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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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Related in: MedlinePlus

The different samples were analyzed by Coomassie blue-stained polyacrylamide gel. The direction of arrow represented the target protein. M represented standard protein molecular weight markers. A. Lane 1, the un-induced BL21 bacteria within pET-32b(+) plasmid; Lane 2, the induced BL21 bacteria within pET-32b(+) plasmid; Lane 3, the un-induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 4, the supernatant of IB with sonication was analyzed by SDS-PAGE, that the IB was harvested from the induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 5, the pellet of IB with sonication was analyzed by SDS-PAGE, that the IB was harvested from the induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 6, the induced Plys bacteria within pET-32b(+)/fgK plasmid; Lane 7, the induced Rosetta bacteria within pET-32b(+)/fgK plasmid. B. Lane 1, the un-induced BL21 bacteria within pET-32b(+) plasmid; Lane 2, the induced BL21 bacteria within pET-32b(+) plasmid; Lane 3, the un-induced BL21 bacteria within pET-32b(+)/tgK plasmid; Lane 4, the induced BL21 bacteria within pET-32b(+)/tgK plasmid; Lane 5, the induced Plys bacteria within pET-32b(+)/tgK plasmid; Lane 6, the induced Rosetta bacteria within pET-32b(+)/tgK plasmid. C. The determination of the optimal induced temperature. Lanes 1, 2 and 3 representing the induced temperatures were 25°C, 30°C and 37°C, respectively. D. The determination of the optimal induced IPTG final concentration. Lanes 1, 2, 3, 4, 5, 6, 7 and 8 representing the induced IPTG final concentrations were 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM and 1.0 mM, respectively. E. The determination of the optimal induced duration. Lanes 1, 2, 3 and 4 representing the induced durations were 2.0 h, 4.0 h, 8.0 h and 16.0 h, respectively. F. Purification of IB and recombinant truncated gK. Lanes 1 and 2 respectively represented the supernatant and pellet of the induced bacteria within pET-32b(+)/tgK plasmid with sonication; Lane 3 represented the purified recombinant truncated gK by a single chromatographic step of IMAC on Ni2+-NTA agarose.
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Figure 4: The different samples were analyzed by Coomassie blue-stained polyacrylamide gel. The direction of arrow represented the target protein. M represented standard protein molecular weight markers. A. Lane 1, the un-induced BL21 bacteria within pET-32b(+) plasmid; Lane 2, the induced BL21 bacteria within pET-32b(+) plasmid; Lane 3, the un-induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 4, the supernatant of IB with sonication was analyzed by SDS-PAGE, that the IB was harvested from the induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 5, the pellet of IB with sonication was analyzed by SDS-PAGE, that the IB was harvested from the induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 6, the induced Plys bacteria within pET-32b(+)/fgK plasmid; Lane 7, the induced Rosetta bacteria within pET-32b(+)/fgK plasmid. B. Lane 1, the un-induced BL21 bacteria within pET-32b(+) plasmid; Lane 2, the induced BL21 bacteria within pET-32b(+) plasmid; Lane 3, the un-induced BL21 bacteria within pET-32b(+)/tgK plasmid; Lane 4, the induced BL21 bacteria within pET-32b(+)/tgK plasmid; Lane 5, the induced Plys bacteria within pET-32b(+)/tgK plasmid; Lane 6, the induced Rosetta bacteria within pET-32b(+)/tgK plasmid. C. The determination of the optimal induced temperature. Lanes 1, 2 and 3 representing the induced temperatures were 25°C, 30°C and 37°C, respectively. D. The determination of the optimal induced IPTG final concentration. Lanes 1, 2, 3, 4, 5, 6, 7 and 8 representing the induced IPTG final concentrations were 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM and 1.0 mM, respectively. E. The determination of the optimal induced duration. Lanes 1, 2, 3 and 4 representing the induced durations were 2.0 h, 4.0 h, 8.0 h and 16.0 h, respectively. F. Purification of IB and recombinant truncated gK. Lanes 1 and 2 respectively represented the supernatant and pellet of the induced bacteria within pET-32b(+)/tgK plasmid with sonication; Lane 3 represented the purified recombinant truncated gK by a single chromatographic step of IMAC on Ni2+-NTA agarose.

Mentions: After sequence confirmation, the recombinant expression plasmids pET-32b(+)/fgK and pET-32b(+)/tgK were transformed into the expression host E. coli BL21. The positive transformants were selected for recombinant protein expression. Initially, the expression of fgK and tgK were induced at 37°C for 4 h by the addition of 0.2 mM IPTG. The induced pET-32b(+)/fgK was compared with uninduced pET-32b(+)/fgK culture and induced and uninduced bacteria carrying an empty pET32b(+) vector by SDS-PAGE. However, a band with a molecular weight approximately 57 kDa was not observed (Fig. 4A, lanes 4 and 5). Therefore, the recombinant pET-32b(+)/fgK expression plasmid was transformed into other expression hosts (Plys, Rosetta) with the same result.


