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.

The Molecular Characterization and Immunity Identification of Microneme 3 of Eimeria acervulina

“…Zhang et al. () showed the MIC5 protein of E. acervulina could be expressed in sporozoite and merozoite stages of the life cycle. In this study, we found that EaMIC3 could be detected in sporozoite stages of the life cycle and was located mainly at the apical tip of the sporozoite.…”

“…() found that EtMIC1 induced significant level of the IFN‐γ and IL‐2 transcript profiles at first immunization, and reached the peak at third immunization, and Zhang et al. () reported that EaMIC5 could provocate high level of the IL‐4 and sCD8. However, in this research, we found that EaMIC3 induced significant level of the IFN‐γ, IL‐10, IL‐17, and TGF‐β at first immunization, and reached the peak at second immunization.…”

“…In addition, the MIC2 and MIC5 of E. acervulina were also identified and reported (Zhang et al. , ).…”

“…Zhang et al. (, ) found EaMIC2 and EaMIC5 protein were expressed in sporozoites and merozoites stages of E. acervulina and could be an effective candidate for the development of new vaccine against this parasite.…”

Toxoplasma gondii Histone 4 Affects Some Functions of Murine Ana‐1 Macrophages In Vitro

“…After induction with IPTG (Sigma-Aldrich, St Louis, MO), the rTgH4 protein fused to the 109 aa Trx. Tag thioredoxin with a C-terminal His-tag (fusion protein called further in the paper rTgH4) was purified using a Ni 2+ -nitrilotriacetic acid (Ni-NTA) column (GE Healthcare, Madison, WI) based on the company's directions (Zhang et al 2014(Zhang et al , 2016. Then, the pET-32a vector Trx.…”