The Construction Of Eimeria Maxima CDNA Expression Library And The Screening For Immune Protective Antigens

Eimeria maxima,with morderate pathogenicity,is one of the most common coccidia in intensive chicken farm.Effective protective antigen is crital for the development of the third-generation anti-coccidiosis vaccine.Hence,Eimeria maxima cDNA expression library was constructed to find more effective protective antigen in this study.After four rounds screening,six positive clones were obtained.One of them was cloned and expressed.The works are shown as following.1 Construction of a cDNA expression library of Eimeria maxima sporulated oocysts using Gateway technologyA cDNA expression library of Eimeria maxima was constructed using Gateway technology.Firstly,the RNA was extracted from fresh sporulated oocysts of E.maxima and the mRNA was isolated and purified.Then the E.maxima entry library was constructed with CloneMinerTM ? cDNA library construction kit.Subsequencly the cDNA was cloned into pVAX1.0 producing the sporulated oocysts expression library of E.maxima.Hereafter,the library was tested by PCR with the primers of known E.maxima genes.The results showed that the total clones of the entry library was 0.92×107 CFU with an average inserts size of 1.63 kb and the total clones of the expression library was 2.32×107 CFU with an average inserts size of 1.64 kb,the positive rates of recombination of them were 100%,and seven known E.maxima genes could be amplified using their specific primers.Overall,the constructed library had high quality and strong representativeness.Our research established a solid foundation for further screening protective antigen genes.2 Immunization screening of Eimeria maxima expression libraryIn this study,Eimeria maxima expression library was screened by four rounds using expression library immunization techniques.The library plasmids were isolated each round.At 14 days of age,chickens of the experimental groups were immunized with library plasmids,meanwhile,the pVAX1.0 controll group was immunized with pVAX1.0 vector,and the challenged and unchallenged controll groups were immunized with sterile PBS.Seven days later,secondary immunizations were given.At 28 days of age,each chicken was orally challenged with 1.5×105 Eimeria maxima sporulated oocysts except the non-immunized and non-challenged controll group.All chickens were sacrificed after 7 days.The indicators including weight gain,the number of oocysts of stool per gram and intestinal lesion scores were statistically analyzed,and then the anticoccidial index per group was figured out synthetically to evaluate the immune protective effect of each group.The group of which ACI is more than 160 was judged to be positive.After four consecutive screening,6 positive clones were obtained,the longest ORF of each gene was inferred and translated into amino acid sequence.The results of sequence analysis from BLASTP search revealed that the encoding amino acid sequence of EmHP-1 had 82%identity with conserved hypothetical protein of Eimeria maxima(GenBank:CDJ56976.1),the encoding armino acid sequence of EmSAG had 100%identity with SAG family member of Eimeria maxima(GenBank:CDJ60815.1),the encoding amino acid sequence of EmRP had 84%identity with rhomboid family domain-containing protein,putative(GenBank:CDJ59262.1),the encoding amino acid sequence of EmHP-2 had 70%identity with hypothetical protein(GenBank:CDJ61108.1),the encoding amino acid sequence of EmCKRS had 100%identity with a putative CAMP-dependent protein kinase regulatory subunit(GenBank:CDJ61187.1),the encoding amino acid sequence EmJS-1 had 66%identity with Eimeria mitis selR domain-containing protein and 69%with Eimeria tenella putative selR domain-containing protein,but had no significant homology with the known genes of Eimeria maxima.3 Cloning and expression of EmRP geneThe positive gene EmRP from.maxima sporulated oocysts expression library was amplified by PCR.The sequence analysis showed that the size of the ORF was 774bp,encoding 257 amino acids,and the theoretical molecular weight was 28.2kDa.The prokaryotic expression vector pCold TF-EmRP was constructed.The recombinant plasmid was expressed successfully in E.coli BL21(DE3).The result of western blot showed that the recombinant protein could be recognized by serum from the naturally infected chicken with E.maxima.