Expressing gK gene of duck enteritis virus guided by bioinformatics and its applied prospect in diagnosis.
BACKGROUND
Duck viral enteritis, which is caused by duck enteritis virus (DEV), causes significant economic losses in domestic and wild waterfowls because of the high mortality and low egg production rates. With the purpose of eliminating this disease and decreasing economic loss in the commercial duck industry, researching on glycoprotein K (gK) of DEV may be a new kind of method for preventing and curing this disease. Because glycoproteins project from the virus envelope as spikes and are directly involved in the host immune system and elicitation of the host immune responses, and also play an important role in mediating infection of target cells, the entry into cell for free virus and the maturation or egress of virus. The gK is one of the major envelope glycoproteins of DEV. However, little information correlated with gK is known, such as antigenic and functional characterization.


RESULTS
Bioinformatic predictions revealed that the expression of the full-length gK gene (fgK) in a prokaryotic system is difficult because of the presence of suboptimal exon and transmembrane domains at the C-terminal. In this study, we found that the fgK gene might not be expressed in a prokaryotic system in accordance with the bioinformatic predictions. Further, we successfully used bioinformatics tools to guide the prokaryotic expression of the gK gene by designing a novel truncated gK gene (tgK). These findings indicated that bioinformatics provides theoretical data for target gene expression and saves time for our research. The recombinant tgK protein (tgK) was expressed and purified by immobilized metal affinity chromatography (IMAC). Western blotting and indirect enzyme-linked immunosorbent assay (ELISA) showed that the tgK possessed antigenic characteristics similar to native DEV-gK.


CONCLUSIONS
In this work, the DEV-tgK was expressed successfully in prokaryotic system for the first time, which will provide usefull information for prokaryotic expression of alphaherpesvirus gK homologs, and the recombinant truncated gK possessed antigenic characteristics similar to native DEV gK. Because of the good reactionogenicity, specificity and sensitivity, the purified tgK could be useful for developing a sensitive serum diagnostic kit to monitor DEV outbreaks.
Zhang
Shunchuan
S
Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, 46# Xinkang Road, Ya'an, Sichuan 625014, China.
Ma
Guangpeng
G
Xiang
Jun
J
Cheng
Anchun
A
Wang
Mingshu
M
Zhu
Dekang
D
Jia
Renyong
R
Luo
Qihui
Q
Chen
Zhengli
Z
Chen
Xiaoyue
X
D018138:Alphaherpesvirinae / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001715:Bird Diseases / Q000175:diagnosis* / Q000821:virology*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004372:Ducks; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D014773:Virology / Q000379:methods*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D004279:DNA, Viral; D006023:Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins