ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2010, Vol. 41 ›› Issue (9): 1147-1153.doi:

• 预防兽医 • Previous Articles     Next Articles

Construction of Recombinant Baculovirus Containing Peste des Petitis Ruminants Virus N Gene and Establishment of Indirect ELISA for Detecting the Serum Antibody

LI Wei1, LI Gang1*, WU Xiao-dong2, QIU Wen-ying1, ZHANG Kun1, Hermann Unger3   

  1. 1. State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. National Exotic Animal Disease Center, China Animal Health & Epidemiology Center, Qingdao 266032, China; 3. FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf, Austria
  • Received:1900-01-01 Revised:1900-01-01 Online:2010-09-20 Published:2010-09-20

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Abstract: This experiment was conducted to diagnose Peste des Petitis Ruminants based on the eukaryo-expressed PPRV nucleoprotein through indirect ELISA. The full-length PPRV nucleoprotein gene was obtained from viral genome RNA by RT-PCR. The amplified fragments were cloned into baculovirus donor vectors pFastHTA of Bac-to-Bac system. These recombinant plasmids, pFastHTA-PPRV-N, were transformed into DH10Bac host bacteria to get recombinant shuttle plasmids, pBacmid-PPRV-N. Recombinant baculovirus, Bacmid-PPRV-N, was generated for expressing PPRV nucleoprotein by transfecting recombinant pBacmid-PPRV-N with LipofectAMINE 2000 into Sf21 insect cells. PPRV nucleoprotein which efficiently expressed by baculovirus in Sf21 cells was verified by SDS-PAGE and Western blot. Furthermore, indirect ELISA was developed using recombinant PPRV nucleoprotein as coating antigen. 37 goats′ sera from epidemic area in Tibet Autonomous Region and 92 goats′ sera from non-infected area in Qinghai Province were detected by indirect ELISA and international standard cELISA kit simultaneously. The sensitivity and specificity of indirect ELISA were 100% and 96.2% compared with cELISA kit. The coincidence rate of the two methods was 96.9%. The results demonstrated that the indirect ELISA established in this study works well in the PPR diagnosis.