Expression and identification of the ADF-linker-3-1E gene of Eimeria acervulina of chicken

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• 作者：Zhao Yuelan ; Liu Yiwei ; Liu Liyuan ; Zhao Yue ; Cao Wenbo…
• 关键词：E. acervulina ; Actin ; depolymerizing factor (ADF) ; 3 ; 1E gene ; Prokaryotic expression
• 刊名：Parasitology Research
• 出版年：2016
• 出版时间：April 2016
• 年：2016
• 卷：115
• 期：4
• 页码：1641-1647
• 全文大小：615 KB
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• 作者单位：Zhao Yuelan (1)
  Liu Yiwei (1)
  Liu Liyuan (1)
  Zhao Yue (1)
  Cao Wenbo (1)
  Bao Yongzhan (1)
  Qin Jianhua (1)
  
  1. College of Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001, China
  
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Biomedicine
  Medical Microbiology
  Microbiology
  Immunology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-1955

文摘

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.