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Transcription phase, protein characteristics of DEV UL45 and prokaryotic expression, antibody preparation of the UL45 des-transmembrane domain.
- Source :
-
Virology journal [Virol J] 2010 Sep 16; Vol. 7, pp. 232. Date of Electronic Publication: 2010 Sep 16. - Publication Year :
- 2010
-
Abstract
- Background: Some UL45 gene function of Herpesvirus was reported. While there was no any report of the duck enteritis virus (DEV) UL45 protein as yet.<br />Results: The UL45 gene and des-transmembrane domain of UL45 (named UL45Δ gene, 295-675bp of UL45) of DEV were amplified by PCR and subcloned into the prokaryotic expression vector pET-32a(+). The constructed recombinant plasmids were transformed into the host strain BL21(DE3) PLysS and induced by IPTG. SDS-PAGE analysis showed the UL45 gene couldn't express while UL45Δ gene was highly expressed. His Purify Kit or salting-out could purify the protein effectively. Using the purified protein to immunize New-Zealand rabbits and produce polyclonal antibody. The agar diffusion reaction showed the titer of antibody was 1:32. Western blot analysis indicated the purified rabbit anti-UL45Δ IgG had a high level of specificity and the UL45 gene was a part of DEV genome. The transcription phase study of UL45 gene showed that expression of UL45 mRNA was at a low level from 0 to 18 h post-infection (pi), then accumulated quickly at 24 h pi and peaked at 42 h pi. It can be detected till 72 h pi. Besides, western blot analysis of purified virion and different viral ingredients showed that the UL45 protein resided in the purified virion and the viral envelope.<br />Conclusions: The rabbit anti-UL45Δ IgG was produced successfully and it can serve as a good tool for penetrating studies of the function of DEV UL45 protein. The transcription phase and protein characteristics analysis indicated that DEV UL45 gene was a late gene and UL45 protein may be a viral envelope protein.
- Subjects :
- Animals
Antibodies, Viral immunology
Antibodies, Viral isolation & purification
Cloning, Molecular
Gene Expression
Rabbits
Recombinant Proteins genetics
Recombinant Proteins immunology
Recombinant Proteins isolation & purification
Time Factors
Transcription, Genetic
Viral Structural Proteins immunology
Virion chemistry
Ducks virology
Gene Expression Profiling
Gene Expression Regulation, Viral
Herpesviridae genetics
Viral Structural Proteins biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 1743-422X
- Volume :
- 7
- Database :
- MEDLINE
- Journal :
- Virology journal
- Publication Type :
- Academic Journal
- Accession number :
- 20843372
- Full Text :
- https://doi.org/10.1186/1743-422X-7-232