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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 construction of cloning plasmids were identified by colony PCR and restriction enzymes (HindIII and XhoI) digestion. M1, DNA marker-DL2000; M2, DNA marker-DL15000. A. Identification of cloning plasmid pMD18-T/fgK. Lane 1, product of the colony PCR; Lane 2, two fragments after digestion of the recombinant DNA with restriction enzymes HindIII and XhoI. B. Identification of cloning plasmid pMD18-T/tgK. Lane 1, two fragments after digestion of the recombinant DNA with restriction enzymes HindIII and XhoI; Lane 2, product of the colony PCR.
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Figure 2: The construction of cloning plasmids were identified by colony PCR and restriction enzymes (HindIII and XhoI) digestion. M1, DNA marker-DL2000; M2, DNA marker-DL15000. A. Identification of cloning plasmid pMD18-T/fgK. Lane 1, product of the colony PCR; Lane 2, two fragments after digestion of the recombinant DNA with restriction enzymes HindIII and XhoI. B. Identification of cloning plasmid pMD18-T/tgK. Lane 1, two fragments after digestion of the recombinant DNA with restriction enzymes HindIII and XhoI; Lane 2, product of the colony PCR.

Mentions: By using high fidelity Taq enzyme to isolate the gK gene, PCR was carried out on DNA from the DEV genome. fgK was amplified by one pair of primers (P1, P2) and tgK was amplified by another primer set (P3, P4). Both pairs of primers were specific to the DEV-gK gene, seen through the screening for expected products (Fig. 2). The fgK PCR product was approximately 1000 bp and that of tgK was approximately 550 bp, which were respectively amplified and cloned into pMD18-T and were identified through colony PCR and restriction enzymes (HindIII and XhoI) digestion. Thus, positive recombinant plasmids was constructed for fgK and tgK, and were designated as pMD18-T/fgK (Fig. 2A) and pMD18-T/tgK (Fig. 2B), respectively. The sequencing results of both cloning plasmids showed that there were no nucleotide errors in the synthetic fgK gene and tgK gene fragment (data not shown).


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 construction of cloning plasmids were identified by colony PCR and restriction enzymes (HindIII and XhoI) digestion. M1, DNA marker-DL2000; M2, DNA marker-DL15000. A. Identification of cloning plasmid pMD18-T/fgK. Lane 1, product of the colony PCR; Lane 2, two fragments after digestion of the recombinant DNA with restriction enzymes HindIII and XhoI. B. Identification of cloning plasmid pMD18-T/tgK. Lane 1, two fragments after digestion of the recombinant DNA with restriction enzymes HindIII and XhoI; Lane 2, product of the colony PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The construction of cloning plasmids were identified by colony PCR and restriction enzymes (HindIII and XhoI) digestion. M1, DNA marker-DL2000; M2, DNA marker-DL15000. A. Identification of cloning plasmid pMD18-T/fgK. Lane 1, product of the colony PCR; Lane 2, two fragments after digestion of the recombinant DNA with restriction enzymes HindIII and XhoI. B. Identification of cloning plasmid pMD18-T/tgK. Lane 1, two fragments after digestion of the recombinant DNA with restriction enzymes HindIII and XhoI; Lane 2, product of the colony PCR.
Mentions: By using high fidelity Taq enzyme to isolate the gK gene, PCR was carried out on DNA from the DEV genome. fgK was amplified by one pair of primers (P1, P2) and tgK was amplified by another primer set (P3, P4). Both pairs of primers were specific to the DEV-gK gene, seen through the screening for expected products (Fig. 2). The fgK PCR product was approximately 1000 bp and that of tgK was approximately 550 bp, which were respectively amplified and cloned into pMD18-T and were identified through colony PCR and restriction enzymes (HindIII and XhoI) digestion. Thus, positive recombinant plasmids was constructed for fgK and tgK, and were designated as pMD18-T/fgK (Fig. 2A) and pMD18-T/tgK (Fig. 2B), respectively. The sequencing results of both cloning plasmids showed that there were no nucleotide errors in the synthetic fgK gene and tgK gene fragment (data not shown).

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