ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2017, Vol. 48 ›› Issue (1): 150-156.doi: 10.11843/j.issn.0366-6964.2017.01.018

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Prokaryotic Expression of N Protein of Caprine Parainfluenza Virus Type 3 and Establishment of Indirect ELISA Antibody Detection Method

WANG Mi1,2，LI Wen-liang1* ，HAO Fei1，MAO Li1，YANG Lei-lei1, ZHANG Wen-wen1, JIANG Jie-yuan1*   

  1. (1.Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China)
  • Received:2016-07-04 Online:2017-01-23 Published:2017-01-23

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Abstract:

Caprine parainfluenza virus type 3 (CPIV) can cause respiratory disease in goats, which could cause extensive morbidity and mortality when co-infected with Mycoplasma, bacteria and other pathogens. In this study, an indirect ELISA detection method based on CPIV3 N protein was established to monitor the disease. Primers were designed to amplify the N gene of CPIV3 JS2013 strain. Target gene was cloned into prokaryotic expression plasmid pET32a(+), to construct the recombinant plasmid pET32a-N, and then pET32a-N was transformed into Escherichia coli BL21 competent cells. Large amount expression of recombinant N protein was induced by IPTG. By optimizing the reaction conditions, an indirect ELISA was established. Clinical sera were tested by indirect ELISA and the results were compared with HI test. The recombinant plasmid was constructed successfully, and the expression of protein was identified by SDS-PAGE and Western blot, which confirmed that the protein was expressed correctly and had good immunogenicity and specificity. The purified protein was used as antigen and the indirect ELISA reaction conditions were optimized as follows: the coating antigen concentration was 1 μg•mL-1, serum dilution was 1:200, serum reaction time was 60 min, HRP-labeled secondary antibody dilution was 1:6 000, reaction time of secondary antibody was 30 min, and substrate chromogenic time was 10 min. By testing of 138 clinical serum samples with the indirect ELISA and HI test, the coincidence rate between these two methods was 88.41%. The indirect ELISA established in this study was sensitive, accurate, which will be suitable and useful for the detection of clinical samples and large scale serological surveys.

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