title: Expression and identification of the ADF-linker-3-1E gene of Eimeria acervulina of chicken.
abstract: Coccidiosis is a widely distributed disease with higher mortality and morbidity, which is caused by several species of protozoan parasites belonging to the genus Eimeria and recognized as a serious challenge for the poultry industry. This research was conducted to construct the recombinant plasmid pET32a(+)-ADF-linker-3-1E of Eimeria acervulina (E. acervulina) of the chicken and test the bioactivity of the ADF-linker-3-1E protein. The ADF-linker-3-1E gene of E. acervulina of the chicken was cloned by splicing by overlap extension by the polymerase chain reaction (SOE-PCR) and then inserted into the pET32a(+) to construct the recombinant plasmid pET32a(+)-ADF-linker-3-1E. The recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) competent cells and then induced by IPTG (0.6 mmol/L). The expressed product in the culture medium was identified by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the ADF-linker-3-1E protein was tested by Western blotting. The result showed that the amplified ADF-linker-3-1E gene was about 1346 bp. The PCR amplification with the recombinant plasmid pET-32a(+)-ADF-linker-3-1E as a template resulted in a special band of 1346 bp. The digested products resulted in two fragments of 1346 bp target fragment and 5.9 kb pET-32a(+)-vector fragment. The results indicated that the ADF-linker3-1E gene was successfully inserted into the pET-32a(+)-vector. The expressed products in the culture medium resulted in a single band of approximately 54.8 kDa by SDS-PAGE. Western blotting analysis indicated that the recombinant protein could be reacted specifically with His-Tag(2A8) Mouse mAb. This study indicated that the ADF-linker-3-1E protein with good bioactivity was successfully obtained, which laid a foundation for the exploitation of the nuclear vaccine by using the ADF-linker-3-1E protein.
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001, China.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Yuelan
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Yiwei
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Liyuan
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001, China.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Yue
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001, China.
authors.forename: Cao
authors.initials: C
authors.lastname: Wenbo
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001, China.
authors.forename: Bao
authors.initials: B
authors.lastname: Yongzhan
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001, China. qjhqqq@126.com.
authors.forename: Qin
authors.initials: Q
authors.lastname: Jianhua
mesh_terms: D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000469:parasitology*; D003048:Coccidiosis / Q000469:parasitology / Q000662:veterinary*; D004539:Eimeria / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051379:Mice; D015800:Protozoan Proteins / Q000235:genetics*; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA / Q000662:veterinary
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins
keywords: 3-1E gene; Actin-depolymerizing factor (ADF); E. acervulina; Prokaryotic expression