Yang Cui, Xu Yu, Jia Renyong, Li Pengfei, Zhang Lei, Wang Mingshu, Zhu Dekang, Chen Shun, Liu Mafeng, Yin Zhongqiong, Cheng Anchun
Avian Disease Research Centre, Sichuan Agricultural University, Chengdu 611130, China; Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan 611130, China.
Avian Disease Research Centre, Sichuan Agricultural University, Chengdu 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan 611130, China.
J Virol Methods. 2017 Sep;247:1-5. doi: 10.1016/j.jviromet.2017.05.003. Epub 2017 May 10.
Duck circovirus (DuCV), as a causative agent of long-term immunosuppressive disease, has caused heavy damage to waterfowl breeding worldwide. In this study, the full-length Cap (capsid) gene of DuCV was expressed in Escherichia coli (E. coli) for the first time by optimizing the codons in its nuclear localization signal (NLS) regions. The recombinant protein was expressed as inclusion bodies, and the quantification of purified Cap protein could reach 0.29mgmL. Moreover, an indirect ELISA method (Cap-ELISA) was established based on the recombinant Cap protein. The results of the optimization for Cap-ELISA revealed that the optimum concentration of the coating antigen and serum dilution ratio were 19.5ng per well and 1:1280, respectively. The cut-off value of the Cap-ELISA for positive sample detection was 0.145, the sensitivity of that reached 1:25600, and it was specific for the detection of DuCV anti-sera. In comparative experiments using 56 clinically suspected DuCV-infection samples, the Cap-ELISA showed a 94.64% coincidence rate with the PCR test. These results indicated that codon optimization is a reasonable strategy to obtain an intact Cap protein, and this Cap-ELISA is suitable for extensive applications in DuCV serological diagnosis.
鸭圆环病毒（DuCV）作为一种长期免疫抑制性疾病的病原体，已给全球水禽养殖业造成了严重损失。在本研究中，通过优化鸭圆环病毒核定位信号（NLS）区域的密码子，首次在大肠杆菌中表达了其全长Cap（衣壳）基因。重组蛋白以包涵体形式表达，纯化后的Cap蛋白定量可达0.29mg/mL。此外，基于重组Cap蛋白建立了一种间接ELISA方法（Cap-ELISA）。Cap-ELISA的优化结果显示，包被抗原的最佳浓度和血清稀释比例分别为每孔19.5ng和1:1280。Cap-ELISA检测阳性样本的临界值为0.145，其灵敏度达到1:25600，且对鸭圆环病毒抗血清检测具有特异性。在使用56份临床疑似鸭圆环病毒感染样本的对比试验中，Cap-ELISA与PCR检测的符合率为94.64%。这些结果表明，密码子优化是获得完整Cap蛋白的合理策略，且这种Cap-ELISA适用于鸭圆环病毒血清学诊断的广泛应用。
