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Expressing gK gene of duck enteritis virus guided by bioinformatics and its applied prospect in diagnosis.

Zhang S, Ma G, Xiang J, Cheng A, Wang M, Zhu D, Jia R, Luo Q, Chen Z, Chen X - Virol. J. (2010)

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, 46# Xinkang Road, Ya'an, Sichuan 625014, China.

ABSTRACT

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.

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The fgK and its full-length amino acid sequence were analyzed by on-line prediction tools and DNASTAR7.0 software. A represented that the optimal exon domain was predicted by GENSCAN http://genes.mit.edu/GENSCAN.html. B represented that the potential antigenic epitopes of the gK was analyzed by DNASTAR7.0 software. C represented hydrophilicity domains of the gK was predicted on line http://mobyle.pasteur.fr/cgi-bin/portal.py?form=toppred.
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Figure 1: The fgK and its full-length amino acid sequence were analyzed by on-line prediction tools and DNASTAR7.0 software. A represented that the optimal exon domain was predicted by GENSCAN http://genes.mit.edu/GENSCAN.html. B represented that the potential antigenic epitopes of the gK was analyzed by DNASTAR7.0 software. C represented hydrophilicity domains of the gK was predicted on line http://mobyle.pasteur.fr/cgi-bin/portal.py?form=toppred.

Mentions: The GENESCAN prediction online indicated that the integral ORF of the DEV-gK gene was divided into 2 parts, which contained an optimal exon domain from 1 to 675 bp and a suboptimal exon domain from 676 to 1032 bp. In addition, the corresponding 225 aa polypeptide chain, encoded by the optimal exon domain shown in blue in Fig. 1A, could be easily expressed, however, the suboptimal exon domain might be hard to express according to the predicted result. To estimate the potential epitopes of DEV-gK, the amino acid sequence of DEV-gK was analyzed using DNASTAR 7.0 software. The putative DEV-gK epitopes thus identified were mainly located from amino acids 25-115, 135-215, and 270-295, with corresponding DNA sequences at nucleotides 73-345, 403-645, and 808-885 (Fig. 1B). Hydrophilicity estimation and transmembrane region assumption were performed using online prediction tools. Hydrophilic domains were mainly located from amino acids 7-27, 119-139, 227-247, 254-274, and 312-332 (Fig. 1C). Moreover, transmembrane regions were identified in 5 amino acid stretches, 7-29, 118-140, 220-242, 252-274, and 313-335. From the above data, the hydrophilic domains and transmembrane regions exist in a one-to-one ratio. To design the tgK gene (91-642 bp), the 4 predicted results were combined together and included the gK amino acids 31-214. Additionally, the tgK possessed good immunogenicity, determined by prediction, and only one potential transmembrane region. These results suggested that expression of tgK might be possible.


Expressing gK gene of duck enteritis virus guided by bioinformatics and its applied prospect in diagnosis.

Zhang S, Ma G, Xiang J, Cheng A, Wang M, Zhu D, Jia R, Luo Q, Chen Z, Chen X - Virol. J. (2010)

The fgK and its full-length amino acid sequence were analyzed by on-line prediction tools and DNASTAR7.0 software. A represented that the optimal exon domain was predicted by GENSCAN http://genes.mit.edu/GENSCAN.html. B represented that the potential antigenic epitopes of the gK was analyzed by DNASTAR7.0 software. C represented hydrophilicity domains of the gK was predicted on line http://mobyle.pasteur.fr/cgi-bin/portal.py?form=toppred.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921365&req=5

Figure 1: The fgK and its full-length amino acid sequence were analyzed by on-line prediction tools and DNASTAR7.0 software. A represented that the optimal exon domain was predicted by GENSCAN http://genes.mit.edu/GENSCAN.html. B represented that the potential antigenic epitopes of the gK was analyzed by DNASTAR7.0 software. C represented hydrophilicity domains of the gK was predicted on line http://mobyle.pasteur.fr/cgi-bin/portal.py?form=toppred.
Mentions: The GENESCAN prediction online indicated that the integral ORF of the DEV-gK gene was divided into 2 parts, which contained an optimal exon domain from 1 to 675 bp and a suboptimal exon domain from 676 to 1032 bp. In addition, the corresponding 225 aa polypeptide chain, encoded by the optimal exon domain shown in blue in Fig. 1A, could be easily expressed, however, the suboptimal exon domain might be hard to express according to the predicted result. To estimate the potential epitopes of DEV-gK, the amino acid sequence of DEV-gK was analyzed using DNASTAR 7.0 software. The putative DEV-gK epitopes thus identified were mainly located from amino acids 25-115, 135-215, and 270-295, with corresponding DNA sequences at nucleotides 73-345, 403-645, and 808-885 (Fig. 1B). Hydrophilicity estimation and transmembrane region assumption were performed using online prediction tools. Hydrophilic domains were mainly located from amino acids 7-27, 119-139, 227-247, 254-274, and 312-332 (Fig. 1C). Moreover, transmembrane regions were identified in 5 amino acid stretches, 7-29, 118-140, 220-242, 252-274, and 313-335. From the above data, the hydrophilic domains and transmembrane regions exist in a one-to-one ratio. To design the tgK gene (91-642 bp), the 4 predicted results were combined together and included the gK amino acids 31-214. Additionally, the tgK possessed good immunogenicity, determined by prediction, and only one potential transmembrane region. These results suggested that expression of tgK might be possible.

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, 46# Xinkang Road, Ya'an, Sichuan 625014, China.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus