ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2019, Vol. 50 ›› Issue (2): 454-460.doi: 10.11843/j.issn.0366-6964.2019.02.025

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Establishment and Clinical Application of an Indirect ELISA for Detection of Antibodies against PCV3 Based on Recombinant ORF2 Protein

WANG Junwei1,2, CHEN Fangzhou1,2, KU Xugang2, LI Chang1,2, HE Qigai1,2,3,4*   

  1. 1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China;
    2. College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China;
    3. Key Lab of Preventive Veterinary Medicine in Hubei Province, Wuhan 430070, China;
    4. The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China
  • Received:2018-05-21 Online:2019-02-23 Published:2019-02-23

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Abstract:

The aim of this study was to establish a sensitive and specific method for detection of antibody against porcine circovirus type 3 (PCV3). The bioinformatical prediction shows that most antigen epitopes of PCV3 Cap protein are clustered at the C terminal (carboxyl terminal) of PCV3-Cap protein, while first 33 amino acids in the N terminal of Cap protein are the nuclear localization sequences. Primers were designed to amplify a part of ORF2 gene which does not contain first 120 amino acids of N terminal and PCV3 positive samples were used as templates for PCR amplification. The recombinant plasmid was constructed by cloning the PCR product into pET-30a vector. The recombinant plasmid was transfected into E. coli BL21 competent cells, and the best induction conditions were determined. The expression protein was purified by Ni-NTA affinity chromatography. Finally, using the Cap protein as a coating protein, an indirect ELISA for detection of antibody against PCV3 was established and then primary applied to clinical detection. As a result:PCV3 ORF2 gene fragment with size of 285 bp was amplified from positive sample. The recombinant plasmid was constructed by inserting the amplicon into pET-30a, followed by enzymatical digestion and sequencing. Expression of the recombinant protein was induced by adding 1 mmol·L-1 IPTG, incubating at 37℃ for 6 h. Western blot result showed that the recombinant protein possessed good reactivity with positive serum against PCV3. The optimal antigen concentration, dilution of testing serum, working dilution of enzyme conjugated antibody of ELISA were 1 μg·mL-1, 1:20 and 1:5 000, respectively. The positive value was 0.273 and the coefficients of inter-and intra-batches were less than 10%, indicating the good repeatability of the method. PCV2 positive serum was negative in this method, indicating its high specificity. Finally, 439 clinical serum samples were detected through this ELISA method and 60.59% (266/439) of them were identified as positive. In summary, a rapid, easy to use, sensitive and specific method for detection of antibody against porcine circovirus type 3 (PCV3) was established.

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