Cloning, expression and characterization of gE protein of duck plague virus.
BACKGROUND
The gE protein of duck plague virus is the important membrane glycoprotein, its protein characterization has not been reported. In this study, we expressed and presented the characterization of the DPV gE product.


RESULTS
According to the sequence of the gE gene, a pair of primers were designed, and the DNA product with 1490bp in size was amplified by using the polymerase chain reaction (PCR). The PCR product was cloned into pMD18-T vector, and subcloned into pET32a(+), generating the recombinant plasmid pET32a/DPV-gE. SDS-PAGE analysis showed that the fusion pET32a/DPV-gE protein was highly expressed after induction by 0.2 mM IPTG at 30 degrees C for 4.5 h in Rosseta host cells. Over expressed 6xHis-gE fusion protein was purified by nickel affinity chromatography, and used to immunize the rabbits for the preparation of polyclonal antibody. The result of the intracellular localization revealed that the gE protein was appeared to be in the cytoplasm region. The real time PCR, RT-PCR analysis and Western blotting revealed that the gE gene was produced most abundantly during the late phase of replication in DPV-infected cells.


CONCLUSIONS
In this work, the DPV gE protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gE product for the first time. These properties of the gE protein provided a prerequisite for further functional analysis of this gene.
Chang
Hua
H
Avian Diseases Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Yaan, Sichuan, China.
Cheng
Anchun
A
Wang
Mingshu
M
Zhu
Dekang
D
Jia
Renyong
R
Liu
Fei
F
Chen
Zhengli
Z
Luo
Qihui
Q
Chen
Xiaoyue
X
Zhou
Yi
Y
D000818:Animals; D003001:Cloning, Molecular; D004372:Ducks; D015870:Gene Expression; D025922:Mardivirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008380:Marek Disease / Q000276:immunology / Q000821:virology; D011201:Poultry Diseases / Q000276:immunology / Q000821:virology; D021381:Protein Transport; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D014759:Viral Envelope Proteins