title: Expression and characterization of duck enteritis virus gI gene.
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.
authors: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China.
authors: Lijuan
authors: L
authors: Li
authors: Anchun
authors: A
authors: Cheng
authors: Mingshu
authors: M
authors: Wang
authors: Jun
authors: J
authors: Xiang
authors: Xiaoyuan
authors: X
authors: Yang
authors: Shunchuan
authors: S
authors: Zhang
authors: Dekang
authors: D
authors: Zhu
authors: Renyong
authors: R
authors: Jia
authors: Qihui
authors: Q
authors: Luo
authors: Yi
authors: Y
authors: Zhou
authors: Zhengli
authors: Z
authors: Chen
authors: Xiaoyue
authors: X
authors: Chen
references: Carbohydr Res. 1996 Oct 4;292:1-9
references: 8870235
references: Hua Xi Yi Ke Da Xue Xue Bao. 1999 Dec;30(4):455-6, 464
references: 11387969
references: Biotechnology. 1992;24:145-9
references: 1422008
references: Anal Biochem. 2000 Oct 15;285(2):194-204
references: 11017702
references: Virus Res. 2008 Sep;136(1-2):152-6
references: 18582977
references: Gene. 2007 Oct 15;401(1-2):88-96
references: 17706377
references: Chembiochem. 2003 Nov 7;4(11):1120-8
references: 14613102
references: Intervirology. 2009;52(3):141-51
references: 19478528
references: Virus Genes. 2006 Oct;33(2):221-7
references: 16972038
references: J Virol. 2001 Jan;75(1):323-40
references: 11119602
references: J Virol. 2002 Jan;76(1):1-8
references: 11739666
references: J Virol. 2009 Sep;83(17):8315-26
references: 19570876
references: J Virol. 2009 Jan;83(1):228-40
references: 18945783
references: Arch Virol. 2006 Jul;151(7):1281-9
references: 16502283
references: J Virol. 1998 Feb;72(2):1542-51
references: 9445058
references: Avian Dis. 2009 Sep;53(3):363-9
references: 19848073
references: Avian Dis. 2008 Jun;52(2):324-31
references: 18646465
references: Virol J. 2010 Jul 17;7:162
references: 20637115
references: Virol J. 2010 Jun 08;7:120
references: 20529349
references: J Virol. 2001 Jan;75(2):821-33
references: 11134295
references: J Virol. 1995 Dec;69(12):7951-9
references: 7494308
references: J Virol. 2003 Aug;77(15):8481-94
references: 12857917
references: J Virol. 2006 Apr;80(7):3167-79
references: 16537585
references: J Virol. 1999 Apr;73(4):2717-28
references: 10074118
references: Virus Genes. 2009 Apr;38(2):243-8
references: 19130202
references: J Virol. 1987 Jul;61(7):2208-16
references: 3035221
references: Virol J. 2009 Feb 10;6:19
references: 19208242
references: EMBO J. 1996 Nov 15;15(22):6096-110
references: 8947032
references: Virus Genes. 2008 Dec;37(3):328-32
references: 18690531
references: Virol J. 2010 Apr 29;7:83
references: 20429885
references: Virus Genes. 2009 Apr;38(2):289-94
references: 19153825
references: Comp Immunol Microbiol Infect Dis. 2005 May;28(3):187-96
references: 15857658
references: J Virol. 2004 Nov;78(21):11519-35
references: 15479793
references: Intervirology. 1993;36(2):99-112
references: 8294188
references: Arch Virol. 2009;154(7):1061-9
references: 19517212
references: Avian Dis. 1998 Jul-Sep;42(3):554-64
references: 9777156
references: J Virol. 1979 Aug;31(2):299-314
references: 225555
references: J Virol. 1998 Nov;72(11):8933-42
references: 9765438
references: J Virol. 1996 Nov;70(11):7878-84
references: 8892910
references: Acta Virol. 2008;52(1):23-30
references: 18459832
references: Intervirology. 2009;52(6):326-34
references: 19786809
references: Virol J. 2010 Jul 21;7:168
references: 20663161
references: J Gen Virol. 2003 Apr;84(Pt 4):939-947
references: 12655095
references: J Virol. 1974 Jul;14(1):8-19
references: 4365321
references: J Virol. 2002 Jun;76(11):5748-58
references: 11992003
references: Exp Hematol. 2002 Jun;30(6):503-12
references: 12063017