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Expression and characterization of duck enteritis virus gI gene.

Li L, Cheng A, Wang M, Xiang J, Yang X, Zhang S, Zhu D, Jia R, Luo Q, Zhou Y, Chen Z, Chen X - Virol. J. (2011)

Bottom Line: DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG).The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully.The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China.

ABSTRACT

Background: At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein.

Results: The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively.

Conclusions: The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.

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Determination of mRNA expression of gI in DEV-infected DEFs. The total RNA were harvested from uninfected(Neg) or DEV-infected DEFs at different times p.i. (0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 60 h), presented on the x-axis. After reverse transcription, the cDNA was used as template for SYBR Green RT-PCR analysis, the data were normalized to the expression level of β-actin, as indicated in the y-axis. Data were analyzed using the iQ5 optical system software (Bio-Rad)
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Figure 4: Determination of mRNA expression of gI in DEV-infected DEFs. The total RNA were harvested from uninfected(Neg) or DEV-infected DEFs at different times p.i. (0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 60 h), presented on the x-axis. After reverse transcription, the cDNA was used as template for SYBR Green RT-PCR analysis, the data were normalized to the expression level of β-actin, as indicated in the y-axis. Data were analyzed using the iQ5 optical system software (Bio-Rad)

Mentions: The mRNA expression of gI gene at different times after infection were determined by normalizing the cycle threshold (CT) values with β-actin gene CT values, and then the histogram reflecting the transcription tendency of gI gene was constructed by iQ5 Optical System Software(Figure 4). It cound be found that, the level of mRNA was low in early phases of infection, presenting slightly increased after 3 h p.i.. Subsequently, signal intensity immediately increased after 12 h p.i., peaked at 48 h p.i., and then declined.


Expression and characterization of duck enteritis virus gI gene.

Li L, Cheng A, Wang M, Xiang J, Yang X, Zhang S, Zhu D, Jia R, Luo Q, Zhou Y, Chen Z, Chen X - Virol. J. (2011)

Determination of mRNA expression of gI in DEV-infected DEFs. The total RNA were harvested from uninfected(Neg) or DEV-infected DEFs at different times p.i. (0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 60 h), presented on the x-axis. After reverse transcription, the cDNA was used as template for SYBR Green RT-PCR analysis, the data were normalized to the expression level of β-actin, as indicated in the y-axis. Data were analyzed using the iQ5 optical system software (Bio-Rad)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3118967&req=5

Figure 4: Determination of mRNA expression of gI in DEV-infected DEFs. The total RNA were harvested from uninfected(Neg) or DEV-infected DEFs at different times p.i. (0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 60 h), presented on the x-axis. After reverse transcription, the cDNA was used as template for SYBR Green RT-PCR analysis, the data were normalized to the expression level of β-actin, as indicated in the y-axis. Data were analyzed using the iQ5 optical system software (Bio-Rad)
Mentions: The mRNA expression of gI gene at different times after infection were determined by normalizing the cycle threshold (CT) values with β-actin gene CT values, and then the histogram reflecting the transcription tendency of gI gene was constructed by iQ5 Optical System Software(Figure 4). It cound be found that, the level of mRNA was low in early phases of infection, presenting slightly increased after 3 h p.i.. Subsequently, signal intensity immediately increased after 12 h p.i., peaked at 48 h p.i., and then declined.

Bottom Line: DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG).The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully.The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China.

ABSTRACT

Background: At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein.

Results: The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively.

Conclusions: The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.

Show MeSH
Related in: MedlinePlus