title: Transcription phase, protein characteristics of DEV UL45 and prokaryotic expression, antibody preparation of the UL45 des-transmembrane domain.
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.
  
  
  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.
authors.affiliation: Avian Diseases Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Ya'an 625014, Sichuan, China.
authors.forename: Ai-Mei
authors.initials: AM
authors.lastname: Shen
authors.forename: Guang-Peng
authors.initials: GP
authors.lastname: Ma
authors.forename: An-Chun
authors.initials: AC
authors.lastname: Cheng
authors.forename: Ming-Shu
authors.initials: MS
authors.lastname: Wang
authors.forename: Dan-Dan
authors.initials: DD
authors.lastname: Luo
authors.forename: Li-Ting
authors.initials: LT
authors.lastname: Lu
authors.forename: Tao
authors.initials: T
authors.lastname: Zhou
authors.forename: De-Kang
authors.initials: DK
authors.lastname: Zhu
authors.forename: Qi-Hui
authors.initials: QH
authors.lastname: Luo
authors.forename: Ren-Yong
authors.initials: RY
authors.lastname: Jia
authors.forename: Zheng-Li
authors.initials: ZL
authors.lastname: Chen
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhou
authors.forename: Xiao-Yue
authors.initials: XY
authors.lastname: Chen
mesh_terms: 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
chemical_list: D000914:Antibodies, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins