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-β-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. Conclusion: Thus, the polyclonal antibody prepared here may serve as a valuable tool to study the functional involvement of VP26 in the DPV life cycle.

Characterization of Synonymous Codon Usage Bias in the Duck Plague Virus UL35 Gene

“…Therefore, the E. coli expression system may be better applied to the production of the DPV UL35 protein. Our hypothesis was verified in our recent study, in which the DPV UL35 protein was successfully expressed in the E. coli expression system [67] . In summary, our work has provided a basic understanding of the evolution and pathogenesis of the DPV UL35 gene and offered some new insights into the mechanisms for codon usage bias, as well as contributing significantly to the area of herpesvirus research and possibly studies with other viruses.…”

Preparation and characterization of an antiserum against truncated UL54 protein of pseudorabies virus

“…For the expression of the truncated UL54 protein, the E. coli strain BL21 (DE3) was used. This strain has the advantage of being deficient in both the lon and ompT proteases and harbors the T7 bacteriophage RNA polymerase gene, which permits the specific expression of heterologous genes driven by the T7 promoter (Studier et al, 1990;Mierendorf et al, 1998;Cai et al, 2009b).…”

Prokaryotic expression and characteristics of duck enteritis virus UL29 gene

“…The urea was removed by the gradient dialysis. Finally, the initially purified recombinant His-tagged ICP8 was again purified by immobilized metal affinity chromatography on Ni 2+ -NTA affinity resin (Cai et al, 2009). To produce anti-DEV ICP8 polyclonal antibody, 6 healthy white rabbits were immunized intradermally with a 1.0 mg of ICP8 with an equal volume of complete Freund's adjuvant (Promega, USA).…”