His6-tagged UL35 protein of duck plague virus: expression, purification, and production of polyclonal antibody.
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.
Cai
Ming-Sheng
MS
Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Yaan, China.
Cheng
An-Chun
AC
Wang
Ming-Shu
MS
Zhao
Li-Chan
LC
Zhu
De-Kang
DK
Luo
Qi-Hui
QH
Liu
Fei
F
Chen
Xiao-Yue
XY
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
D000914:Antibodies, Viral; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins