Transcription phase, protein characteristics of DEV UL45 and prokaryotic expression, antibody preparation of the UL45 des-transmembrane domain.
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.


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.


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.
Shen
Ai-Mei
AM
Avian Diseases Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Ya'an 625014, Sichuan, China.
Ma
Guang-Peng
GP
Cheng
An-Chun
AC
Wang
Ming-Shu
MS
Luo
Dan-Dan
DD
Lu
Li-Ting
LT
Zhou
Tao
T
Zhu
De-Kang
DK
Luo
Qi-Hui
QH
Jia
Ren-Yong
RY
Chen
Zheng-Li
ZL
Zhou
Yi
Y
Chen
Xiao-Yue
XY
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology / Q000302:isolation & purification; D003001:Cloning, Molecular; D004372:Ducks / Q000821:virology*; D015870:Gene Expression; D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral; D006564:Herpesviridae / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013997:Time Factors; D014158:Transcription, Genetic; D015678:Viral Structural Proteins / Q000096:biosynthesis* / Q000276:immunology; D014771:Virion / Q000737:chemistry
D000914:Antibodies, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins