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The Molecular Characterization and Immunity Identification of Microneme 3 of Eimeria acervulina
Author(s) -
Zhang ZhenChao,
Liu XinChao,
Yang XinChao,
Liu LianRui,
Wang Shuai,
Lu MingMin,
Ehsan Mohammad,
Gadahi Javaid A.,
Song XiaoKai,
Xu LiXin,
Yan RuoFeng,
Li XiangRui
Publication year - 2016
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/jeu.12318
Subject(s) - eimeria acervulina , microneme , biology , recombinant dna , eimeria , open reading frame , microbiology and biotechnology , complementary dna , antibody , sequence analysis , rapid amplification of cdna ends , gene , peptide sequence , apicomplexa , genetics , immunology , malaria , plasmodium falciparum
The gene of Eimeria acervulina microneme protein 3 (Ea MIC 3) was cloned and characterized. According to the conserved sequence, the 3′‐ and 5′‐ends of Ea MIC 3 were amplified by the rapid amplification of cDNA ends ( RACE ). The full length cDNA of this gene was obtained by overlapping the sequences of 3′‐ and 5′‐extremities and amplification by reverse transcription PCR . The sequence analysis revealed that the opening reading frame ( ORF ) of Ea MIC 3 was 2,607 bp and encoded a protein of 868 amino acids with 93.04 kDa. Western blotting assay showed that the recombinant protein was successfully recognized by the sera of chickens experimentally infected with E. acervulina , whereas the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of Ea MIC 3. Immunofluorescence analysis indicated that Ea MIC 3 was expressed in the sporozoites and merozoites stages of E. acervulina . Animal challenge experiments demonstrated that the recombinant protein of Ea MIC 3 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented anticoccidial index ( ACI ) more than 165. Moreover, Ea MIC 3 protein produced significantly high level of IgG antibody, IFN ‐γ, IL ‐10, IL ‐17 TGF ‐β, CD 4 + , and CD8 + .