Construction And Immunoprotective Effect Of Chicken Eimeria Tenella Multimeric Vaccine EtDLC8a-GST-MIC2

In organisms,the homodimeric structure of proteins is a ubiquitous phenomenon.The fusion of two proteins having a homodimeric structure can form a homomultimer having a linear structure or a net structure.The homomultimer has a wide range of applications,and can be used as a vaccine platform for presenting antigenic proteins,by inserting different antigenic epitopes to increase load of antigens,and stimulating the body to produce better immune protection.In this study,the homologous polymer vaccine platform was used as the train of thought.The EtDLC8a-GST vaccine platform was constructed by cloning the amplified dynamic protein light chain 8a(EtDLC8a)gene of Eimeria tenella into the PGEX-6p-1 vector containing glutathione S transferase(GST)gene using genetic engineering technology.The EtDLC8a-GST constructed by Native PAGE and Western blot can form polymers.The non-structural region of EtDLC8a gene was selected as the antigen fragment insertion site to further improve the EtDLC8a-GST vaccine platform.At the same time,part of the antigen fragments of MIC2,the antigen of Eimeria tenella,were inserted into the vaccine platform to construct the EtDLC8a-GST-MIC2 polyprotein complex vaccine,and the protective evaluation of the polyprotein complex was conducted.Analysis by Native-page and Western-blot showed that the constructed EtDLC8a-GST vaccine platform could form protein complexes with poly structure,while the antigen fragment EtMIC2 inserted into the vaccine platform would not affect the formation of poly structure.The immunoprotective effect of recombinant vaccine was evaluated by animal experiments.210 1-day-old male chicks were randomly divided into 7 groups,Negative control group(group I),positive control group(group II),ETMIC2 antigen fragment immunization group(group III),TgDLC8a-GST immunization group(group IV),EtDLC8a-GST immunization group(group V),TgDLC8a-GST-MIC2 immunization group(group VI)and EtDLC8a-GST-MIC2 immunization group(group VII).The chicks were immunized at the initial age of 7 days,at the second age of 14 days,and at the age of 21 days,except the negative control group(group I),the rest groups were infected with the parasite.The effects of immune protection were evaluated by weight gain,cecal lesions,oocyst output,lymphocyte level,serum and mucosal antibody levels.The results showed that the constructed EtDLC8a-GST polymeric vaccine platform could form a polymeric structure,and the inserted EtMIC2 antigen fragments had no effect on the polymeric structure.The results of animal experiments showed that,compared with the control group,the ETMIC2 antigen fragment immunization group(group III)(ACI=92.70)showed relatively weak immune protection provided by some antigen fragments.Comparison between TgDLC8a-GST-MIC2 immune group(group VI)(ACI=137.00)and EtDLC8a-GST-MIC2 immune group(group VII)(ACI=136.44)and ETMIC2 antigen fragment immune group(group III)(ACI=92.70)shows that the immune protection effect provided by polycomplex is better than that of protein monomer(ACI evaluation criteria:ACI<120 is invalid,120<ACI<160 is medium effective,160<ACI<180 is good,and ACI>180 is excellent).The results of serum antibody and mucosal antibody levels showed that there were significant differences between EtMIC2antigen fragment immunization group(group III),TgDLC8a-GST-MIC2 immunization group(group VI)and EtDLC8a-GST-MIC2 immunization group(group VII)and control group(P<0.05).The differences between TgDLC8a-GST-MIC2 group(group VI)and EtDLC8a-GST-MIC2 group(group VII)and EtMIC2 antigen fragment group(group III)were significant(P<0.05).In terms of cytokine level,TgDLC8a-GST-MIC2 group(group VI)and EtDLC8a-GST-MIC2 group(group VII)were significantly different from EtMIC2 antigen fragment group(group III)(P<0.05).In addition,the results of lymphocyte subsets showed that at the level of CD4~+and CD8~+T lymphocyte subsets,there were significant differences between the TgDLC8a-GST-MIC2 immune group(group VI)and EtDLC8a-GST-MIC2immune group(group VII)and the EtMIC2 antigen fragment immune group(group III)(P<0.05).The polymeric vaccine platform can obviously enhance the immune protection of some antigens.