title: In vitro expression and development of indirect ELISA for Capsid protein of duck circovirus without nuclear localization signal.
abstract: Duck circovirus was a newly discovered pathogen that causing ducks immunosuppression in recent years, but it can not been cultured in vitro that limited its depth study. In the present study, the Cap gene that defect the nuclear localization signal (NLS) of DuCV was amplified and connected to the express vector pET-32a (+), to express the recombinant Cap protein in the bacterium Escherichia coli Rosetta. The recombinant Cap protein was purified and an indirect ELISA method was developed based on the recombinant Cap protein. The results showed that the truncated Cap gene was 567 bp and cloned into pET-32a (+) vector successfully. The recombinant Cap protein was expressed as inclusion bodies. The results of optimization for indirect ELISA revealed that the optimal antigen and serum dilutions were selected to be 4 μg/well and 1:40, respectively; the coating condition was 37°C for 1 h and 4°C overnight; the blocking time in 1% BSA was 1 h at 37°C and the second antibody dilution was 1:800, the cut-off value was 0.352. Known anti-sera samples of other duck common pathogens were tested by the developed ELISA and the results showed it was specific for DuCV anti-sera detection. The sensitivity of indirect ELISA reached 1:2560, and the coefficient of variation between intra-assay and inter-assay were less than 10%. Compared the PCR and indirect ELISA methods, the positive compliance rate was 95.6% for detected 59 duck samples.
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University Chengdu, 611130, China ; Institute of Preventive Veterinary Medicine, Sichuan Agricultural University Chengdu, 611130, China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of 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, 611130, China.
authors.forename: Renyong
authors.initials: R
authors.lastname: Jia
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University Chengdu, 611130, China ; Institute of Preventive Veterinary Medicine, Sichuan Agricultural University Chengdu, 611130, China.
authors.forename: Zhilong
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of 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, 611130, China.
authors.forename: Mingshu
authors.initials: M
authors.lastname: Wang
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University Chengdu, 611130, China ; Institute of Preventive Veterinary Medicine, Sichuan Agricultural University Chengdu, 611130, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University Chengdu, 611130, China ; Key Laboratory of Animal Disease and Human Health of Sichuan Province Chengdu, 611130, China.
authors.forename: Dekang
authors.initials: D
authors.lastname: Zhu
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University Chengdu, 611130, China ; Institute of Preventive Veterinary Medicine, Sichuan Agricultural University Chengdu, 611130, China.
authors.forename: Shun
authors.initials: S
authors.lastname: Chen
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University Chengdu, 611130, China ; Institute of Preventive Veterinary Medicine, Sichuan Agricultural University Chengdu, 611130, China.
authors.forename: Mafeng
authors.initials: M
authors.lastname: Liu
authors.affiliation: Key Laboratory of Animal Disease and Human Health of Sichuan Province Chengdu, 611130, China.
authors.forename: Zhongqiong
authors.initials: Z
authors.lastname: Yin
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University Chengdu, 611130, China ; Key Laboratory of Animal Disease and Human Health of Sichuan Province Chengdu, 611130, China.
authors.forename: Xiaoyue
authors.initials: X
authors.lastname: Chen
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of 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, 611130, China.
authors.forename: Anchun
authors.initials: A
authors.lastname: Cheng
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000175:diagnosis*; D036022:Capsid Proteins / Q000032:analysis*; D018173:Circoviridae Infections / Q000175:diagnosis*; D017925:Circovirus; D004372:Ducks; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D066298:In Vitro Techniques; D012680:Sensitivity and Specificity
chemical_list: D036022:Capsid Proteins
keywords: Duck circovirus; cap gene; indirect ELISA; prokaryotic expression