title: Identification and molecular characterization of microneme 5 of Eimeria acervulina.
abstract: In the present study, the microneme 5 gene of Eimeria acervulina (E. acervulina) (EaMIC5) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expressed sequence tag (EST, GenBank Accession No. EH386430.1) to amplify the 3'- and 5'-ends of EaMIC5. The full length cDNA of this gene was obtained by overlapping the sequences of 3'- and 5'-extremities and amplification by reverse transcription PCR. Sequence analysis revealed that the open reading frame (ORF) of EaMIC5 was 336 bp and encoded a protein of 111 amino acids with 12.18 kDa. The ORF was inserted into pET-32a (+) to produce recombinant EaMIC5. Using western blotting assay, the recombinant protein was successfully recognized by the sera of chicks experimentally infected with E. acervulina, while the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of EaMIC5. Immunofluorescence analysis using antibody against recombinant protein EaMIC5 indicated that this protein was expressed in the sporozoites and merozoites stages of E. acervulina. Animal challenge experiments demonstrated that the recombinant protein of EaMIC5 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens, and presented anti-coccidial index (ACI) more than 160. All the above results suggested that the EaMIC5 was a novel E. acervulina antigen and could be an effective candidate for the development of a new vaccine against this parasite.
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: ZhenChao
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: JingWei
authors.initials: J
authors.lastname: Huang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: MengHui
authors.initials: M
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: YuXia
authors.initials: Y
authors.lastname: Sui
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: LianRui
authors.initials: L
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: LiXin
authors.initials: L
authors.lastname: Xu
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: RuoFeng
authors.initials: R
authors.lastname: Yan
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: XiaoKai
authors.initials: X
authors.lastname: Song
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
authors.forename: XiangRui
authors.initials: X
authors.lastname: Li
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D002645:Chickens; D003001:Cloning, Molecular; D003048:Coccidiosis / Q000469:parasitology / Q000517:prevention & control*; D004539:Eimeria / Q000235:genetics* / Q000276:immunology*; D020224:Expressed Sequence Tags; D005260:Female; D005455:Fluorescent Antibody Technique; D007124:Immunoenzyme Techniques; D052940:Merozoites / Q000276:immunology; D033661:Oocysts / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D011201:Poultry Diseases / Q000469:parasitology / Q000517:prevention & control*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D034101:Sporozoites / Q000276:immunology; D015430:Weight Gain / Q000276:immunology
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins