Eukaryotic Expression Of Gametophyte Protein GAM22 Of Eimeria Tenella And Its Immunological Protection

Chicken coccidiosis is a kind of protozoa disease with global distribution.Of seven chicken coccidia,E.tenella and E.necatrix are the most pathogenic of the chicken coccidia,causing acute cecal coccidiosis and small intestinal coccidiosis,respectively.At present,the primary control measures against coccidiosis are the ubiquitous use of anti-coccidial drugs and live oocyst vaccines.However,long term andcontinuous feeding of drugs is easy to cause drug resistance,contaminate environment,and impact on food safety.The live oocyst vaccine has series defects,including coccidial virulence reversibility,pathogenicity and high production expenses.Genetic engineering vaccines such as recombinant subunit vaccines overcome these limitations.In a previous study,our lab has cloned and expressed Etgam22 and Etgam59 genes of E.tenella in prokaryotic cells,and found that the rEtGAM22 and rEtGAM59 could be recognized specifically by the serum of chickens infected with E.tenella and E.necatrix,respectively.Animal challenge experiments demonstrated that the rEtGAM22 and rEtGAM59 could increase the weight gains,and decrease the production of oocysts.In the present study,EtGAM22 protein was expressed using baculovirus expression system,the immune protection against E.tenella and E.necatrix afforded by EtGAM22 were evaluated,and compared with the recombinant prokaryotic expression protein.Finally,the protective effect of combined immunization with rEtGAM22 and rEtGAM59 was evaluated.These results laid a foundation for the development of recombinant subunit vaccine.1.Construction of eukaryotic expression vector of gam22 gene of Eimeria tenella and analysis of its reactivityThe optimized Etgam22 gene was inserted into the transposon vector pFastbacl to constract the recombinant plasmid named pFastbac-Etgam22.Then the pFastbac-Etgam22 was transformed into E.coli DH10 Bac to constract the recombinant baculovirus shuttle vector named Bacmid-Etgam22.The Bacmid-Etgam22 was transfected into Sf9 cells to generate recombinant baculovirus named reBac-Etgam22.The recombinant baculovirus was induced to express in Sf9 cells,and the recombinant protein rEtGAM22-e was about 35 kDa.Immunofluorescence assay(IFA)showed that the recombinant protein could be recognized by polyclonal antibody from the mice immunized with prokaryotic expression protein rEtGAM22-p.Western blot analysis showed that the recombinant protein could be recognized specifically by anti-His monoclonal antibodies,anti-recombinant protein polyclonal antibodies from mice,and the serum of chickens infected with E.tenella and E.necatrix,respectively.All the results showed that the recombinant protein rEtGAM22-e has a good reactogenicity and cross-reactivity.2.Analysis of immunoprotective efficacy of recombinant protein rEtGAM22 expressed in eukaryotic cellsThe yellow feather broiler was used as experimental animal.The immunoprotective efficacy of rEtGAM22-e expressed in eukaryotic cells against E.tenella and E.necatrix infection was assessed by survival rate,weight gain,lesion scores,oocyst reduction rate,anticoccidial index(ACI),and serum antibody level,respectively.Meanwhile,the immunoprotective efficacy was compared with rEtGAM22-p and rEtGAM59 expressed in prokaryotic cells.The results showed as follows:(1)Protection against E.tenella:the survival rate of the immunized group was 100%,and the unimmunized and challenge group(UC)was 90%.The weight gain of the group(B1)immuned with rEtGAM22-e expressed in:’eukaryotic cells was higher than that of the group(A1)immuned with rEtGAM22-p expressed in prokaryotic cells,but was slightly lower than that of the group(C1)immuned with rEtGAM59 expressed in prokaryotic cells.The lesion of B1 group was higher than that of A1 and C1 group.The oocyst reduction rate of B1 group was higher than that of A1 group,but was lower than that of C1 group.The ACI of C1 group was 158.83,which was the hightest among three immunized groups and UC group.The serum antibody level of B1 g:roup was significantly higher than that of A1 group(P<0.05),but lower than that of C1 group.(2)Protection against E.necatrix:The results were similar to the test against E.tenella,but the value of ACI was slightly lower.Conclusion:Both of the rEtGAM22 and rEtGAM59 had the protective efficacy against E.tenella and E.necatrix.The immune protection of rEtGAM22 expressed in eukaryotic cells was slightly higher than in prokaryotic cells.The protective efficacy immunized with rEtGAM59 was better than that with rEtGAM22.3.Analysis of immunoprotective efficacy of co-immunization with recombinant gametocyte proteins rEtGAM22-e and rEtGAM59The evaluation method of immunoprotective efficacy of co-immunization with rEtGAM22-e and rEtGAM59 was same as previous study.The results showed as follows:(1)Protection against E.tenella:the survival rate was 100%in all the groups.Except for the group with rEtGAM22-e alone,the weight gains of the immunized groups were significantly higher than that of the UC group(P<0.05),and was no significant difference compared with the UU group(P>0.05).The lesion scores of the co-immunization group with low dosage were lowest than that of the other immunized groups.The oocyst reduction rates in the co-immunization groups was similar,46.21%and 44.70%,respectively,and were higher than that in the groups with rEtGAM22-e or rEtGAM59 alone.The ACI of the groups co-immunized with rEtGAM22-e and rEtGAM59 at high and low dosage were 153.43 and 157.65,respectively,which were higher than that of the groups with rEtGAM22-e or rEtGAM59 alone.The serum antibody levels in the co-immunizied groups were significantly higher than that in the groups with rEtGAM22-e or rEtGAM59 alone(P<0.05).(2)Protection against E.necatrix:The results were similar to the test against E.tenella.The ACIs of the groups co-immunized with rEtGAM22-e and rEtGAM59 at high and low dosage were 158.28 and 160.34,respectively.Conclusion:The protective efficacy of co-immunization with rEtGAM22-e and rEtnGAM59 was higher than that of immunization with rEtGAM22-e or rEtnGAM59 alone,in which the protective efficacy was higher with immnizing dosage at 100 μg(50+50)than at 200 μg(100+100).