragments at the gRNA locus,while ?NcMYR1 and ?NcMYR2 strains no longer expressed NcMYR1 and NcMYR2 proteins,indicating that ?NcMYR1 and ?NcMYR2 strains were successfully generated.3.Functional study of NcMYR1 and NcMYR2 genesThe role of NcMYR1 and NcMYR2 proteins in invasion,growth and pathogenicity was investigated by plaque assay,invasion,proliferation,egression assay and virulence experiment.The results showed that the plaque area of ?NcMYR1 strain decreased compared with the wild-type strain(P<0.001),invasion rate decreased(P<0.001);the replication rate was decreased(P<0.001),the egression rate decreased(P<0.05).?NcMYR2 strain showed no significant difference in plaque area,invasion,replication and egression rate compared with wild-type strain(P>0.05).BALB/c mice were infected with ?NcMYR1,?NcMYR2 and wild-type strains respectively,and the survival rate of the mice was recorded.Cytokines,parasite tissue burden and pathological changes were detected.The results showed that compared with mice infected with wild-type strains,the survival rate of mice infected with ?NcMYR1 strain was 100%,and that of mice infected with ?NcMYR2 strain was 0%,indicating that the virulence of ?NcMYR1 strain was reduced,while the virulence of ?NcMYR2 strain was not affected.Serum IL-6,IL-12 and IFN-? secretion in mice infected with ?NcMYR1 strain decreased significantly(P<0.01);serum IL-6 secretion in mice infected with ?NcMYR2 strain decreased significantly(P<0.05),while both IFN-? and IL-12 secretion increased but the difference was not significant(P>0.05).qPCR results showed that the parasite tissue burden of heart,liver,spleen,lung,kidney and brain in ?NcMYR1 infected mice decreased(P<0.05).The parasite tissue burden of heart,liver,spleen,lung,kidney and brain in ?NcMYR2 strain mice was not significantly different from that in wild strain mice(P>0.05).The pathological changes of heart,liver,spleen,lung,kidney and brain in mice infected with ?NcMYR1 strain were alleviated compared with mice infected with wild-type strain,while the pathological changes of the above organs in mice infected with ?NcMYR2 strain were not significantly different from those in mice infected with wild-type strain.This indicated that ?NcMYR1 strain was less pathogenic to mice.The above studies showed that NcMYR1 was the invasion and virulence-associated gene of N.caninum.After knocking out the NcMYR1 gene,the growth,reproduction,pathogenicity and other aspects of the strain were significantly reduced,and the virulence was significantly weakened.Therefore,NcMYR1 could be used as a candidate vaccine molecule.There were no significant changes in the growth,replication,pathogenicity and virulence after NcMYR2 gene knocked out.4.Protective effects of ?NcMYR1 strain on mice against wild-type strain infectionThe ?NcMYR1 strain with weakened virulence in the above experiment was immunized in mice as inactivated vaccine,and the tail vein blood was collected for cytokine detection on 1d,10 d,20 d,30 d,40 d,respectively,and the survival rate of the mice was observed by infection of wild-type strain.IFN-? infection peaked at 40 days after ?NcMYR1 strain was immunized,and IL-12 peaked at 10 days after ?NcMYR1 strain was immunized.and wild-type strain was infected after ?NcMYR1 strain was immunized for 10 d,20 d,30 d,40 d.The immune protection rate in the 10-day group reached 30%,10-day group reached 20%,10-day group reached 10%,40-day group reached 80%,indicating that mice immunized with ?NcMYR1 strain had immune protection against N.caninum infection.Mice immunized with the recombinant NcMYR1 protein as vaccine antigen were then infected with N.caninum,the survival rate of the mice was observed to evaluate the immune effect of recombinant NcMYR1 protein.The results showed that the secretion of IL-12 and IFN-? in mice immunized with recombinant NcMYR1 protein was significantly increased compared with the control group(P<0.001),and the immune protection of mice immunized with recombinant NcMYR1 protein against N.caninum was up to 20%.To sum up,the NcMYR1 and NcMYR2 proteins were localized in the cytoplasm of N.caninum.CRISPR-cas9 genome editing system was used to generate NcMYR1 gene knockout strains(?NcMYR1)and NcMYR2 gene knockout strains(?NcMYR2).The NcMYR1 gene knockout strain was significantly weakened in the aspects of growth,replication,virulence and pathogenicity.The immune protection effect of ?NcMYR1 strain was 80%.It lays a foundation for the screening of drug targets of N.caninum and the study of antigenic vaccine of N.caninum. |
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