Identification and immunogenicity of microneme protein 2 (EbMIC2) of Eimeria brunetti

• Published in: Experimental parasitology Vol. 162; pp. 7 - 17
• Main Authors: Hoan, Tran Duc, Zhang, Zhenchao, Huang, Jingwei, Yan, Ruofeng, Song, Xiaokai, Xu, Lixin, Li, Xiangrui
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2016
• Subjects: Animals; Antigens, Protozoan - genetics; Antigens, Protozoan - immunology; Chicken; Chickens - parasitology; China - epidemiology; Cloning, Molecular; Coccidiosis - epidemiology; Coccidiosis - parasitology; Coccidiosis - veterinary; Disease Outbreaks - veterinary; DNA, Complementary - chemistry; DNA, Complementary - metabolism; Eimeria; Eimeria - immunology; Eimeria - isolation & purification; Eimeria brunetti; Escherichia coli; Gene Expression Regulation; Immune Sera - immunology; Microneme-2; Poultry Diseases - epidemiology; Poultry Diseases - parasitology; Poultry Diseases - prevention & control; Protozoan Proteins - genetics; Protozoan Proteins - immunology; Protozoan Proteins - isolation & purification; Protozoan Vaccines - standards; Recombinant plasmid; Recombinant protein; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Reverse Transcription; RNA, Protozoan - isolation & purification; Sequence Alignment - veterinary; Vaccination - veterinary; Vaccines, Synthetic - standards; China; Recombinant plasmid; Microneme-2; Chicken; Recombinant protein; Eimeria brunetti
• ISSN: 0014-4894; 1090-2449
• DOI: 10.1016/j.exppara.2015.12.015

There have been only a few antigen genes of Eimeria brunetti reported up to now. In this study, the gene encoding the microneme protein 2 (EbMIC2) was isolated from oocysts of E. brunetti by RT-PCR and the immunogenicity of recombinant EbMIC2 was observed. The EbMIC2 was cloned into vector pMD19-T for sequencing. The sequence was compared with the published EbMIC2 gene from GenBank revealed homology of the nucleotide sequence and amino acids sequence were 99.43 and 98.63%, respectively. The correct recombinant pMD-EbMIC2 plasmid was inserted into the pET-28a (+) expressing vector and transformed into competent Escherichia coli BL21 cells for expression. The expressed product was analyzed using SDS-PAGE and Western-blot. The results indicated that the recombinant EbMIC2 protein was recognized strongly by serum from naturally infected chicken with E. brunetti. Rat rcEbMIC2 antisera bound to bands of about 36 kDa in the somatic extract of E. brunetti sporozoites. The recombinant plasmid pVAX1-EbMIC2 was constructed and then the efficacies of recombinant plasmid and recombinant protein were evaluated. The results of IgG antibody level and cytokines concentration suggested that recombinant EbMIC2 could increase the IgG antibody level and induce the expressions of cytokines. Animal challenge experiments demonstrated that the recombinant EbMIC2 protein and recombinant plasmid pVAX1-EbMIC2 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented high anti-coccidial index. All results suggested that EbMIC2 could become an effective candidate for the development of new vaccine against E. brunetti infection. [Display omitted] •The clone had no significant differences with the EbMIC2 deposited in the GenBank.•The expressed recombinant protein could have high immunogenicity.•Recombinant EbMIC2 could protection against homologous challenge in chicken.•Serum from challenged chickens showed significantly compared with control groups.•Parameters of challenged groups showed significantly compared with unchallenged.