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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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Intracellular location and distribution of DEV gI proein analyzed by IIF. Mock-infected (A-C) and DEV-infected (D-R). DEFs were fixed at different stages (4 h p.i., 12 h p.i., 36 h p.i., and 48 h p.i.) as described in materials and methods. The samples were stained with the gI antiserum (A-C, G-R) or preimmune serum (D-F), and reacted with anti-rabbit IgG-conjugated FITC, and then counter-stained with DAPI (blue is representative the cell nuclei). A, D, G, J, M, and P show the FITC staining; and B, E, H, J, M, and Q show the DAPI staining. C, F, I, L, O, and R are the merged images of FITC and DAPI staining. The arrows indicate the DEV gI FITC fluorescence staining. (Images were acquired by using 20× objective)
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Figure 5: Intracellular location and distribution of DEV gI proein analyzed by IIF. Mock-infected (A-C) and DEV-infected (D-R). DEFs were fixed at different stages (4 h p.i., 12 h p.i., 36 h p.i., and 48 h p.i.) as described in materials and methods. The samples were stained with the gI antiserum (A-C, G-R) or preimmune serum (D-F), and reacted with anti-rabbit IgG-conjugated FITC, and then counter-stained with DAPI (blue is representative the cell nuclei). A, D, G, J, M, and P show the FITC staining; and B, E, H, J, M, and Q show the DAPI staining. C, F, I, L, O, and R are the merged images of FITC and DAPI staining. The arrows indicate the DEV gI FITC fluorescence staining. (Images were acquired by using 20× objective)

Mentions: Intracellular distribution of DEV gI protein could be visualized by IIF experiments utilizing rabbit immune serum against expressed gI protein or pre-immune serum. As shown in Figure 5, infected cells (Figure 5G-R) showed a specific green fluorescent cytoplasmic staining pattern, whereas essentially no signal was detected in mock-infected cells(Figure 5A-C) or corresponding preimmune serum (Figure 5D-F). The faint fluorescence could be detected in the cytoplasm of infected cells as early as 4 h p.i. (Figure 5G-I), and then a strong fluorescence was found intensively distributed in the cytoplasm and especially in the juxtanuclear region at 12 h p.i.. A typical pattern of staining is shown in Figure 5J-L. After that, following by a series of morphological changes, the cytoplasm disintegration and nuclear fragmentation in DEV-infected cells, fluorescence was gently dispersed at 36 h p.i.(Figure 5M-O) and 48 h p.i. (Figure 5P-R).


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)

Intracellular location and distribution of DEV gI proein analyzed by IIF. Mock-infected (A-C) and DEV-infected (D-R). DEFs were fixed at different stages (4 h p.i., 12 h p.i., 36 h p.i., and 48 h p.i.) as described in materials and methods. The samples were stained with the gI antiserum (A-C, G-R) or preimmune serum (D-F), and reacted with anti-rabbit IgG-conjugated FITC, and then counter-stained with DAPI (blue is representative the cell nuclei). A, D, G, J, M, and P show the FITC staining; and B, E, H, J, M, and Q show the DAPI staining. C, F, I, L, O, and R are the merged images of FITC and DAPI staining. The arrows indicate the DEV gI FITC fluorescence staining. (Images were acquired by using 20× objective)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Intracellular location and distribution of DEV gI proein analyzed by IIF. Mock-infected (A-C) and DEV-infected (D-R). DEFs were fixed at different stages (4 h p.i., 12 h p.i., 36 h p.i., and 48 h p.i.) as described in materials and methods. The samples were stained with the gI antiserum (A-C, G-R) or preimmune serum (D-F), and reacted with anti-rabbit IgG-conjugated FITC, and then counter-stained with DAPI (blue is representative the cell nuclei). A, D, G, J, M, and P show the FITC staining; and B, E, H, J, M, and Q show the DAPI staining. C, F, I, L, O, and R are the merged images of FITC and DAPI staining. The arrows indicate the DEV gI FITC fluorescence staining. (Images were acquired by using 20× objective)
Mentions: Intracellular distribution of DEV gI protein could be visualized by IIF experiments utilizing rabbit immune serum against expressed gI protein or pre-immune serum. As shown in Figure 5, infected cells (Figure 5G-R) showed a specific green fluorescent cytoplasmic staining pattern, whereas essentially no signal was detected in mock-infected cells(Figure 5A-C) or corresponding preimmune serum (Figure 5D-F). The faint fluorescence could be detected in the cytoplasm of infected cells as early as 4 h p.i. (Figure 5G-I), and then a strong fluorescence was found intensively distributed in the cytoplasm and especially in the juxtanuclear region at 12 h p.i.. A typical pattern of staining is shown in Figure 5J-L. After that, following by a series of morphological changes, the cytoplasm disintegration and nuclear fragmentation in DEV-infected cells, fluorescence was gently dispersed at 36 h p.i.(Figure 5M-O) and 48 h p.i. (Figure 5P-R).

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