Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (16): 3460-3469.doi: 10.3864/j.issn.0578-1752.2013.16.017

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Development of iELISA for Detection and Application of Brucella abortus Virb8

 ZHANG Jian1,2, MA Wei-ming2, ZHANG Liang1, HE Hong-bin1, DING Jia-bo3, YANG Hong-jun1   

  1. 1.Cow Research Center, Shandong Academy of Agricultural Science, Ji,nan 250100
    2.College of Veterinary Medicine, Shangdong Agricultural University, Tai’an 271018, Shandong
    3.China Institute of Veterinary Drug Control, Beijing 100081
  • Received:2013-01-04 Online:2013-08-15 Published:2013-05-31

Abstract: 【Objective】 Brucella abortus Virb8 is an important Brucella virulence factor. The aim of this study was to construct a prokaryotic expression vector for Brucella Virb8. Then, an indirect enzyme-linked immunosorbent assay (iELISA) for detecting antibodies to Brucella abortus was established using the purified recombinant protein. 【Method】 Brucella S2 genome sequence was extracted and used as a templet in polymerase chain reaction to amplify Virb8 gene. The PCR product was cloned into the pEASY-T3 vector and subsequently sequenced. Then, the gene was cloned into the expression vector pET-32a to construct the recombinant plasmid pET-32a-Virb8. The fused protein pET-32a-Virb8 was expressed and purified by Ni-NTA Spin Kit. Serum samples (n=235) were simultaneously tested by the iELISA and the rose bengal plate test (RBPT). 【Result】 In this study, Brucella Virb8 was successfully cloned and highly purified protein was obtained. The established iELSIA was stable and sensitive. The coincidence of iELISA with RBPT was 97.02%. 【Conclusion】 The iELISA could be used as a method for serological diagnosis of brucellosis and a foundation for advance study on immunogenicity and protection of brucellosis.

Key words: Brucella abortus , Virb8 , iELISA

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