Preliminary Diagnostic Evaluation Of The Recombination Antigens Sag4、5、7、8、11 In Eimeria Stiedai

Rabbit hepatic coccidiosis is a parasitic protozoa caused by Eimeria spp.and settled in the epithelial cells of rabbit bile ducts,which is destroying to rabbit production.The disease widely distributed in worldwide,mostly in warm and humid area.Rabbits can be infected easily,particularly young rabbits from weaning to 11 weeks of age are most susceptible,and the symptoms are the most obvious,which can seriously intervene in the growth of rabbits,weakened immunity lead to otherdiseases,and the mortality rate is extremely high.To solve this problem,effective prevention,accurate diagnosis and timely treatment are important means to control the disease.In this study,Es.SAG4,Es.SAG5,Es.SAG7,Es.SAG8,and Es.SAG11 in the surface antigen(SAG)genes were screened out from the transcription data of Eimeria stiedai.Gene amplification and prokaryotic expression,using relative fluorescence quantitative PCR(RT-PCR)method forthe above 5 surface antigen genes,the transcription levels of 4 different life cycle stages of Eimeria stiedai were analyzed and compared.The serological diagnostic value of recombinant surface antigens rEs.SAG4,rEs.SAG5,rEs.SAG7,rEs.SAG8 and rEs.SAG11 were made using immune enzyme-linked adsorption assay(ELISA)and western blotting methods forpreliminary assessment.In addition,it provides important data reference forthe rapid and accurate diagnosis of hepatic coccidiosis in the future.1.Prokaryotic expression and characterization of Eimeria stiedai surface antigen genes SAG4,SAG5,SAG7,SAG8 and SAG11Surface Antigens are a class of secreted proteins located on the surface of protozoan cell membranes.Such proteins play an important role in host cell recognition,invasion,adhesion,and immune evasion.Different surface antigens are secreted and protozoa have different life stages.Each surface antigen plays an important role in parasites.In this study,Es.SAG4,Es.SAG5,Es.SAG7,Es.SAG8,and Es.SAG11 in the surface antigen genes were screened from the transcription data of Eimeria stiedai.By gene clone,sequencing and prokaryotic expression,these results show that the length of the above genes are 444 bp,444bp,531 bp,663bp and 765 bp,respectively.The corresponding genes encode 147,147,176,220 and 254 amino acids.Utilized online software prediction,these protein molecularweights were found around 15.3k Da,16.18 k Da,19.85 k Da,23.63 k Da and 27.3k Da,respectively.The bioinformatics analysis software predicts that Es.SAG8 and Es.SAG11 have signal peptide sites,while Es.SAG4,Es.SAG5,Es.SAG7 do not have signal peptide sites;Es.SAG4 has a transmembrane domain;Es.SAG5,Es.SAG7 and Es.SAG11 has a N-glycosylation site.Using RT-PCR to compare the transcription levels of these five genes in the fourdifferent life cycle stages of Eimeria stiedai,it was found that Es.SAG4,Es.SAG7 Es.SAG8 and Es.SAG11 were The transcription level in the merozoite stage of coccidia is higher,while Es.SAG8 has highertranscription in the sporulation stage of Eimeria stiedai.2.Diagnostic evaluation of the surface antigens rEs.SAG4,rEs.SAG5,rEs.SAG7,rEs.SAG8 and rEs.SAG11The five purified recombinant proteins could react with positive serums of Eimeria stiedai by immunoblotting.Based theirimmunoreactivity,We evaluated the serological diagnostic potential of five antigens,rEs.SAG4,rEs.SAG5,rEs.SAG7,rEs.SAG8,and rEs.SAG11 to establish indirect ELISA.The results showed that the sensitivity of five recombination proteins is 97.92%,87.5%,91.67%,93.75% and 97.92%,the specificity is 100%,95.83%,97.92%,100% and 85.42%,respectively.Using three rabbit intestinal coccidia positive serum and three rabbit Sarcoptes scabiei.The positive sera were subjected to immunoblotting,and it was found that rEs.SAG5,rEs.SAG7 and rEs.SAG11 strongly cross-reacted with rabbit intestinal coccidia positive serum,but did not cross-react with rabbit Sarcoptes scabiei positive serum,rEs.SAG4 and rEs.SAG8 There is no cross reaction with rabbit intestinal coccidia and rabbit Sarcoptes scabiei.The above experiments show that the recombinant proteins rEs.SAG4,rEs.SAG5,rEs.SAG7,rEs.SAG8,and rEs.SAG11 can be used as the diagnostic antigens forrabbit coccidiosis.Furthermore,rEs.SAG4 have highersensitivity and specificity which are potential and valuable diagnostic antigens.