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 different samples were analyzed by Coomassie blue-stained polyacrylamide gel. The direction of arrow represented the target protein. M represented standard protein molecular weight markers. A. Lane 1, the un-induced BL21 bacteria within pET-32b(+) plasmid; Lane 2, the induced BL21 bacteria within pET-32b(+) plasmid; Lane 3, the un-induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 4, the supernatant of IB with sonication was analyzed by SDS-PAGE, that the IB was harvested from the induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 5, the pellet of IB with sonication was analyzed by SDS-PAGE, that the IB was harvested from the induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 6, the induced Plys bacteria within pET-32b(+)/fgK plasmid; Lane 7, the induced Rosetta bacteria within pET-32b(+)/fgK plasmid. B. Lane 1, the un-induced BL21 bacteria within pET-32b(+) plasmid; Lane 2, the induced BL21 bacteria within pET-32b(+) plasmid; Lane 3, the un-induced BL21 bacteria within pET-32b(+)/tgK plasmid; Lane 4, the induced BL21 bacteria within pET-32b(+)/tgK plasmid; Lane 5, the induced Plys bacteria within pET-32b(+)/tgK plasmid; Lane 6, the induced Rosetta bacteria within pET-32b(+)/tgK plasmid. C. The determination of the optimal induced temperature. Lanes 1, 2 and 3 representing the induced temperatures were 25°C, 30°C and 37°C, respectively. D. The determination of the optimal induced IPTG final concentration. Lanes 1, 2, 3, 4, 5, 6, 7 and 8 representing the induced IPTG final concentrations were 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM and 1.0 mM, respectively. E. The determination of the optimal induced duration. Lanes 1, 2, 3 and 4 representing the induced durations were 2.0 h, 4.0 h, 8.0 h and 16.0 h, respectively. F. Purification of IB and recombinant truncated gK. Lanes 1 and 2 respectively represented the supernatant and pellet of the induced bacteria within pET-32b(+)/tgK plasmid with sonication; Lane 3 represented the purified recombinant truncated gK by a single chromatographic step of IMAC on Ni2+-NTA agarose.
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Figure 4: The different samples were analyzed by Coomassie blue-stained polyacrylamide gel. The direction of arrow represented the target protein. M represented standard protein molecular weight markers. A. Lane 1, the un-induced BL21 bacteria within pET-32b(+) plasmid; Lane 2, the induced BL21 bacteria within pET-32b(+) plasmid; Lane 3, the un-induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 4, the supernatant of IB with sonication was analyzed by SDS-PAGE, that the IB was harvested from the induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 5, the pellet of IB with sonication was analyzed by SDS-PAGE, that the IB was harvested from the induced BL21 bacteria within pET-32b(+)/fgK plasmid; Lane 6, the induced Plys bacteria within pET-32b(+)/fgK plasmid; Lane 7, the induced Rosetta bacteria within pET-32b(+)/fgK plasmid. B. Lane 1, the un-induced BL21 bacteria within pET-32b(+) plasmid; Lane 2, the induced BL21 bacteria within pET-32b(+) plasmid; Lane 3, the un-induced BL21 bacteria within pET-32b(+)/tgK plasmid; Lane 4, the induced BL21 bacteria within pET-32b(+)/tgK plasmid; Lane 5, the induced Plys bacteria within pET-32b(+)/tgK plasmid; Lane 6, the induced Rosetta bacteria within pET-32b(+)/tgK plasmid. C. The determination of the optimal induced temperature. Lanes 1, 2 and 3 representing the induced temperatures were 25°C, 30°C and 37°C, respectively. D. The determination of the optimal induced IPTG final concentration. Lanes 1, 2, 3, 4, 5, 6, 7 and 8 representing the induced IPTG final concentrations were 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM and 1.0 mM, respectively. E. The determination of the optimal induced duration. Lanes 1, 2, 3 and 4 representing the induced durations were 2.0 h, 4.0 h, 8.0 h and 16.0 h, respectively. F. Purification of IB and recombinant truncated gK. Lanes 1 and 2 respectively represented the supernatant and pellet of the induced bacteria within pET-32b(+)/tgK plasmid with sonication; Lane 3 represented the purified recombinant truncated gK by a single chromatographic step of IMAC on Ni2+-NTA agarose.
Mentions: After sequence confirmation, the recombinant expression plasmids pET-32b(+)/fgK and pET-32b(+)/tgK were transformed into the expression host E. coli BL21. The positive transformants were selected for recombinant protein expression. Initially, the expression of fgK and tgK were induced at 37°C for 4 h by the addition of 0.2 mM IPTG. The induced pET-32b(+)/fgK was compared with uninduced pET-32b(+)/fgK culture and induced and uninduced bacteria carrying an empty pET32b(+) vector by SDS-PAGE. However, a band with a molecular weight approximately 57 kDa was not observed (Fig. 4A, lanes 4 and 5). Therefore, the recombinant pET-32b(+)/fgK expression plasmid was transformed into other expression hosts (Plys, Rosetta) with the same result.

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