title: His6-tagged UL35 protein of duck plague virus: expression, purification, and production of polyclonal antibody.
abstract: OBJECTIVE
  Duck plague virus (DPV), the causative agent of duck plague (DP), is an alphaherpesvirus that causes an acute, febrile, contagious, and septic disease of waterfowl. UL35 protein (VP26) is a major capsid protein encoded by the UL35 gene, which is located in the unique long (UL) region of the DPV genome. To investigate the specific roles of VP26, the UL35 gene was amplified from the DPV DNA by polymerase chain reaction (PCR) and subcloned into pET-32a(+).
  
  
  METHODS
  The resultant prokaryotic expression vector, pET-32a(+)/UL35, includes an amino-terminal His6 as a fusion partner. Escherichia coli BL21 (DE3) competent cells were transformed with the construct and protein expression was subsequently induced by the addition of isopropyl-beta-D-thiogalactopyranoside to the culture medium. Protein lysates were submitted to SDS-PAGE to evaluate recombinant protein expression.
  
  
  RESULTS
  The band that corresponded to the predicted protein size (33 kDa) was observed on the SDS-PAGE gel. The recombinant His6-tagged VP26 fusion protein was expressed at a high level in an insoluble form (inclusion bodies) and constituted about 24% of the total cellular protein. Then, the fusion protein was purified to near homogeneity using single-step immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid affinity resin, yielding about 620 mg per liter culture. After purification, New Zealand white rabbits were immunized with purified His6-tagged VP26 in order to raise polyclonal antibody against this recombinant protein. Using the resultant sera, Western blot analysis showed that the recombinant protein was recognized by the polyclonal antibody.
  
  
  CONCLUSIONS
  Thus, the polyclonal antibody prepared here may serve as a valuable tool to study the functional involvement of VP26 in the DPV life cycle.
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Yaan, China.
authors.forename: Ming-Sheng
authors.initials: MS
authors.lastname: Cai
authors.forename: An-Chun
authors.initials: AC
authors.lastname: Cheng
authors.forename: Ming-Shu
authors.initials: MS
authors.lastname: Wang
authors.forename: Li-Chan
authors.initials: LC
authors.lastname: Zhao
authors.forename: De-Kang
authors.initials: DK
authors.lastname: Zhu
authors.forename: Qi-Hui
authors.initials: QH
authors.lastname: Luo
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.forename: Xiao-Yue
authors.initials: XY
authors.lastname: Chen
mesh_terms: D018138:Alphaherpesvirinae / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D001715:Bird Diseases / Q000821:virology*; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004372:Ducks / Q000821:virology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins