ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2006, Vol. 37 ›› Issue (7): 676-680.doi:

• 预防兽医 • Previous Articles     Next Articles

Fusion Expression of MPB70，MPB83 and ESAT-6 of Mycobacterium Bovis and Its Use as Diagnostic Reagent

GUO She-ping; LIU Si-guo;ZHANG Xiu-hua;WANG Chun-lai;GONG Qiang;GUO Yang;SHAO Mei-li
  

  1. 1.National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001,China; 2.Graduate School, CAAS, Beijing 100081, China; 3.Shenyang Agricultural University, Shenyang 110161, China; 4.Northeast Agricultural University, Harbin 150001, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2006-07-25 Published:2006-07-25

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Abstract: The DNA fragments of mpb70, mpb83 and esat-6 were amplified by PCR from M. bovis Vallee genome DNA. Mpb70 and mpb83 were spliced by overlapping extension (SOE). Then the DNA fragments were inserted into the plasmid pET32a(+) and the recombinant plasmid pET70-83-E6 was accquired. The recombinant protein was expressed in the form of solution in pET70-83-E6 transformed BL21（DE3）induced by IPTG. Then the recombinant protein was purified by affinity chromatography. Western blot analysis shows that the protein could be recognized by sera from M. bovis infected bovines but not by sera from M.paratuberculosis infected bovines. The purified protein was used as diagnostic reagent and found to be useful for immunodiagnostic of bovine tuberculosis (BTB) by enzyme linked immunosorbent (ELISA). In field test, 46 out of 117 bovine sera (67 of them were positive in PPD skin test) were positive (39.32%). 82.05% (96/117) of them were consistent with PPD skin test.