Proteomics Of Three Kinds Eimeria Spp. And Identification Of Common Antigens

Coccidiosis,a widespread protozoosis,is seriously harmful to poultry industry.At present,prevention and control of coccidiosis mainly rely on chemical drugs.However,with the widespread use of anticoccidial drugs,new issues continue to be exposed,such as drug resistance,anticoccidials residues,and animal foodborne safety issues,which made veterinary scientists into a larger puzzle.With the deepening in the immune mechanism of coccidian,researchers set their sights on the research and development of the coccidiosis vaccine.In this study,to identify the immunogenic common antigen of Eimeria spp.,the soluble protein of oocysts from three Eimeria spp.were analyized through immunoproteomics approach.After that,eucaryotic experesson plasmides were constructed with the genes of the identified common antigens.Subsequently,animal experiments were performed to evaluate the protective efficacy of the common antigens against mixed infection of three Eimeria spp.Our results provide reference for the clinical prevention of mixed infection of chicken coccidian.1.Immunoproteomics analysis of sporulated oocysts in Eimeria tenella,Eimeria acervulina and Eimeria maximaUsing the method of immunoproteomics,we analyzed the soluble protein of E.tenella,E.acervulina and E.maxima.After analyzing sporulated oocysts protein in three kinds of Eimeria spp.,we find that there are 626 protein spots in E.acervulina,632 protein spots inE.maxima,629 protein spots in E.tenella.Western blot shows that E.tenella,E.acervulina and E.maxima have 50,64,57 protein spots respectively which can be recognized by the antiserum of chicken coccidiosis.We analyzed these protein spots by MALDI-TOF-MS/MS and the result shows that,112 protein spots were matched 96 kinds of protein.A series of protein which is known including Enolase2,elongation factor 2,lactate dehydrogenase,Glyceraldehyde-3-phosphate dehydrogenase,14-3-3 protein,Microneme protein2,ubiquitin-conjugating enzyme domain-containing protein were been searched.59 protein spots were not been searched.On line analysis and sequence alignment of immunogenicity proteins,the nucleotide sequence whose homology among 3 species is above 91%was identified as a common antigen.Finally,we got four kinds of common antigens including elongation factor 2,ubiquitin-conjugating enzyme domain-containing protein,14-3-3 protein,and Glyceraldehyde-3-phosphate dehydrogenase.2.The cloning of common antigen gene and construction of eukaryotic expression plasmid pVAX-EaUCE and pVAX-Ea14-3-3We cloned two protein gene including ubiquitin-conjugating enzyme domain-containing protein,putative[Eimeria acervulina]and 14-3-3 protein,putative[Eimeria acervulina].Specific primers were designed according to the nucleotide sequence included in their GenBank.The common antigen gene was amplified by PCR with the template of Eimeria acervulina cDNA,444bp and 837bp fragments were amplified respectively,and the amplified products were connected into pVAX1.0 vector.The positive clones were identified by double enzyme digestion and sent to the company for sequencing,and the results were compared with the sequence online.Double enzyme digestion and identification shows they were 450,900bp strip,they were consistent with the intended purpose strip size.Sequence analysis showed that the resulting nucleotide sequence compared with the nucleotide sequence of GenBank（XM₀13391686,XM₀13394831）were 100%homology,it indicates that we have successfully cloned the common antigen gene EaUCE and Ea14-3-3,and successfully constructed the eukaryotic expression plasmid pVAX-EaUCE and pVAX-Ea14-3-3.3.Eukaryotic expression plasmid pVAX-EaUCE and pVAX-Ea14-3-3 induce the reaction of T lymphocyte subsets and the change of cytokineAt 14 days old,10 chickens were intarmuscluarly immunized with 100 μg pVAX-EaUCE and pVAX-Eal4-3-3 seperately,the PBS and pVAX control were designed.Seven days later,a booster immunization was given as the same method of the primary immunization.Seven days post the primary immunization and booster immunization,the splens of the immunized chickens were collected to isolate splenic lymphocyte sperately.Subsequently,CD4+/CD3 +,CD8 +/CD3 + ratio of the splenic lymphocytes were dected by flow cytometry.The results showed that CD4 +/CD3 +,CD8 +/CD3 + ratio of immunized group splenic T cell was significantly higher than that of PBS and pVAX1 empty plasmid control groups（P<0.05）.The results demonstrated that the common eukaryotic expression plasmid can effectively induce chicken’s T lymphocyte responses.Detecting the mRNA content of IFN,IL-2,IL-4,TNFSF15,IL-17D and TGF-β,we found that the mRNA content in the recombinant plasmid group is more than other groups（P<·0.05）.In a word,the eukaryotic expression plasmid of common antigen protein can effectively induce the secretion of cytokines in chicken.4.The reaction of antibody induced by eukaryotic expression plasmid pVAX-EaUCE and pVAX-Ea14-3-3At 14 days old,10 chickens were intarmuscluarly immunized with 100 μg pVAX-EaUCE and pVAX-Eal4-3-3 seperately,the PBS and pVAX control were designed.Seven days later,a booster immunization was given as the same method of the primary immunization.A week later,we collected the blood from heart and isolated serum.Blood samples were collected once a week for six weeks.We use Eimeria acervulina sporulated oocysts soluble protein as coating antigen,detect serum IgG levels by indirect ELISA method.The results show that the first week after the second immunization to the sixth week,the antibody titers of pVAX-EaUCE and pVAX-Eal 4-3-3 group were significantlyhigher than PBS group and pVAX1 empty plasmid group.The antibody titers of immunized group rised slowly from the first week to the third week,and peaked at the fourth week,after then the antibody titers decreased gradually.PBS group and pVAXl empty plasmid group were not detected any specific antibodies.Tests showed that the eukaryotic expression plasmid pVAX-EaUCE and pVAX-Ea14-3-3 could induce chicken to produce specific antibodies.5.The protective immunity of eukaryotic expression plasmid pVAX-Ea14-3-3 a pVAX-EaUCEAt 14 days of age,chickens were weighed and randomly distributed to 17 groups of 30 each.Experimental group chickens were immunized with 100 μg pVAX-EaUCE and pVAX-Ea14-3-3 by leg intramuscular injection respectively.The challenged control group and unchallenged control chickens were injected with sterile PBS and empty vector control group was intramuscularly immunized with 100 μg pVAXl at the same injection site.A booster immunization was given 7 days post the first immunization.At 28 days of age,the chickens were challenged with fresh E.tenella,E.acervulina,and E.maxima or mixed sporulated oocysts except the non-immunized and non-challenged group.Six days post challenge,all the chickens were slaughtered.Average body weight gain,oocyst decrease ratio,lesion score,and ACI were calculated.The results showed that after infected with E.tenella,E.acervulina,E.maxima and mixed sporulated oocysts,the ACI of each pVAX-Ea14-3-3 immunized group were more than 160.It means that eukaryotic expression plasmid pVAX-Ea 14-3-3 has good protective efficacy when chicken suffered E.tenella,E.acervulina,and E.maxima infection.Compared with the control group,intestinal lesions value,oocysts number are reduced,and the relative growth rate of chickens are raised in eukaryotic expression plasmid pVAX-EaUCE immunized group.This shows that our eukaryotic expression plasmid pVAX-EaUCE and pVAX-Eal4-3-3 have some cross-protective efficacy to chicken when resist E.tenella,E.acervulina,E.maxima and mixed sporulated oocysts infection.