Yuelan Zhao, Yiwei Liu, Liyuan Liu, Yue Zhao, Wenbo Cao, Yongzhan Bao, Jianhua Qin
College of Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001, China.
Parasitol Res. 2016 Apr;115(4):1641-7. doi: 10.1007/s00436-016-4903-y. Epub 2016 Jan 15.
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.
球虫病是一种分布广泛、死亡率和发病率较高的疾病，由艾美耳属的几种原生动物寄生虫引起，被认为是家禽业面临的严峻挑战。本研究旨在构建鸡堆型艾美耳球虫（E. acervulina）的重组质粒pET32a(+)-ADF-linker-3-1E，并检测ADF-linker-3-1E蛋白的生物活性。通过聚合酶链反应（PCR）的重叠延伸剪接（SOE-PCR）克隆鸡堆型艾美耳球虫的ADF-linker-3-1E基因，然后将其插入pET32a(+)中构建重组质粒pET32a(+)-ADF-linker-3-1E。将重组质粒转化到大肠杆菌Rosetta (DE3)感受态细胞中，然后用IPTG（0.6 mmol/L）诱导。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定培养基中的表达产物。通过蛋白质免疫印迹法检测ADF-linker-3-1E蛋白的生物活性。结果显示，扩增得到的ADF-linker-3-1E基因约为1346 bp。以重组质粒pET-32a(+)-ADF-linker-3-1E为模板进行PCR扩增，得到一条1346 bp的特异条带。酶切产物产生1346 bp的目标片段和5.9 kb的pET-32a(+)-载体片段两个片段。结果表明ADF-linker3-1E基因成功插入到pET-32a(+)-载体中。SDS-PAGE分析显示培养基中的表达产物产生一条约54.8 kDa的单一条带。蛋白质免疫印迹分析表明重组蛋白能与His-Tag(2A8)小鼠单克隆抗体特异性反应。本研究表明成功获得了具有良好生物活性的ADF-linker-3-1E蛋白，为利用ADF-linker-3-1E蛋白开发核酸疫苗奠定了基础。
