| E.tenella is one of the most widely distributed coccidia with the strongest pathogenicity,which seriously damages the poultry industry and causes great economic losses.At present,drug control is an effective way to control coccidiosis,but with the emergence of coccidiosis resistant strains and the formation of the concept of green food,the research of new vaccines against coccidia has become a hot spot.E.tenella SAG13 surface antigen protein is an up-regulated protein screened by secretory proteome analysis based on DF-1 cell infection model.In order to explore the immunoprotective effect of EtSAG13 recombinant protein as a subunit vaccine,we prokaryotic expressed EtSAG13 gene,amplified it with E.Tenella sporozoite cDNA as template,synthesized primers according to GenBank gene sequence,connected the synthesized EtSAG13 gene sequence to pET28a(+)vector,constructed pET28a-SAG13recombinant plasmid,and constructed pET28a the expression of EtSAG13 gene was induced by IPTG,and the protein was analyzed and identified by SDS-PAGE and Western blot.The recombinant protein expressed in EtSAG13 was immunized to BALB/c mice.The polyclonal antibody was prepared and subcellular localization was carried out.It was located on the surface of E.tenella sporozoite,and part of EtSAG13 proteins could be secreted out of merozoites,so it was identified as a new secretory protein.In order to explore the immunogenicity of EtSAG13 protein,this study took pET28a-SAG13 subunit vaccine group as the experimental group,and evaluated the immune effect of EtSAG13 protein recombinant subunit vaccine through the chicken coccidium challenge test.90 chicks with little individual difference were randomly divided into group 1(EtSAG13 immune and challenged group),group 2(non immune and non challenged group)and group 3(non immune but challenged group).Each group had 30chicken to compare the immune effect of pET28a-SAG13 subunit vaccine.At the age of 7days,one group was injected subcutaneously with protein fully emulsified with complete Freund's adjuvant.The immune dose was 100?g/animal,and the other two groups were injected with PBS of the same amount.At the age of 14 days,the second enhanced immunization was conducted with EtSAG13 protein emulsified with incomplete Freund's adjuvant of the same proportion.At the age of 21 days,one and three groups were inoculated with PBS Twenty thousand fresh E.tenella sporulated oocysts with infective activity.The results showed that the CD3~+CD4~+and CD3~+CD8~+of chicken body after immunization with pET28a-SAG13 subunit vaccine.The percentage of CD3~+was significantly increased,which indicated that the immune system could induce significant cellular and humoral immune responses.In the cecal lesion score(LS),the cecal lesion score of group 1 immunized with EtSAG13 was significantly lower than that of group 3immunized with EtSAG13(P<0.05);the weight of the experimental group was significantly increased compared with that of the control group,and the amount of oocysts excreted was significantly increased The IgG level of the chickens immunized with EtSAG13 was significantly higher than that of the control group and the non immunized group.The results showed that EtSAG13 protein was located on the surface of the sporozoite and part of EtSAG13 could be secreted to the outside of the cell.In this study,the secretory characteristics and immunogenicity of EtSAG13 protein were preliminarily identified,which will provide the basis for the further study of the specific molecular functions of EtSAG13 protein in the invasion process and the development of a new chicken coccidial vaccine. |
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